PROPOSED: 9/ 2003 Styrene ( CAS Reg. No. 100 42 5)

PROPOSED ACUTE EXPOSURE GUIDELINE LEVELS ( AEGLs) PREFACE Under the authority of the Federal Advisory Committee Act ( FACA) P. L. 92 463 of 1972, the National Advisory Committee for Acute Exposure Guideline Levels for Hazardous Substances ( NAC/ AEGL Committee) has been established to identify, review and interpret relevant toxicologic and other scientific data and develop AEGLs for high priority, acutely toxic chemicals.

AEGLs represent threshold exposure limits for the general public and are applicable to emergency exposure periods ranging from 10 minutes to 8 hours. Three levels   AEGL 1, AEGL 2 and AEGL 3   are developed for each of five exposure periods ( 10 and 30 minutes, 1 hour, 4 hours, and 8 hours) and are distinguished by varying degrees of severity of toxic effects. The three AEGLs are defined as follows:

AEGL 1 is the airborne concentration ( expressed as parts per million or milligrams per cubic meter [ ppm or mg/ m3]) of a substance above which it is predicted that the general population, including susceptible individuals, could experience notable discomfort, irritation, or certain asymptomatic, non sensory effects. However, the effects are not disabling and are transient and reversible upon cessation of exposure.

AEGL 2 is the airborne concentration ( expressed as ppm or mg/ m3) of a substance above which it is predicted that the general population, including susceptible individuals, could experience irreversible or other serious, long lasting adverse health effects or an impaired ability to escape.

AEGL 3 is the airborne concentration ( expressed as ppm or mg/ m3) of a substance above which it is predicted that the general population, including susceptible individuals, could experience life threatening health effects or death.

Airborne concentrations below the AEGL 1 represent exposure levels that could produce mild and progressively increasing but transient and nondisabling odor, taste, and sensory irritation or certain asymptomatic, non sensory effects. With increasing airborne concentrations above each AEGL, there is a progressive increase in the likelihood of occurrence and the severity of effects described for each corresponding AEGL. Although the AEGL values represent threshold levels for the general public, including susceptible subpopulations, such as infants, children, the elderly, persons with asthma, and those with other illnesses, it is recognized that individuals, subject to unique or idiosyncratic responses, could experience the effects described at concentrations below the corresponding AEGL level. Styrene NAC: 02/ 2004 iii TABLE OF CONTENTS PREFACE ........................................................................................................................................ 1 EXECUTIVE SUMMARY............................................................................................................ VI 1 INTRODUCTION.................................................................................................................... 1 2 HUMAN TOXICITY DATA................................................................................................... 3 2.1 Acute Lethality.................................................................................................................. 3 2.2 Nonlethal Toxicity............................................................................................................. 3 2.2.1 Case Reports.......................................................................................................... 3 2.2.2 Occupational exposure........................................................................................... 4 2.2.3 Experimental Studies............................................................................................. 5 2.3 Developmental/ Reproductive Toxicity............................................................................ 13 2.4 Genotoxicity .................................................................................................................... 13 2.5 Carcinogenicity ............................................................................................................... 14 2.6 Summary......................................................................................................................... 14 3 ANIMAL TOXICITY DATA................................................................................................ 17 3.1 Acute Lethality................................................................................................................ 17 3.1.1 Rats...................................................................................................................... 17 3.1.2 Mice ..................................................................................................................... 21 3.1.3 Guinea pigs .......................................................................................................... 23 3.1.4 Hamsters.............................................................................................................. 23 3.2 Nonlethal Toxicity........................................................................................................... 24 3.2.1 Nonhuman primates............................................................................................. 24 3.2.2 Rats...................................................................................................................... 24 3.2.3 Mice ..................................................................................................................... 27 3.2.4 Guinea pigs .......................................................................................................... 29 3.2.5 Rabbits................................................................................................................. 29 3.3 Developmental/ Reproductive Toxicity............................................................................ 30 3.3.1 Rats...................................................................................................................... 30 3.3.2 Mice ..................................................................................................................... 31 3.3.3 Rabbits................................................................................................................. 32 3.3.4 Hamsters.............................................................................................................. 32 3.4 Genotoxicity .................................................................................................................... 32 3.5 Carcinogenicity ............................................................................................................... 34 3.6 Summary......................................................................................................................... 37 4 SPECIAL CONSIDERATIONS............................................................................................ 41 4.1 Toxicokinetics ................................................................................................................. 41 4.2 Mechanism of Toxicity .................................................................................................... 48 4.3 Other relevant information............................................................................................. 48 4.3.1 PBPK Modelling.................................................................................................. 48 4.3.2 Species variability................................................................................................ 49 4.3.3 Susceptible populations ....................................................................................... 49 4.3.4 Concurrent exposure issues ................................................................................. 50 Styrene NAC: 02/ 2004 iv 5 DATA ANALYSIS AND PROPOSED AEGL 1 ................................................................... 50 5.1 Summary of Human Data Relevant to AEGL 1............................................................. 50 5.2 Summary of Animal Data Relevant to AEGL 1 ............................................................. 50 5.3 Derivation of AEGL 1 .................................................................................................... 51 6 DATA ANALYSIS AND PROPOSED AEGL 2 ................................................................... 51 6.1 Summary of Human Data Relevant to AEGL 2............................................................. 51 6.2 Summary of Animal Data Relevant to AEGL 2 ............................................................. 52 6.3 Derivation of AEGL 2 .................................................................................................... 54 7 DATA ANALYSIS AND PROPOSED AEGL 3 ................................................................... 55 7.1 Summary of Human Data Relevant to AEGL 3............................................................. 55 7.2 Summary of Animal Data Relevant to AEGL 3 ............................................................. 56 7.3 Derivation of AEGL 3 .................................................................................................... 56 8 SUMMARY OF PROPOSED AEGLS.................................................................................. 58 8.1 AEGL Values and Toxicity Endpoints ........................................................................... 58 8.2 Comparison with Other Standards and Guidelines ........................................................ 60 8.3 Data Adequacy and Research Needs .............................................................................. 62 9 REFERENCES....................................................................................................................... 62 APPENDIX A: DERIVATION OF AEGL VALUES................................................................... 73 APPENDIX B: DERIVATION OF EXPONENTIAL FUNCTION FOR TEMPORAL SCALING 77 APPENDIX C: BENCHMARK CALCULATIONS FOR STYRENE........................................ 79 APPENDIX D: DERIVATION SUMMARY FOR STYRENE AEGLS...................................... 83 APPENDIX E: DERIVATION OF THE LEVEL OF DISTINCT ODOR AWARENESS......... 87 LIST OF TABLES TABLE 1: CHEMICAL AND PHYSICAL PROPERTIES ........................................................... 2 TABLE 2: SUMMARY OF ACUTE NON LETHAL EFFECTS IN CONTROLLED HUMAN STUDIES FOLLOWING INHALATION OF STYRENE.................................................... 9 TABLE 3: SUMMARY OF LETHAL EFFECTS IN ANIMALS AFTER ACUTE INHALATION EXPOSURE TO STYRENE.................................................................................................. 18 TABLE 4: SUMMARY OF ACUTE NON LETHAL EFFECTS IN ANIMALS AFTER INHALATION EXPOSURE TO STYRENE........................................................................ 25 TABLE 5: SUMMARY OF RESULTS ON STUDIES OF CANCER IN RATS TREATED WITH STYRENE * ........................................................................................................................... 35 Styrene NAC: 02/ 2004 v TABLE 6: SUMMARY OF RESULTS ON STUDIES OF CANCER IN MICE TREATED WITH STYRENE * ........................................................................................................................... 37 TABLE 7: EXPOSURE CONCENTRATIONS AND BLOOD LEVEL OF STYRENE IN HUMANS AND RATS...................................................................................................... 44 TABLE 8: AEGL 1 VALUES FOR STYRENE........................................................................... 51 TABLE 9: AEGL 2 VALUES FOR STYRENE........................................................................... 55 TABLE 10: AEGL 3 VALUES FOR STYRENE ......................................................................... 57 TABLE 11: SUMMARY/ RELATIONSHIP OF PROPOSED AEGL VALUES......................... 58 TABLE 12: EXTANT STANDARDS AND GUIDELINES FOR STYRENE............................. 60 LIST OF FIGURES FIGURE 1: SYMPTOM RATINGS AT OR AFTER ACUTE EXPOSURE OF HUMANS TO STYRENE ................................................................................................................................ 8 FIGURE 2: CONCENTRATION RESPONSE CURVE FOR ACUTE LETHALITY FOLLOWING INHALATION OF STYRENE IN RATS.................................................... 18 FIGURE 3: STYRENE CONCENTRATION IN ARTERIAL BLOOD OF HUMANS DURING AND AFTER A 2 HOUR EXPOSURE TO 69 PPM STYRENE IN AIR............................ 42 FIGURE 4: OBSERVED ( CIRCLES) AND SIMULATED CONCENTRATIONS OF STYRENE IN ARTERIALIZED CAPILLARY BLOOD FROM TWO HUMAN VOLUNTEERS..... 42 FIGURE 5: STYRENE CONCENTRATION IN BLOOD OF RATS DURING A 5 HOUR EXPOSURE TO DIFFERENT CONCENTRATIONS OF STYRENE IN AIR.................. 43 FIGURE 6: PATHWAYS FOR THE METABOLISM OF STYRENE IN HUMANS AND RODENTS.............................................................................................................................. 47 FIGURE 7: CATEGORICAL REPRESENTATION OF STYRENE INHALATION DATA... 59 Styrene NAC: 02/ 2004 vi EXECUTIVE SUMMARY Styrene is a colorless or slightly yellow, viscous liquid, soluble in ethanol, benzene and petroluem ether and slightly soluble in water. Owing to its volatility, low flash point, and the range of explosive limits in air ( lower: 1.1 %, upper: 6.3 % v/ v), styrene poses an acute fire and explosion hazard. Due to its tendency to polymerize at room temperature in the presence of oxygen and to oxidize on exposure to light and air, styrene is normally stabilized by the addition of < 0.006  0.01% w/ w tertiary butylcatechol ( 4 tert butylbenzene 1,2 diol) as an inhibitor. Styrene is one of the most important monomers in industry worldwide. It is predominantly used for the production of polymers ( polystyrene and copolymers of styrene with acrylonitrile and/ or butadiene). Worldwide production reached 17 945 thousand tonnes in 1998.

In humans, the observed effects associated with acute exposure to styrene are irritation of eyes and mucous membranes and central nervous system ( CNS) depression. Limited data in humans provide no evidence that ( occupational) styrene exposure causes lesions of the nasal epithelia or decrements in olfactory function ( Dalton et al. 2003; Ödkvist et al. 1985). No data were available indicating reproductive or developmental effects of styrene in humans following acute exposure. Epidemiological studies revealed no sound evidence for an association between repeated occupational exposure to styrene and reproductive or developmental effects. Genotoxicity was observed in human cells in vitro; in vivo, no data were available with respect to genotoxicity following acute exposure of humans. In epidemiological studies, evidence for an association of occupational exposure to styrene and genotoxic effects were observed. With respect to carcinogenicity in humans, in its latest evaluation IARC ( 2002) concluded that there is " limited evidence in humans for the carcinogenicity of styrene" and, taking into account the results from animal carcinogenicity studies, that styrene is " possibly carcinogenic to humans ( Group 2B)". Styrene is being reassessed under the IRIS Program of the US EPA, no quantitative carcinogenicity assessment for lifetime exposure is currently proposed. US EPA ´ s Office of Research and Development has updated previous assessments on the carcinogenic potential of styrene and concluded that styrene is appropriately classified as a Group C ( possible human carcinogen) ( US EPA 2003).

Animal studies were mostly carried out with rats and mice, limited data are available for guinea pigs, hamsters and an unspecified species of monkeys. As in humans, irritation and CNS effects are also observed in animals following acute inhalation exposure. In mice, RD50 values for sensory irritation of 156 ppm  980 ppm were reported. Signs indicating irritation were also reported in toxicity studies with rats at concentrations as low as 200 ppm, immediate irritation in rats was noted at 1300 ppm. CNS depression in rats and mice was observed at higher concentrations. Rats lost consciousness at 2000 ppm after 5 hours of exposure and showed reduced attention at 6 hour exposures to 1500 ppm. In mice, signs of CNS depression during a 4 hour exposure were staggered gait at 1420 ppm and apathy and finally narcosis at higher concentrations of 2983 and 3766 ppm. In rats, death was observed when animals were exposed for 4 hours to 4814 ppm and higher concentrations ( BASF 1979b). Death was mostly rapid due to CNS depression but some delayed deaths with signs of pulmonary lesions were observed in rats at concentrations also causing severe CNS effects. Mice were much more sensitive than rats ( and, based on a limited number of data, guinea pigs and monkeys). Death of mice was observed following a single 6 hour exposure to 250 ppm or 500 ppm. Also, at these concentrations, respiratory toxicity with lesions of the nasal epithelia and of the bronchioles were observed in mice but not in rats.

With respect to developmental or reproductive effects, no embryo/ fetotoxicity or malformations were observed in rats after a single oral treatment on the 11th or 17th day of gestation, respectively. In mice, decreased postnatal survival was observed after signe oral administration of a maternally toxic dose on day 17 of gestation, while no effect was noted at a lower dose. Following repeated exposure of rats through gestation day 6   20 to 300 ppm, an increased neonatal death rate and delayed postnatal Styrene NAC: 02/ 2004 vii development was observed compared to pair fed controls. Fetotoxicity was also seen in hamsters exposed to 1000 ppm 6 hours/ day from gestation day 6  18, but not at 750 ppm. In other studies with repeated oral or inhalation exposure of rats, mice, and rabbits, no significant developmental effects were observed. Styrene is genotoxic in vitro, provided there is sufficient activation to styrene oxide ( SO), and in vivo. Data from laboratory animals indicate that styrene exposure may lead to the formation of DNA adducts, sister chromatid exchange, and chromosomal aberrations. With respect to carcinogenicity, no clear effect was observed in rats. In mice, an increase of lung tumors was observed.

Styrene has a pungent, slightly sweetish odor. The derivation of the level of distinct odor awareness ( LOA) was based on results from human studies presented in the report of Van Doorn et al. ( 2002) and follows the guidance as described in the same report. The LOA represents the concentration above which it is predicted that more than half of the exposed population will experience at least a distinct odor intensity, while about 10 % of the population will experience a strong odor intensity. The LOA should help chemical emergency responders in assessing the public awareness of the exposure due to odor perception. For styrene, the calculated level of distinct odor awareness ( LOA) is 0.54 ppm.

The AEGL 1 derivation is based on irritating effects of styrene in humans. In a study on psychological reactions related to chemosensory irritation, ratings for odor and annoyance increased similarily with increasing styrene concentrations ranging from 0.5   40 ppm, while there was only a marginal increase for irritation. Effects sizes comparing the ratings between exposure to 20 ppm and preexposure were higher for odor, irritation, and annoyance. Effects sizes were also higher compared to " clean air only" exposure. However, the ratings for irritation indicated only marginal effects in this respect ( Seeber et al. 2002). No increase in irritation or headaches compared to control was noted at 20 ppm in a further study ( Hake et al. 1983). Subjective signs and symptoms of irritation and CNS effects were not negatively influenced during a 6 hour exposure at 25 ppm or 50 ppm or at 50 ppm with 4 peak exposures of 15 minutes at 100 ppm ( Ska et al. 2003). At 50 ppm, a further study indicated a slight increase in subjective symptoms ratings for eye and nose irritation, headache, and fatigue ( Oltramare et al. 1974). At 100 ppm, Oltramare et al. ( 1974) further reported that signs of irritation and of mild subjective CNS effects ( headaches, fatigue, poor concentration, sleepiness) were felt more often than at 50 ppm. Complaints of mild eye and throat irritation at 99 ppm in one test but not in another at 116 ppm were reported by Stewart et al. ( 1968). Complaints of eye and nose irritation were frequent at about 200 ppm ( Oltramare et al. 1974; Stewart et al. 1968).

A concentration of 20 ppm ( Seeber et al. 2003) was selected to derive AEGL 1. Because this concentration represents a NOAEL for local ( as well as CNS) effects and in other studies effects at 50 ppm and 100 ppm were only weak or absent, an intraspecies factor of 1 is applied. The value of 20 ppm was used for all timepoints since slight irritation and subjective discomfort that were reported at higher concentrations did not increase within several hours of exposure.

The derivation of AEGL 2 is based on human studies. Irritation and CNS effects have to be considered for the derivation of AEGL 2. Nasal and mild eye irritation were reported by volunteers exposed to 376 ppm ( Stewart et al. 1968). In their study of styrene exposed workers, Götell et al. ( 1972) reported that they themselves suffered from immediate lacrymation and irritation of the nasopharynx when exposed to 300   400 ppm, and concentrations of 500   800 ppm caused irritation intolerable to the investigators within 1 or 2 minutes. Strong eye and nasal irritation was also reported by volunteers exposed to concentrations   600 ppm ( Carpenter et al. 1944; Wolf et al. 1956).

With respect to effects on the CNS, a 6 hour exposure at 50 ppm with 4 repeated 15 minute peaks at 100 ppm had no negative influence on performance to neuropsychological tests ( Ska et al. 2003). Styrene NAC: 02/ 2004 viii At 99 ppm, intermittent difficulties in performing a modified Romberg test were observed in 3/ 6 subjects exposed for 7 hours with a 30 minute break in between. Other tests on coordination and on manual dexterity were normal, and no effects were noted at the end of exposure. No CNS effects were seen in another experiment with 116 ppm exposure for 2 hours or 216 ppm for 1 hour in the same study ( Stewart et al. 1968). Headaches, but no effects on equilibrium and cognitive function tests were noted in male and female volunteers at repeated exposures to 100 and 125 ppm for at least one hour ( Hake et al. 1983). Oltramare et al. ( 1974) noted slight difficulties in balance performance at 50   200 ppm ( 1.5 hours), but there was no concentration response, and slight difficulties in balance performance at 200 ppm ( 1 hour), but the variation of data was large. No effects on simple and choice reaction time was seen following exposure to 250 ppm for 30 minutes. However, when the concentration was raised to 350 ppm for 30 minutes directly afterwards, both simple and choice reaction time were increased ( Gamberale and Hultengren 1974). More pronounced effects were observed during exposure to 376 ppm for one hour: One subject complained of nausea that persisted one hour after the end of exposure, 2 subjects had a feeling of being inebriated, 3 of 5 subjects exposed were unable to normally perform a modified Romberg test, and also 3 subjects had significant decrements in other tests of coordination and manual dexterity ( Stewart et al. 1968). In a toxicokinetic study, 2 subjects were exposed to 386 ppm styrene for 2 hours while performing light physical exercise of 50 W ( Löf and Johanson 1993). In that study, no information was presented as to the presence or absence of subjective or objective signs of intoxication or irritation. However, it may reasonably be assumed that no severe CNS effects will have occurred in such a study. At higher concentrations, the irritation becomes very strong ( see above), and only one controlled study was located that was conducted at this level ( Carpenter et al. 1944). In this study, 2 subjects exposed to 800 ppm for 4 hours suffered from listlessness, drowsiness, impairment of balance, and, after cessation of exposure, muscular weakness and unsteadiness with inertia and depression. A " steadiness test" measuring manual dexterity indicated a marked decreased of performance compared to pre exposure level. Besides CNS depression, the subjects complained of eye and throat irritation.

The AEGL 2 is based on the CNS effects observed in humans following exposure to 376 ppm for 1 hour: nausea in one subject; feeling of being inebriated in two, and unability to normally perform the modified Romberg test and significant decrements in other tests of coordination and manual dexterity in three of five subjects ( Stewart et al. 1968). The effects described address a level of CNS depression that seems still below a level for an impairment of the ability to escape and therefore a concentration of 376 ppm is considered a NOAEL. However, this level is close to concentrations causing intolerable irritation in humans that may limit the ability to escape and thus are above AEGL 2.

Generally, for volatile substances with CNS depressant effects an intraspecies factor of 3 is applied to account for sensitive individuals because the effective concentration range does not differ more than 2 3fold between individuals. In case of styrene, it must be taken into account that physical activity has a marked effect on the uptake of styrene and its level in blood. In the studies used to derive AEGL 2, the subjects were at rest. In controlled studies, the observed increase of styrene in arterial blood at exposure to about 150 ppm styrene was approximately 3fold when the physical activity was increased from rest to light exercise ( 50 W), 5fold at moderate exercise ( 100 W), and 10fold at heavy exercise ( 150 W) ( Astrand 1975). Therefore, it could be argued that an intraspecies uncertainty factor of 10 to account for sensitive subgroups would be necessary to protect individuals at heavy physical exercise. Application of a factor of 10 would lead to a 1 hour AEGL 2 of 38 ppm and similar values at longer time periods. On the other hand, the following two points which indicate that a factor of 3 is justified, are believed to outweigh the above rationale: Firstly, due to physiological limitations, heavy physical exercise ( 150 W) cannot be performed continuously for longer periods of time. Therefore, it is unrealistic to consider an exposure scenario with heavy exercise for one or several hours. In contrast, light exercise ( 50 W) may be performed over a longer period of time. In this case, the increase of the styrene concentration in blood will be about 3fold which is Styrene NAC: 02/ 2004 ix within the range of an uncertainty factor of three. Secondly, an AEGL 2 value in the range of 38 ppm as mentioned above would be in conflict with styrene exposure data at occupational workplaces. At workplaces, such concentrations are or were frequently observed ( IARC 2002) without workers showing signs of CNS depression that would have limited their ability to escape.

Therefore, an intraspecies uncertainty factor of 3 is considered adequate to protect sensitive subgroups including groups exposed to styrene during longer periods of light exercise. This leads to a value of 130 ppm as AEGL 2 for 1 hour.

This experimentally derived exposure value was scaled to shorter periods of time using the equation cn x t = k ( Ten Berge et al. 1986). In accordance with NRC ( 2001), a default of n = 3 for shorter periods of time ( 30 minutes and 10 minutes) was applied, due to the lack of suitable experimental data for deriving the concentration exponent. The " n" value of 1.2 used for calculations of AEGL 3 ( see below) was not used for AEGL 2 for following reasons: Firstly, the exponent was derived from lethality studies in which delayed mortality was observed that was not related to narcotic effects on the CNS ( which are relevant for AEGL 2) but probably to pulmonary lesions observed at these very high concentrations ( in addition to CNS effects which are the major cause of death). Secondly, toxicokinetics at high exposure concentrations over several hours of exposures ( as in the lethality studies) is different from that at lower concentrations for shorter time periods.

Toxicokinetic studies with humans exposed to styrene concentrations at 70   200 ppm show that most of the increase of the styrene concentration in blood is seen during the first 30 minutes of exposure and that there is no or very little increase at 1   3 hours at these concentrations. Therefore, no additional extrapolation is necessary and the AEGL 2 of 130 ppm derived for 1 hour is applied to longer periods of time.

The AEGL 3 values are derived from a lethality study with rats ( BASF 1979b). In rats, exposure to high concentrations of styrene leads to progressive CNS depression with narcosis and, finally, death. At concentrations leading to severe CNS effects, delayed deaths with pulmonary lesions were also described in these studies. In humans, the acute effects on the CNS are well described. However, no reports of lethal intoxications following styrene exposure were identified in the literature; therefore, it is not known if the pulmonary lesions observed in rats may also occur in humans exposed to life threatening or potentially lethal concentrations of styrene.

For a conservative approach, data from studies with rats taking into account delayed deaths with pulmonary lesions were used to derive AEGL 3. From the data of the 4 hour exposure study of BASF ( 1979b), a benchmark calculation was performed with the lethality data using different models. A BMDL05 for female rats of 3409 ppm ( rounded to 3400 ppm) was used as a starting point to derive AEGL 3.

A total uncertainty factor of 10 was applied. This total factor may formally be split up into an interspecies factor of 3 and an intraspecies factor also of 3. For volatile solvents like styrene with a CNSdepressant effect, an interspecies uncertainty factor of 3 has been applied in the derivation of AEGL for several substances. This is based on the similarity of effects manifested in rodents compared to humans. In case of styrene, limited data indicate no gross differences in the concentration of styrene in blood between rats and humans. According to a toxicokinetic model, at concentrations exceeding 200 ppm styrene in air, the non steady state concentration of styrene in blood of humans ( calculated for 6 hours of exposure) will always be lower than that in blood of rats ( Ramsey and Andersen 1984). Styrene levels in human blood were in accordance with this model up to 376 ppm in air, however, no experimental human data are available for validation of the model at higher concentrations. Styrene NAC: 02/ 2004 x An intraspecies uncertainty factor of 3 was applied to account for sensitive individuals since the threshold for CNS impairment is not expected to vary much among individuals. As in case of the derivation of AEGL 2, an intraspecies uncertainty factor of 3 is considered adequate to protect sensitive subgroups including groups exposed to styrene during longer periods of light exercise.

Extrapolation was made to the relevant AEGL time points of 30 minutes and 1 hour using the relationship Cn x t = k with a value of n = 1.2 which was derived from extrapolation of the LC50 in rats for 4 and 6 hours ( BASF 1979b; Bonnet et al. 1982a). The 10 minutes AEGL 3 was assigned the same value as that for the 30 minutes AEGL 3 as it was considered inappropriate to extrapolate from an experimental period of 4 hours to 10 minutes. The 8 hour AEGL 3 was assigned the same value as that for the 4 hour AEGL 3 as toxicokinetic data indicate that there is at most little increase of internal dose after 4 hours of exposure; moreover, lower values which would be derived by default calculations are not supported by toxicological data for humans.

Individual cases of respiratory sensitization to styrene were described. Taking into account the wide use of styrene both in industry and in do it yourself products, sensitization seems to be an exceptionally rare event. Although the risk of sensitization following a single exposure at AEGL is considered negligible, individuals already sensitized to styrene may not be able to tolerate styrene concentrations that are without effect in non sensitized individuals and may not be protected by the AEGL developed for styrene in this TSD.

SUMMARY TABLE OF AEGL VALUES FOR STYRENE a Classification 10 Minute 30 Minute 1 Hour 4 Hour 8 Hour Endpoint ( Reference)

AEGL 1 ( Nondisabling) 20 ppm ( 85 mg/ m ³ ) 20 ppm ( 85 mg/ m ³ ) 20 ppm ( 85 mg/ m ³ ) 20 ppm ( 85 mg/ m ³ ) 20 ppm ( 85 mg/ m ³ ) NOAEL for slight irritation ( Seeber et al. 2002)

AEGL 2 ( Disabling) 230 ppm ( 980 mg/ m ³ ) 160 ppm ( 680 mg/ m ³ ) 130 ppm ( 550 mg/ m ³ ) 130 ppm ( 550 mg/ m ³ ) 130 ppm ( 550 mg/ m ³ ) CNS effects in humans ( Gamberale and Hultengren 1974; Stewart et al. 1968)

AEGL 3 ( Lethality) 1900 ppm * ( 8090 mg/ m ³ ) 1900 ppm * ( 8090 mg/ m ³ ) 1100 ppm ( 4690 mg/ m ³ ) 340 ppm ( 1450 mg/ m ³ ) 340 ppm ( 1450 mg/ m ³ ) No lethality in rats ( BASF 1979b)

a: Since liquid styrene is an eye irritant, eye contact must be avoided. *: The lower explosive limit ( LEL) of styrene in air is 1.1 % ( 11,000 ppm). The AEGL 3 value of 1900 ppm ( 8090 mg/ m ³ ) for 10 minutes and 30 minutes are higher than 1/ 10 of the LEL. Therefore, safety considerations against hazard of explosion must be taken into account.

References Astrand, I. 1975. Uptake of solvents in the blood and tissues of man. A review. Scandinavian Journal of Work, Environment and Health 1: 199 218.

BASF. 1979b. Bericht über die Bestimmung der akuten Inhalationstoxizität LC50 von Styrol als Dampf bei 4stündiger Exposition an Sprague Dawley Ratten. Unveröfffentlichte Untersuchung. [ Report on the Styrene NAC: 02/ 2004 xi determination of the acute inhalation toxicity LC50 of styrene as vapor following a 4 hour exposure in Sprague Dawley rats. Unpublished study.] BASF AG, Ludwigshafen, Germany.

Bonnet, P., Y. Morele, G. Raoult, D. Zissu, and D. Gradiski. 1982a. Détermination de la concentration léthales50 des principaux hydrocarbures aromatiques chez le rat. Archives des Maladies Professionnelles de Medecine du Travail et de Securite Sociale 43: 261 265.

Carpenter, C. P., H. F. Jr. Smyth, and D. C. Pozzani. 1944. Studies on the inhalation of 1: 3 butadiene; with a comparison of its narcotic effect with benzol, toluol, and styrene, and a note on the elimination of styrene by the human. Journal of Industrial Hygiene and Toxicology 26: 69 78.

Dalton, P., B. Cowart, D. Dilks, M. Gould, P. S. Lees, A. Stefaniak, and E. Emmett. 2003. Olfactory function in workers exposed to styrene in the reinforced plastics industry. American Journal of Industrial Medicine 44: 1 11.

Gamberale, F. and M. Hultengren. 1974. Exposure to styrene. II. Psychological functions. Work, Environment and Health 11: 86 93.

Götell, P., O. Axelson, and B. Lindelöf. 1972. Field Studies on Human Styrene Exposure. Work, Environment and Health 9: 76 83.

Hake, C. L., R. D. Stewart, A. Wu, S. Graff, H. V. Forster, W. H. Keeler, A. J. Lebrun, P. E. Newton, and R. J. Soto. 1983. Styrene  Development of a Biologic Standard for the Industrial Worker by Breath Analysis. NIOSH MCOW ENVM STY 77 2. Medical College of Wisconsin, Milwaukee. Cited in NIOSH ( 1983).

IARC. 2002. Some Traditional Herbal Medicines, some Mycotoxins, Naphthalene and Styrene. Summary of Data Reported and Evaluation. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans 82, p. 437 550. IARC ( International Agency for Research on Cancer), WHO ( World Health Organization), Lyon, France.

Löf, A. and G. Johanson. 1993. Dose dependent kinetics of inhaled styrene in man. IARC Scientific Publications 89 99.

NIOSH. 1983. Criteria for a Recommended Standard  Occupational Exposure to Styrene. DHHS ( NIOSH) Report. 83 119. National Institute for Occupational Safety and Health ( NIOSH), Cincinnati, OH.

Ödkvist, L. M., C. Edling, and H. Hellquist. 1985. Influence of vapours on the nasal mucosa among industry workers. Rhinology 23: 121 127.

Oltramare, M. E., E. Desbaumes, C. Imhoff, and W. Michiels. 1974. Toxicologie du styrene monomere [ Toxicology of Monomeric Styrene  Experimental and Clinical Studies on Man.]. Editions Medicine et Hygienique. Geneva, Switzerland. Cited in NIOSH ( 1983) and WHO ( 1983).

Seeber, A., C. van Thriel, K. Haumann, E. Kiesswetter, M. Blaszkewicz, and K. Golka. 2002. Psychological reactions related to chemosensory irritation. International Archives of Occupational and Environmental Health 75: 314 325. Styrene NAC: 02/ 2004 xii Ska, B., A. Vyskocil, R. G. Tardif, G. Carrier, R. Thuot, K. Muray, and C. Viau. 2003. Effects of peak concentrations on the neurotoxicity of styrene in volunteers. Human and Experimental Toxicology 22: 407 415.

Stewart, R. D., H. C. Dodd, E. D. Baretta, and A. W. Schaffer. 1968. Human exposure to styrene vapor. Archives of Environmental Health 16: 656 662.

US EPA. 2003. Technology Transfer Network Air Toxics Website. Styrene. Air Toxics Website. U. S. Environmental Protection Agency, Washington, DC. http:// www. epa. gov/ ttn/ atw/ htlhef/ styrene. htm.

van Doorn, R., M. W. Ruijten, and T. van Harreveld. 2002. Guidance for the Application of Odor in Chemical Emergency Response. Version 2.1; August, 29., 2002. Presented at the NAC/ AEGL Meeting September 2002, Washington DC.

WHO. 1983. Styrene. Environmental Health Criteria ( EHC) 26. International Programme on Chemical Safety ( IPCS); World Health Organization, Geneva, Switzerland.

Wolf, J. R., V. K. Rowe, D. D. McCollister, R. L. Hollingsworth, and F. Oyen. 1956. Toxicological studies of certain alkylated benzenes and benzene. Experiments on laboratory animals. Archives of Industrial Health 14: 387 398. Styrene NAC: 02/ 2004 1 1 INTRODUCTION Styrene is a colorless or slightly yellow, viscous liquid. It is slightly soluble in water, soluble in ethanol and very soluble in benzene and petroleum ether. Due to its tendency to polymerize at room temperature in the presence of oxygen and to oxidize on exposure to light and air, styrene is normally stabilized by the addition of < 0.006  0.01% w/ w tertiary butylcatechol ( 4 tert butylbenzene 1,2 diol) as an inhibitor ( WHO 1983).

Pure styrene has a pungent, slightly sweetish odor. However, oxidation may lead to the formation of peroxides, certain aldehydes and ketones giving a sharp, penetrating, disagreeable odor. When emitted into the air, its half time is estimated to be about 2 hours, and chemical transformation products include benzaldehyde and formaldehyde, both of which are odorous air pollutants ( WHO 2000).

Styrene is one of the most important monomers in industry worldwide. The first step in its industrial production is the catalytic alkylation of benzene with ethylene leading to ethylbenzene. In the second step, ethylbenzene is dehydrogenated to styrene. In an alternative process, styrene is formed as a coproduct in the synthesis of propylene oxide from ethylbenzene and propene via ethylbenzene hydroperoxide and 1 phenylethanol ( WHO 1983). Purified products typically are 99.7% to greater than 99.9% w/ w styrene with less than 0.1 % ethylbenzene, cumene, phenylpropene, phenyl acetate and p xylene.

Styrene is predominantly used for the production of polymers ( polystyrene, copolymers of styrene with acrylonitrile and/ or butadiene) that find wide application in latex paints and coatings, synthetic rubbers, polyesters and styrene alkyd coatings. Styrene is a HPV ( high production volume) chemical with a worldwide production of 17 945 thousand tonnes in 1998. Small amounts of styrene can be found in gum exudate from the damaged trunk of certain trees, probably being produced by decomposition of cinnamic acid derivatives that are present in such exudates in large quantities. Styrene also occurs in many agricultural products and foods, however, it is not clear whether styrene is naturally produced within plants ( IARC 2002).

Owing to its volatility, low flash point, and the range of explosive limits in air ( lower: 1.1 %, upper: 6.3 % v/ v), styrene poses an acute fire and explosion hazard. Chemical and physical properties of styrene are presented in Table 1. Styrene NAC: 02/ 2004 2 TABLE 1: CHEMICAL AND PHYSICAL PROPERTIES Parameter Data Reference Synonyms Vinylbenzene, phenylethene, ethenylbenzene, cinnamene WHO 1983 Chemical formula C8H8 Molecular weight 104.14 g/ mol WHO 1983 CAS Reg. No. 100 42 5 ATSDR 1992 Physical state Liquid at room temperature Weast 1973 Solubility 300 mg/ l in water ( at 20 ° C), soluble in alcohol, ether, acetone, miscible with benzene and petrol ether Weast 1973; WHO 1983 Vapor pressure 3.1 hPa ( at 10 ° C), 6.67 hPa ( at 20 ° C), 8.67 hPa ( at 25 ° C), 13.3 hPa ( at 35 ° C) ATSDR 1992; NIOSH 1983; WHO 1983 Vapor density ( air = 1) 3.6 Liquid density ( g/ cm ³ ) 0.9060 ( at 20 ° C) Weast 1973 Melting point  30.63 ° C Weast 1973 Boiling point 145.2 ° C ( at 1013 hPa) Weast 1973 Explosive limits in air 1.1   6.3 % ATSDR 1992 Flash point ( closed cup) 31 ° C ATSDR 1992 Autoignition temperature 490 ° C ATSDR 1992 Conversion factors ( at 25 ° C) 1 ppm = 4.26 mg/ m ³ 1 mg/ m ³ = 0.234 ppm Calculated according to NRC 2001 Styrene NAC: 02/ 2004 3 2 HUMAN TOXICITY DATA 2.1 Acute Lethality No reports of lethal intoxication following styrene exposure were located in the literature.

2.2 Nonlethal Toxicity 2.2.1 Case Reports The investigators of a field study on styrene exposure of workers noted that they could not withstand styrene concentrations of 500   800 ppm for more than 1   2 minutes, whereas the workers exposed to this level complained of only minor to moderate irritation of eyes and nasopharynx. The authors further report that they themselves ( five unadapted persons) suffered from lacrymation and irritation of the nasopharynx at about 300   400 ppm ( Götell et al. 1972).

By degassing the tank of a ship on a river, styrene was blown into the surrounding air without sufficient dilution. 15 employees of a nearby power plant and 3 river police men who were exposed to an unknown concentration of styrene complained of immediate eye irritation and tickle in the throat, dizziness, headache and nausea ( Hahn et al. 2000).

After using a polyester resin canoe building kit, a 36 year old man twice suffered from neurologic symptoms ( MacFarlane et al. 1984). The work had been carried out in an unventilated shed for about 4   5 hours during which styrene evaporated from the construction kit. The man developed severe postural hypotension, neurological signs ( slurred speech, nystagmus, limb ataxia) and conjunctivitis.

Moscato et al. ( 1987) described two cases of workers employed in plastics factories that had bronchial asthma or runny nose, dry irritating cough and chest tightness. They were exposed to styrene and ethyl benzene and one of them to polyester resin. However, specific inhalation challenges revealed an immediate bronchospastic response only after provoked inhalation exposure to styrene ( 15 ppm for 15 minutes). In both subjects, symptoms completely disappeared after changing their job. A further case of asthma in a subject occupationally exposed to styrene and showing a positive reaction to styrene in a provoked exposure test was reported by Hayes et al. ( 1991). A case of skin dermatitis following dermal exposure to styrene was reported by Sjöborg et al. ( 1982), skin patch tests revealed a strong reaction to styrene and a cross reaction to vinyl toluene, but a weak one to benzoyl peroxide ( used in hardeners for styrene based plastics) and no reaction to styrene polymerization inhibitors and typical styrene impurities.

Non inhalation exposure Repare of a water tank led to contamination of tap water with styrene and subsequent oral and inhalation exposure ( Arnedo Pena et al. 2003). Residents of 27 apartments in two buildings using the contaminated water were contacted. A questionnaire on subjective symptoms was administered to 84 out of 93 persons living in affected apartments at the time of the accident. Styrene measured in samples of water collected two days after the accident reached concentrations up to 900 µ g/ L. Symptoms were reported by 46 persons, most frequently irritation of the throat ( 26%), nose ( 19%), eyes ( 18%) and skin ( 14%). General gastrointestinal symptoms were observed with 11% reporting abdominal pain and 7% diarrhea. The factors most strongly associated with symptoms were drinking tap water, exposure to vapors from the basement and eating foods prepared with tap water. All residents in the ground floor reported symptoms. Styrene NAC: 02/ 2004 4 2.2.2 Occupational exposure A great number of studies on workers with occupational exposure to styrene in different workplaces have been carried out. These studies have been repeatedly reviewed and summarized ( ACGIH 1997; ATSDR 1992; Cohen et al. 2002; DFG 1987; Government Canada 1993; IARC 2002; OEHHA 1999; Sherrington and Routledge 2001; US EPA 1998; WHO 1983; WHO 2000). Workers are exposed to styrene in a number of industries, e. g. in the production of styrene and styrene polymers. In the fabrication of reinforced polyester plastics composites, 8 hour average samples in breathing zones often exceed styrene concentrations of 100 ppm ( IARC 2002). Here, the highest exposure concentrations were observed in chopper gun operators where 8 hour mean concentrations in personal breathing zone of 564 mg/ m ³ ( range 307   938 mg/ m ³ ) ( 132 ppm; range 72   219 ppm) were measured ( Truchon et al. 1992). In previous studies on workers in the manufacture of reinforced plastics, 8 hours TWA concentrations in the breathing zone of up to 292 ppm were reported, with peaks of about 1500 ppm during shorter periods of work for about 5   10 minutes ( Götell et al. 1972).

In workers exposed to styrene, central and peripheral nervous systems effects have been observed. Especially, reversible decrease in color discrimination has been described in many studies. Decrements of auditory function ( threshold for hearing at high frequencies, hearing acuity) was also observed in several smaller cross sectional studies, however, in the largest study on workers in the glass fibre reinforced plastics industry, no evidence was observed that exposure to styrene had an effect on hearing acuity when both lifetime styrene exposure and noise were taken into account. Studies of effects on the immune and hematopoetic system, liver, and kidney did not reveal consistent changes ( IARC 2002). Generally, in these studies effects on workers with long term exposure to styrene were investigated. A detailed description of the findings from these studies is beyond the scope of this document because they do not provide data that can be used for the derivation of AEGL. Therefore, only studies are described here in which effects following acute occupational exposure to styrene were investigated.

Acute behavioral effects and symptoms of exposure to styrene were investigated in a crosssectional study ( Edling and Ekberg 1985). 12 workers ( mean age 30 years) with a mean exposure to styrene of 2.5 years took part in the study. Neuropsychiatric symptoms ( questionnaire) and a reaction time test were conducted after an exposure free intervall of at least 24 hours before and after the morning and the afternoon shift. A reference group of 10 non exposed men was available for the morning shift. The mean 8 hour TWA of breathing zone personal samples was 43 ± 28 mg/ m ³ ( 10 ± 6.5 ppm) in the morning shift and 54 ± 37 mg/ m ³ ( 13 ± 9 ppm) in the afternoon shift. No significant differences in neuropsychiatric symptoms and reaction time were observed between pre and postshift evaluations and between exposed and controls.

Acute ( and chronic) effects of styrene on the nervous system were investigated in a further crosssectional study ( Triebig et al. 1989). A total of 36 workers from companies handling polyester resin materials for 1   16 ( median: 7) years and two control groups were each examined on a Monday. One control group formed to compare acute effects consisted of 20 men from two companies with no exposure to neurotoxic chemicals. To compare chronic effects, a second control group was formed by " one to one matching" with respect to age, socio economic status, and pre exposure intelligence level. Ambient air monitoring using active sampling ( short time) and passive samplers ( long time) showed styrene in air of 3   251 ppm ( median: 18 ppm) and 140   600 ppm during lamination of the inside of boats. Clinical examination revealed no signs or symptoms of peripheral neuropathy or encephalopathy. Acute eye irritation was noted after exposure to about 200 ppm or more. Neurobehavioural tests showed neither significant differences in acute effects between the two groups nor between pre and postshift testing nor significant differences in relevant neurobehavioural variables between the styrene workers and controls. Styrene NAC: 02/ 2004 5 Limited data on the effects of styrene vapors on the nasal mucosa are available from a crosssectional study ( Ödkvist et al. 1985). 11 ship builders ( mean age 39 years, range 26   57 years) exposed to styrene for a mean of 7 years ( range 1   16 years) took part in the study. Air levels in the plant were in the range of 200   250 mg/ m ³ ( 47   59 ppm) ( no details reported). 25 men matched for age and smoking habits and without industrial exposure served as controls. Nasal biopsies were taken from the mucosa of the inferior turbinates, and morphological findings were graded according to a scoring system evaluating histological characteristics. No statistically significant differences between the mean scores of both groups were found.

2.2.3 Experimental Studies Two male subjects were exposed to 800 ppm styrene for 4 hours in a 4000 cubic ft. room ( about 110 m ³ ) in which fans were arranged to produce rapid and thorough mixing of the air ( Carpenter et al. 1944). Styrene was evaporated at room temperature from large wicks in air stream and the vapor concentration was monitored ( using an " interferometer" developed for the iodometric determination of organic vapors) and controlled manually. Psycho motor response was followed by means of a " steadiness" test. The test was performed by the subject holding at arm ´ s length a small wire in a hole drilled in a copper strip. The number of contacts and the time the wire was in contact with the periphery of the hole was recorded during a 3 minute period. Exposure to styrene caused immediate eye and throat irritation, increased nasal mucous secretion, pronounced and persistent metallic taste, and CNS depression with listlessness, drowsiness, impairment of balance, and, after termination of exposure, muscular weakness and unsteadiness that were accompanied by inertia and depression. In the steadiness test, the contact time was 630 % of the day ´ s normal value. There was apparently no control without exposure so it cannot completely be ruled out that some of the effects described might not be related to the styrene exposure but to the experimental conditions. However, this seems unlikely since the styrene concentration was very high and the effects noted were very pronounced. Furthermore, during exposure to lower concentrations of other chemicals ( butadiene, toluene) in the same experiment, weaker or no effects were observed.

An unspecified number of humans were exposed to a range of analytically determined concentrations of styrene in an enclosed, tightly sealed room ( Wolf et al. 1956). The subjects qickly entered the room and noted their reactions with respect to odor, eye irritation, and nasal irritation. Probably, there was no unexposed control, but experimental details ( esp., number of subjects, duration of exposure) were not reported by the authors. At 60 ppm, there was a " detectable odor but no irritation". 100 ppm were " tolerated without excessive discomfort" though the odor was " strong". An " objectionably strong odor" was felt between 200 and 400 ppm, while 600 ppm or more caused strong eye and nasal irritation.

In a toxicokinetic study, two volunteers were exposed to styrene at concentrations up to 386 ppm for 2 hours while performing light physical exercise of 50 W ( Löf and Johanson 1993, see section 4.1). No information was presented with respect to subjective or objective signs of intoxication or irritation, but it may reasonably be assumed that no severe effects will have occurred in such a study.

Local irritation and effects on the nervous system were studied by Stewart et al. ( 1968). The study was conducted with a group of 9 healthy male technical employees ( 32   55 years old) with no known exposure to styrene for at least a year. In 5 experiments, a number of 1   5 subjects were exposed for 1, 2 or ( with a 30 minute break at half time) 7 hours in an exposure chamber of about 50 m ³ to analytically ( infrared analysis and gas chromatography) confirmed styrene concentrations of 51.4 ppm ( 1 hour), 99.4 ppm ( 7 hours), 116.7 ppm ( 2 hours), 216.1 ppm ( 1 hour), and 376 ppm ( 1 hour). During exposure, subjective and objective responses of each individual were recorded every 15 minutes. A neurological examination was performed every 15 minutes during exposures lasting up to 2 hours and every hour at Styrene NAC: 02/ 2004 6 longer lasting exposures. This examination included a modified Romberg test ( balancing on one foot with eyes closed and both arms at a side), heel and toe, and finger to nose test. Additionally a manual dexterity and a Flannigan coordination test were performed the morning and afternoon during the 7 hour exposure and after 30 minutes during the 1 hour exposure to 216 and 376 ppm.

No untoward subjective symptoms or objective signs of illness were recorded during a 1 hour exposure to 51 ppm ( 3 subjects) or during a 2 hour exposure to 117 ppm ( 1 subject). The odor was strong, but not judged to be objectionable. At 216 ppm ( 3 subjects), the odor was initially strong, and one subject noted nasal irritation after 20 minutes. No signs of CNS effects were observed during the 1 hour exposure.

Effects were seen at 376 ppm ( 5 subjects). Three of the subjects previously exposed to 216 ppm reported they were able to discern that they were now exposed to a higher concentration. " Mild" eye irritation occurred within 3 minutes. All subjects complained of nasal irritation within 15 minutes and one of them of a burning sensation of the skin of his face. Neurological alterations also were seen at this concentration. After 25 minutes, one subject, after 60 minutes two subjects were unable to normally perform the modified Romberg test. After 50 minutes, significant decrements were found in 3 of 5 subjects in other tests of coordination and manual dexterity. Furthermore, nausea after 45 minutes in one subject ( persisting one hour post exposure), and feeling of being inebriated ( 2 subjects) and headache ( one subject) after one hour were reported.

Exposure to 99 ppm for 2 x 3.5 hours caused complaints of mild eye and throat irritation in 3 of 6 subjects after 20   30 minutes which later subsided. 3 of 6 subjects reported intermittent difficulties in performing the Romberg test one or two times at the eight trials of this test during exposure. Tests of coordination and manual dexterity were normal. At the end of exposure, there were no reports of subjective symptoms. Throughout the study, clinical and laboratory data were normal and not altered compared to preexposure ( Stewart et al. 1968).

Effects of styrene on psychological functions were studied in 12 healthy male volunteers ( age 21   31 years) ( Gamberale and Hultengren 1974). They were exposed in groups of six to either air ( control) or to nominal but analytically ( gas chromatography) monitored concentrations of 50, 150, 250 and 350 ppm styrene via mouthpiece in four continuous 30 minute periods. After each 30 minute period, the concentration of styrene was raised to the next higher level without interruption of exposure. In a second set of experiments, the control group was exposed to styrene and vice versa. Performance tests were carried out during each period of eposure. Care was taken that the volunteers were unaware of the exposure status by introducing menthol into the inhaled air, breathing through a mouthpiece only, and use of nose clips. Additionally, control experiments were initiated with a a relatively strong smell of styrene in the mouthpiece and ended with a short exposure to styrene after completion of the final test. All subjects believed that they had been exposed on both trial days. Local irritation was almost completely absent because the subjects were exposed via mouthpiece so that eyes and nose were spared from direct exposure. Nevertheless, compared to control exposure, subjects felt slight discomfort ( feeling of tension and being affected) after exposure to styrene. In the performance tests, the performance level in the two perceptual tests ( Identical Numbers and Spokes), was affected by training, both under control and exposure conditions. In both tests, the training effect in exposure to styrene was somewhat less pronounced than in control conditions, especially at the two higher concentrations. This could indicate that training was less effective under styrene exposure, however, the differences between the mean performance values for control and styrene exposure were not significantly different in any case. There was a clear effect of styrene on reaction time. The reaction time was significantly impaired in two tests ( simple and choice reaction time) at 350 ppm but not at lower concentrations. Styrene NAC: 02/ 2004 7 In a further study, 6 volunteers were exposed to analytically ( Beckman hydrocarbon analyzer) monitored concentrations of styrene in room of about 15 m ³ ( Oltramare et al. 1974). 3 of the 6 volunteers had been exposed occpationally to styrene but not during the last 1 days prior to the experiment. Altogether, 42 exposure sessions were held each lasting 1   3 hours and usually exposing one or two subjects at a time. 2 subjects were exposed to styrene once at 300 ppm, all were exposed once or twice to 100 and 200 ppm, and most were exposed at 3   5 ppm (" odor blinded" control) and 50 ppm.

Psychomotor functions of the three subjects with previous occupational exposed to styrene were studied in sessions each lasting 90 minutes. Volunteers were individually exposed. All were exposed to 3   5 ppm first, then to 50 ( only two subjects), 100, or 200 pm in random order, and finally to 3   5 ppm again. Reaction time was determined before, 1 hour after start, and 30 minutes after termination of exposure. Simple reaction time was about the same as pre exposure at 3   5 ppm but was lengthened by 12   37 % at 50, 100, and 200 ppm during exposure. Similar results were obtained in an audiovisual reaction test. However, there was no concentration response trend. In a multiple stimulus reaction test, no effect on performance was seen at 50 ppm. As the ability to perform this test improved with repeated trials, both during each session and from session to session, the authors comment that an effect of styrene at 50 ppm might have been masked. A decrement of about 2 % at 100 ppm and of 10 % at 200 ppm during and after exposure were seen ( Oltramare et al. 1974).

Difficulties in balance performance were also studied by Oltramare et al. ( 1974) in 3 of 6 subjects. Statistically significant differences in a modified Romberg test on a swaying platform were observed after 1 hour exposure to 200 ppm compared to control. No difference was seen when results from control and the 100 ppm group were compared. The authors noted that   due to the small sample size and the large variation of data   the results should be confirmed before definite conclusions are drawn.

Also, the 6 volunteers were asked to note the occurrence of 12 subjective symptoms ( irritation: lips, nose, eyes; gastralgia, CNS effects: nausea, dizziness, headaches, sleepiness, poor concentration, intoxication, fatigue, malaise) during and after the exposure ( FIGURE 1). A total of 55 individual responses were available for analysis from all of the exposure sessions. For each of the 12 symptoms, the number of positive responses was presented as numerator and the total number of exposures at this concentration as the denominator of a ratio. Since a given subject could have been exposed more than once at a given concentration, it is not evident if multiple positive responses for each individual symptom mean that several subjects experienced a symptom or that one subject experienced that symptom at several occasions. Also, it cannot be deduced from the data which symptoms were reported by the 3 previously exposed workers. However, the authors state that the workers reported irritation at 3   5 ppm, and the authors considered that this may have been due to chronic inflammation from working with styrene. On the other hand, the symptoms noted for CNS effects were consistently fewer for the subjects with previous exposure. For the parameters indicating CNS effects and also for gastralgia, there was a clear increase in positive symptom reports at 100 ppm and higher concentrations. The authors reported that at 50 ppm about half of the subjects experienced what was described as a prenarcotic discomfort. For irritation, an increase in symptom reports seems evident only for eye irritation at 200 ppm, and, less so, for irritation of the lips at 200 ppm ( Oltramare et al. 1974). Styrene NAC: 02/ 2004 8 FIGURE 1: SYMPTOM RATINGS AT OR AFTER ACUTE EXPOSURE OF HUMANS TO STYRENE ( Table adopted from Oltramare et al. 1974)

Vestibulo oculomotor disturbances were studied by Ödkvist et al.( 1982). 10 healthy nonsmoking volunteers ( 5 man, 5 women, age 20   30 years) inhaled styrene via mouth tube at an analytically confirmed concentration between 87 and 139 ppm ( fluctuating < 2 % during each individual exposure) during light exercise ( 50 W) for one hour. Vestibulo oculomotor tests ( swing test, optovestibular test, visual suppression test, optokinetic test, saccade test, slow pursuit moving test) were performed before, during and 1 hour after exposure. Each individual served as its own control. There were no effects on any test except the saccade test in which 8 of 10 subjects showed an enhanced maximum speed of the saccade during exposure. The authors conclude that the results suggest an effect of styrene on the vestibulo ocular system by blocking inhibitory mechanisms in the CNS.

Pierce et al. ( 1998) exposed 4 healthy male non smoking volunteers ( 26   30 years old, no known history of solvent exposure) in a 13.8 m ³ chamber to analytically ( infrared spectrophotometry) confirmed concentrations of 15   99 ppm styrene in different exposure scenarios. No changes were Styrene NAC: 02/ 2004 9 observed in a digit recognition test performed after 35 minutes of exposures and in electroencephalogram performed after each 100 minute exposure.

TABLE 2: SUMMARY OF ACUTE NON LETHAL EFFECTS IN CONTROLLED HUMAN STUDIES FOLLOWING INHALATION OF STYRENE Exposure duration Concentration ppm ( mg/ m ³ ) Effects and remarks Reference 1   2 minutes 500   800 ppm 300   400 ppm Intolerable irritation of previously non exposed subjects; lacrymation, irritation of nasopharynx Götell et al. 1972 4 hours 800 Immediate eye and throat irritation, CNS depression with listlessness, drowsiness, impairment of balance, and, after termination of exposure, muscular weakness and unsteadiness with inertia and depression Carpenter et al. 1944 Not reported 60 ppm 100 ppm 200   400 ppm   600 pm Rapid onset of effects: " detectable odor but no irritation" " tolerated without excessive discomfort", " strong" odor " objectionably strong odor" strong eye and nasal irritation Wolf et al. 1956 4 x 30 minutes with stepwise increasing concentration 50 ppm 150 ppm 250 ppm 350 ppm Exposure via mouthpiece ( avoiding eye irritation); slight increase of simple and choice reaction time at 350 ppm Gamberale and Hultengren 1974 1 hour 87   139 ppm No effect on vestibulo oculomotor parameters except saccade test where 8 of 10 subjects showed an enhanced maximum speed of the saccade Ödkvist et al. 1982 35 minutes 100 minutes 15   99 ppm 15   99 ppm No changes in digit recognition test No changes in electroencephalogram Pierce et al. 1998 1 hour 2 hours 20 minutes 1 hour 3 minutes 15 minutes 25 min.   1 hour 2 x 3.5 hours ( with 30 minutes break) 51 ppm 117 ppm 216 ppm 216 ppm 376 ppm 376 ppm 376 ppm 99 ppm No subjective symptoms or objective signs of illness; strong, but not objectionable odor Odor initially strong, nasal irritation No signs of CNS effects " Mild" eye irritation Nasal irritation CNS effects: difficulties in balance performance tests, decrements in manual dexterity test, nausea, inebriation, headaches Complaints of mild eye and throat irritation after 20   30 minutes, subsiding later; intermittent difficulties in performing Romberg test in 3/ 6 subjects. No subjective symptoms or signs of CNS effects at the end of exposure. Clinical and laboratory data normal. Stewart et al. 1968 Styrene NAC: 02/ 2004 10 TABLE 2: SUMMARY OF ACUTE NON LETHAL EFFECTS IN CONTROLLED HUMAN STUDIES FOLLOWING INHALATION OF STYRENE Exposure duration Concentration ppm ( mg/ m ³ ) Effects and remarks Reference 1   3 hours 1 hour 1.5 hours 50, 100, 200 ppm 200 ppm 50, 100, 200 ppm Rating scores for subjective symptoms of CNS effects ( headaches, sleepiness, nausea, fatigue, poor concentration)

in rating score at   100 ppm Scores for irritation at 50 ppm also  , but no evident concentration response Slight difficulties in balance performance, but large variation of data Possibly slight increase in reaction time, but no dose response Oltramare et al. 1974 6 hours 25   50 ( with/ without 4 peaks 15 min 100 ppm) Neither performance to neuropsychological tests nor subjective signs and symptoms of irritation or CNS effects negatively influenced Ska et al. 2003; Vyskocil et al. 2002a 1  7.5 hours 0 ppm 20 ppm 75   125 ppm* 100 ppm 125 ppm Men: 3 days at 20 ppm, 4 days at 100 ppm, 4 days at 75   125 ppm ( average 100 ppm), 5 days at 125 ppm, 7 days at 0 ppm Women: 4 days at 100 ppm, 2 days at 0 ppm No CNS effects in equilibrium and cognitive testing Subjective symptoms ( exposure times not reported):

Irritation ( eyes, nose, throat) Headache: Men/ Women Men/ Women 13 %/ 8 % 3 %/ 0 % 17 % 0 % 20 % 0 % 33 %/ 32 % 13 %/ 35 % 45 % 12 % Hake et al. 1983 3   4 hours 20 ppm 0.5   40 ppm ( peak) Ratings for odor, annoyance and, marginally, for irritation increase with concentration; ratings of irritation verbally labelled as " hardly at all" Seeber et al. 2002 *: fluctuating exposure concentration, average 100 ppm.

Acute effects of styrene on the CNS were also studied in a total group of healthy male volunteers ( 20   50 years old, smokers and non smokers) not previously exposed to styrene and with no documented exposure to neurotoxicants during the study ( Ska et al. 2003; Vyskocil et al. 2002a; 2002b). The volunteers were exposed to styrene at rest to 5 different scenarios that lasted 6 hours each: a) continuously to 106 mg/ m ³ ( 25 ppm), b) variable exposure with a mean of 25 ppm and four 15 minutes peak exposures up to 213 mg/ m ³ ( 50 ppm), c) exposure to 1 ppm, control), d) exposure to 50 ppm, e) mean exposure to 50 ppm with four 15 minutes peaks up to 426 mg/ m ³ ( 100 ppm). The sequence of exposures was c a b c d e. Exposure was carried out in an 18 m ³ chamber and the styrene concentration was monitored by gas chromatography and infrared analysis. Before and after exposure, the volunteers were submitted to a battery of test proposed by the World Health Organization to detect neurotoxic effects of chemicals: sensory tests ( visual: Lanthony D 15 and vision contrast test, olfactory: smell test), neuropsychological tests ( reaction time, attention, memory, psychomotricity), and self evaluation questionnaires for mood ( seven category response scale) and symptoms ( four point scale for 17 items regarding irritation and CNS effects) in a test retest design. The testings were performed before exposure Styrene NAC: 02/ 2004 11 ( Base line) and within 1 hour after the end of exposure. Initially, 42 subjects took part in the study. However, only data from subjects who had taken part in all scenarios were retained for further analyses. Missing data were due to absence of subjects at a given scenario or to factual problems during testing. Therefore, complete data were available for 24 subjects. The different exposure scenarios negatively influenced neither the performance to any test nor the subjective signs and symptoms.

Psychological reactions related to chemosensory irritation during exposure to a number of chemicals including styrene were investigated by Seeber et al. ( 2002). Exposure studies were conducted in a ventilated room of 28 m ³ ( air exchange about 250 m ³ / h) with continuous control of the concentration of the test substance ( deviations < 3 %). In all experiments, 4 young healthy male volunteers who had no knowledge of the experimental conditions, were investigated simultaneously. The concentrations of styrene were 20 ppm for 3 hours or 0,5 ppm periods for 50 minutes followed by 40 ppm peaks for 30 minutes during a total 4 hours. At control and at 20 ppm each, a total number of 16 volunteers were exposed, at 0.5/ 40 ppm, the total number of volunteers was 24. Ratings for irritation, odor and annoyance were assessed and mean values were calculated from 2   5 repeated ratings for a given exposure level ( total observations 16   246). For odor and annoyance, ratings increased similarly with increasing styrene concentration while there was only a marginal report for irritation. Thus, annoyance was more closely associated with odor than with irritation. Effect sizes comparing the ratings during exposure to 20 ppm and during the pre exposure test were higher for odor, irritation and annoyance. Effect sizes were also higher compared to " clean air only" exposure. However, the ratings for irritation ( in case of styrene and all other solvents investigated) reached only levels verbally labelled " hardly at all".

Studies with repeated inhalation exposure In a study conducted for and summarized by NIOSH ( 1983), 10 men were exposed in groups of 2   4 for 1, 3, or 7.5 hours/ day to 0, 20, 100, or 125 ppm styrene ( Hake et al. 1983). 8 women were exposed in groups of 1   4 at 0 or 100 ppm. For men, there were 3 days of exposure at 20 ppm, 4 days at 100 ppm, 4 days at 100 ppm with concentrations fluctuating between 75 and 125 ppm, 5 days at 125 ppm, and 7 days at 0 ppm. For women, there were 4 days at 100 ppm and 2 days at 0 ppm. In control exposures, the chamber was odorized with 10 ppm styrene upon entry of the subjects after which exposure was reduced to 0 ppm within 10 minutes. Each subject was exposed to more than one concentration, non exposure weekends or control exposures were interspersed with exposure to styrene.

There were no deleterious effects on equilibrium as measured by Romberg and heel to toe tests. Some changes in visual evoked response and amplitude of electroencephalogram ( EEC) were observed in 3 of 6 subjects studied that   according to the authors   were consistent with CNS depression. However, the changes were neither consistent between subjects nor in magnitude within subjects. Furthermore, there was no significant variance in cognitive testing scores related to styrene exposure. Respiratory parameters generally showed no effects of styrene exposure; however, the authors observed decrements in maximal expiration values in subjects repeatedly exposed to 100 ppm for 7.5 hours ( no details reported).

With respect to subjective symptoms noted on a checklist during exposure, the overall data indicated some dose response for irritation ( eyes, nose, and throat) and headaches. For men, the reported incidences of irritation were 13 % ( 0 ppm), 17 % ( 20 ppm); 20 % ( 100 ppm), 33 % ( 100 ppm fluctuating), 45 % ( 125 ppm); for headaches, incidences were 3 %, 0 %, 0 %, 13 %, 12 %. For women, the incidence of irritation was 8 % ( 0 ppm) ad 32 % ( 100 ppm), for headaches, incidences were 0 % and 35 %. There was no specific indication as to which exposure time the various subjective responses were elicited at a given exposure concentration ( Hake et al. 1983). Styrene NAC: 02/ 2004 12 Odor perception The odor of styrene has been described as solventy, rubbery, and plasticy ( Leonardos et al. 1969; Ruth 1986) and also as strongly metallic ( Gamberale and Hultengren 1974). A wide range of odor thresholds is reported in the literature. This wide range may be due to different degrees of purities of the test substances used, the presence or absence of polymerization inhibitors, different methodology used, different bases used ( median, mean, range), individual variability or an adaptation to odor perception following repetitive exposure.

The olfactory function and the styrene odor detection threshold were compared between a group of workers exposed to styrene at least 4 years ( reinforced plastics industry, current mean personal air sampling concentrations of styrene about 11   66 ppm) and a group of age and gender matched naïve controls ( Dalton et al. 2003). Absolute odor threshold concentration values were not presented in the study. The styrene odor detection threshold for workers was on average 32 fold higher than for controls. Furthermore, when the results were stratified by age, the most pronounced increase in odor threshold was observed in workers who were in their 5th or 6th decade, while duration of exposure was not related to the effect. No differences were found betweeen workers and controls with respect to the odor threshold for an olfactory standard, phenylethyl alcohol, and for the ability to identify a variety of 20 different aroma compounds in an odor identification test. The results do not provide evidence that styrene is an olfactory toxicant in humans.

Van Doorn et al. ( 2002) present results of odor threshold determinations for styrene that were a) measured by olfactometry methods considered compatible with a precursor of the NVN2820 and EN13725 method or b) were measured by TNO in the Netherlands using a precursor of the NVN2820 and EN 13725 methods, with a mean n butanol threshold of 25 ppb. Results of both were converted to the reference agreed in EN13725 of 400 ppb n butanol by using a factor of 40: 25 = 1.6. Thereby, odor thresholds of 0.049 ppm and 0.025 ppm, respectively, were obtained. Taking into account the threshold value of 0.033 ppm obtained by the Japanese method ( see below Hoshika et al. 1993), Van Doorn et al. ( 2002) calculated a mean odor threshold of 0.0345 ppm for styrene.

A comparison of odor threshold values determined by different methods in Japan ( triangle olfactometer method, odor room, 20 trained male perfumers 30   45 years old) and in the Netherlands ( olfactometer, 4 men, 4 women 18   40 years old), showed that the " barely perceptible or detectable odor thresholds" of 0.033 ppm and 0.016 ppm, respectively, are quite similar ( Hoshika et al. 1993). The Japanese " triangle olfactometer method" produces an n butanol threshold of 38 ppb that is compatible with the value ( 40 ppb) of the method according to EN13725 ( Van Doorn et al. 2002).

In accordance with these data, WHO ( 1983) reported an odor perception threshold of 0.05   0.08 ppm. In other older studies and compilations, substantially higher values were reported. Odor thresholds ranging from 0.1   201 ppm ( 0.43   860 mg/ m ³ ) for styrene ( inhibited) and from 0.047   201 ppm ( 0.2021   860 mg/ m ³ ) for styrene ( uninhibited) were reported by Ruth ( 1986). Based on 10 original literature references which were not explicitly reported, a geometric mean odor threshold of 0.32 ppm styrene ( standard error 2.0 ppm) was calculated ( Amoore and Hautala 1983).

The odor recognition threshold was determined for 53 odorant chemicals including styrene under controlled laboratory conditions using a standardized and defined procedure ( Leonardos et al. 1969). The odor threshold represents that concentration at which all four trained panelists could positively recognize the odor. Different threshold values were obtained for styrene without inhibitor ( 0.047 ppm) or with inhibitor Styrene NAC: 02/ 2004 13 ( 0.10 ppm) and for inhibited styrene additionally purified by gas liquid chromatography ( 0.21 ppm). The chemical nature of the inhibitor was not reported.

2.3 Developmental/ Reproductive Toxicity No data regarding developmental or reproductive toxicity in humans following single exposure to styrene have been found in the available literature.

Studies with repeated inhalation exposure The epidemiological data have been extensively reviewed recently ( Brown et al. 2000; IARC 2002). In case reports, malformations in children of styrene exposed mothers and spontaneous abortion in female workers occupationally exposed to styrene were described. However, these observations could not be confirmed in epidemiological studies. According to the reviews mentioned above, there is no sound evidence for an association between workplace exposure to styrene and spontaneous abortions, malformations or decreased male fecindity.

2.4 Genotoxicity Genotoxicity studies have been extensively evaluated and summarized in a number of reviews ( ATSDR 1992; Bonassi et al. 1996; Cohen et al. 2002; IARC 1994; IARC 2002; Vodicka et al. 2002; WHO 1983; WHO 2000). Since a detailed description of the findings from these studies is beyond the scope of this TSD, findings as described in these reviews are summarized.

In in vitro systems with human cells, styrene induced chromosomal aberrations ( CA), sister chromatid exchanges ( SCE), micronuclei, and hypoploidy in whole blood cultures in the absence of exogenous metabolic activation system were observed. CA and SCE were also observed in lymphocyte cultures in the absence of exogenous metabolic activation system.

In vivo, no data regarding genotoxic effects in humans following single exposure to styrene have been found in the available literature.

Studies with repeated inhalation exposure A number of cytogenetic studies have been conducted on workers with occupational exposure to styrene, especially in the reinforced plastics industry. The workers in the individual studies had been exposed between less than one year and about 30 years to widely differing concentrations of styrene as estimated from air sample or monitoring or biological monitoring of urinary styrene metabolites. The number of workers included in individual studies mostly was less than 50. With respect to chromosomal aberrations, the majority of studies revealed a significant increase in CA ( including gaps), and doseresponses were observed in several studies. A cross studies evaluation found a positive association among studies between the level of styrene exposure and the frequency of CA. Fewer studies have looked at sister chromatide exchange ( SCE), and the percentage of positive studies was smaller than the percentage of positive studies of CA. However, two regression analyses revealed significant associations between styrene in air or urinary mandelic acid excretion and SCE frequency. There is less evidence of an association between styrene exposure and the frequency of micronuclei, and in a cross studies evaluation, no such association could be found. Other studies in workers have provided evidence that occupational exposure to exposure may lead to a several fold increase in the formation of DNA adducts ( O6 deoxyguanosine and N7 deoxyguanosine adducts), DNA single strand breaks, and gene mutations at the HPRT and the glycophorin A locus. Styrene NAC: 02/ 2004 14 Recently, physiological modeling of the relative contributions of styrene 7,8 oxide ( SO) derived from direct inhalation and from styrene metabolism to the systemic dose in humans has been performed. From these calculations, it has been suggested that SO which is present in the air at workplaces in the reinforced plastics industry could present a greater hazard of cytogenetic damage than inhalation of styrene ( Tornero Velez and Rappaport 2001).

2.5 Carcinogenicity No data regarding the development of cancer in humans following single exposure have been found in the available literature.

Studies with repeated inhalation exposure The cancer epidemiology data have been reviewed recently ( Cohen et al. 2002; IARC 2002).

Retrospective cohort mortality studies and nested case control studies were conducted in three types of industy: in the production of styrene monomer and polystyrene, of glass fibre reinforced plastics, and of styrene butadiene rubber.

Because workers in the reinforced plastics industry have higher styrene exposure and less potential for exposure to other substances than the other cohorts studied, the most informative data with regard to an association between styrene exposure and cancer come from studies of these cohorts. In three studies in such cohorts, an excess of lung or respiratory cancer was found. However, the excess occurred in those groups of workers with lower exposure. An excess of lymphatic and hematopoietic ( LH) cancers was observed in some epidemiological studies in the reinforced plastics industry, but not in others. Such an association also was found in two studies of workers in styrene production, but exposure was poorly documented and may have been also to other chemicals beside styrene. Studies in workers of the styrenebutadiene rubber production also found a small excess of leukemia mortality. However, these findings are difficult to evaluate because of the high correlation between exposure to styrene and butadiene ( Cohen et al. 2002).

Reports of increased risks of other cancers ( rectal, pancreatic, nervous system) are also reported in some studies. Mostly, the numbers of cases are small, and these findings are not supported from data of larger cohort studies.

2.6 Summary A great number of studies on workers with occupational exposure to styrene in different workplaces have been carried out. These studies have been repeatedly reviewed and summarized ( ACGIH 1997; ATSDR 1992; Cohen et al. 2002; DFG 1987; Government Canada 1993; IARC 2002; OEHHA 1999; Sherrington and Routledge 2001; US EPA 1998; WHO 1983; WHO 2000). The highest exposure occurs in the fabrication of reinforced polyester plastics composites, where 8 hour average samples in breathing zones often exceeded 100 ppm styrene ( IARC 2002). In older studies on workers in the manufacture of reinforced plastics, 8 hours TWA concentrations in the breathing zone of up to 292 ppm were reported, with peaks of about 1500 ppm during shorter periods of work for about 5   10 minutes ( Götell et al. 1972).

In workers with chronic exposure to styrene, effects on the central and peripheral nervous systems that were described in many studies include a reversible decrease in color discrimination. Decrements of auditory function was also observed, though findings made in several smaller cross sectional Styrene NAC: 02/ 2004 15 studies could not be confirmed in the largest study. Studies of effects on the immune and hematopoetic system, liver, and kidney did not reveal consistent changes ( IARC 2002).

No reports of lethal intoxication following styrene exposure were located in the literature.

Pure styrene has a pungent, slightly sweetish odor. A wide range of odor thresholds has been reported. Van Doorn et al. ( 2002) presented results of odor threshold determinations for styrene and calculated an n butanol corrected mean odor threshold of 0.0345 ppm for styrene.

Styrene is irritating to eyes and the respiratory tract. In a number of controlled studies with human volunteers, irritation and effects on the CNS were investigated.

In a study on psychological reactions related to chemosensory irritation, ratings for odor and annoyance increased similarily with increasing styrene concentrations ranging from 20   40 ppm, while there was only a marginal increase for irritation. Effects sizes comparing the ratings between exposure to 20 ppm and pre exposure were higher for odor, irritation, and annoyance. Effects sizes were also higher compared to " clean air only" exposure. However, the ratings for irritation reached only levels verbally labelled as " hardly at all" ( Seeber et al. 2002). No increase in irritation or headaches compared to control was noted at 20 ppm in a further study ( Hake et al. 1983). At 50 ppm, one study indicated a marginal increase in subjective symptoms ratings for eye and nose irritation, headache, and fatigue ( Oltramare et al. 1974). In that study, signs of irritation and of mild subjective CNS effects ( headaches, fatigue, poor concentration, sleepiness) were reported more often at 100 ppm. Complaints of mild eye and throat irritation at 100 ppm in one test but not in another were reported by Stewart et al. ( 1968). In a recent study, subjective signs and symptoms during 6 hour exposure to 25   50 ppm styrene with 4 peaks of 15 minutes at 100 ppm indicated no irritation ( Vyskocil et al. 2002a, b). At about 200 ppm, most subjects noted irritation of eye and nose ( Oltramare et al. 1974; Stewart et al. 1968) and the severity increased with a further increase in concentration to 376 ppm. In their study on styrene exposed workers, Götell et al. ( 1972) noted that they themselves suffered from immediate lacrymation at 300   400 ppm and could not withstand 500   800 ppm for more than 1   2 minutes although the workers tolerated such concentrations. In two further studies with controlled exposure of volunteers, concentrations   600 ppm caused strong eye, nasal, and throat irritation ( Carpenter et al. 1944; Wolf et al. 1956).

No lesions of the nasal mucosa were observed in a cross sectional study on styrene exposed workers. Furthermore, the ability to detect and identify different odors in a controlled odor test was not affected in workers with long term exposure to styrene. These limited data provide some evidence that   in contrast to rats and especially mice   styrene does not seem to be an olfactory or upper respiratory tract toxicant in humans. Support for this conclusion also comes from toxicokinetic studies in vitro in which the metabolic capacity of nasal epithelia from humans, rats, and mice was compared ( see section 4.1, page 47).

With respect to CNS effects, one study reported higher ratings of headaches, poor concentration, and fatigue at 50 ppm compared to   odor blinded" control exposed to 3   5 ppm ( Oltramare et al. 1974). In another study, headaches did not occur when subjects were repeatedly exposed to fluctuating concentrations of 75   125 ppm ( average: 100 ppm), but were reported at 125 ppm ( Hake et al. 1983). Pierce et al. ( 1998) found no changes in a digit recognition test after 35 minutes of exposure and in electroencephalogram after 100 minutes of exposure to 15   99 ppm styrene in different exposure scenarios. At 100 ppm, intermittent difficulties in performing a modified Romberg test were observed in 3/ 6 subjects exposed for 7 hours with a 30 minute break in between. Other tests on coordination and on manual dexterity were normal, and no effects were noted at the end of exposure. In the same study, no CNS effects were seen in another experiment with 100 ppm exposure for 2 hours or 216 ppm for 1 hour ( Stewart et al. 1968). Also, exposure Styrene NAC: 02/ 2004 16 for 6 hours at 50 ppm with 4 peaks of 15 minutes at 100 ppm had no negative influence on performance to neuropsychological tests ( Vyskocil et al. 2002a, b). No effects on equilibrium and cognitive function tests were noted in male and female volunteers at repeated exposures to 100 and 125 ppm for at least one hour ( Hake et al. 1983). Oltramare et al. ( 1974) noted that slight difficulties in balance performance at 50   200 ppm ( 1.5 hours), but there was no concentration response, and slight difficulties in balance performance at 200 ppm ( 1 hour), but the variation of data was large. No effects on simple and choice reaction time was seen following exposure to 250 ppm for 30 minutes. However, when the concentration was raised to 350 ppm for 30 minutes directly afterwards, both simple and choice reaction time were increased ( Gamberale and Hultengren 1974). More pronounced effects were observed during exposure to 376 ppm for one hour: one subject complained of nausea that persisted one hour after the end of exposure, 2 had a feeling of being inebriated, and 3 of 5 subjects exposed were unable to normally perform the modified Romberg test and also 3 subjects ( unclear, if the same 3 subjects) had significant decrements in other tests of coordination and manual dexterity ( Stewart et al. 1968). Only one controlled study was located in which CNS effects were followed at a higher concentration than 376 ppm. In that study, two subjects exposed to 800 ppm for 4 hours reported that they suffered from listlessness, impairment of balance, drowsiness, and, after termination of exposure, from muscular weakness and unsteadiness with inertia and depression. CNSdepression was also indicated by a marked decrease in performance in a " steadiness test" ( measuring manual dexterity) ( Carpenter et al. 1944).

No data are are available indicating reproductive or developmental toxicity of styrene in humans after acute exposure.

In in vitro systems with human cells, styrene induced chromosomal aberrations ( CA), sister chromatid exchanges ( SCE), micronuclei, and hypoploidy. No data regarding genotoxic effects in humans following single exposure to styrene are available. Epidemiological studies provide evidence for genotic effects ( chromosomal aberrations, mutations, DNA adducts) in occupationally exposed workers.

With respect to carcinogenicity, IARC ( 2002) concluded that the increased risks for cancers of the lymphatic and hematopoetic system are small, statistically unstable and often based on subgroup analyses, the findings are not very robust and that it cannot be ruled out that the observations are the results of chance, bias or confounding. Cohen et al. ( 2002) conclude that, although the balance of epidemiologic studies do not suggest a causal association between styrene and any human cancer, because of the limited power of these studies, the inconclusive results do not rule out the possibility that the observed increase of lung tumors in mice are of relevance to humans.

In its latest evaluation, IARC ( 2002) concluded that there is " limited evidence in humans for the carcinogenicity of styrene" and, taking into account the results from animal carcinogenicity studies ( see 3.5), that styrene is " possibly carcinogenic to humans ( Group 2B)" ( IARC 2002). US EPA ´ s Office of Research and Development has also updated previous assessments on the carcinogenic potential of styrene and concluded that styrene is appropriately classified as a Group C, possible human carcinogen ( US EPA 2003).

Based on the body burden of styrene 7,8 epoxide or its adducts with hemoglobin and DNA, and taking into account the results from carcinogenicity studies with styrene in animals, a cancer risk has been estimated in the range of 1.7   7.5 per 100,000 persons exposed for 40 years to 20 ppm styrene, 8 hours/ day, 5 days/ week, 48 weeks/ year ( Greim 2003). Styrene NAC: 02/ 2004 17 3 ANIMAL TOXICITY DATA 3.1 Acute Lethality Data on acute lethality after inhalation exposure to styrene are available for rats, mice, and guinea pigs ( TABLE 3). Non lethal effects observed in these studies are described in section 3.2.2.

3.1.1 Rats Female and male Sprague Dawley rats ( 10 of each sex/ group; 20 of each sex at the highest concentration) were exposed to analytically ( gas chromatography) determinded concentrations of 2983, 3766, 4814, 5911, 6621, 7218 and 8407 ppm styrene in a 180 L dynamic exposure chamber for 4 hours ( BASF 1979b). Survival of animals was followed for 14 days after the exposure. No deaths were observed at 2983 and 3766 ppm. At the other conentrations, deaths were observed up to three days after exposure. No differences in LC50 between female and male rats were observed. A combined LC50 of 6410 ppm ( 95 % conf. limit 6025   6769 ppm) for male and female rats was determined. Necroscopy revealed acute dilation and congestive hyperemia in the heart, enlarged lung, and centrilobular liver changes with fatty degeneration. Other, non lethal effects are described in section 3.2.2.

The LC50 values were determined for a number of benzene derivatives in male Sprague Dawley rats ( Bonnet et al. 1982a). Groups of 12 rats each were exposed ( as more precisely described in Gradiski et al.( 1978)) in 170 L dynamic exposure chambers to analytically ( gas chromatography) confirmed vapor concentrations of styrene for 6 hours. Animals were observed for 14 days after the end of exposure. The 6 hour LC50 for styrene was 4618 ppm ( 95 % confidence interval 4399   4894 ppm). Death was preceded by somnolence, tremors, and muscular seizures but no lacrymation was observed. From the figure presented in the publication, it can be estimated that 90 % of the animals died ( LC90) at about 5000 ppm and 10 % died ( LC10) at about 3300 ppm ( FIGURE 2) indicating a steep concentration response curve. The authors also reported the occurrence of delayed deaths ( more than 24 hours after the end of exposure) and that surviving animals showed growth retardation betweeen day 7 and 14 post exposure. However, no detailed data were presented.

A total number of 405 rats ( sex, strain and number of animals per exposure group not reported) were exposed to styrene concentrations ranging from 1300 ppm to 10,000 ppm for one hour to up to more than 30 hours ( Spencer et al. 1942). After the exposure the animals were observed for 2   4 weeks. No LC50 but only LC0 and LC100 were reported in the study. The highest concentration that could be reached without observed condensation of the chemical out of the atmosphere was 10,000 ppm1. At this concentration, no deaths were observed after one hour of exposure, but all rats exposed for 3 hours died. At 5000 ppm, no animal died after exposure for 2 hours but all animals exposed for 8 hours died. At 2500 ppm, all animals survived an 8 hour exposure and death of all animals only was observed when the exposure lasted 21 hours. All animals survived 16 hours of exposure to 2000 ppm. Immediate deaths during or shortly after the end of exposure were due to the effect of styrene on the CNS ( see 3.2.2). However, there were also delayed deaths with pulmonary edema and hemorrhages that were considered to develop as a result of the acute irritating effect of styrene on the lung.

1 [ Note: from the vapor pressure data   see TABLE 1   a saturated vapor concentration of 6580 ppm at 20 ° C and of 8560 ppm at 25 ° C can be calculated]. Styrene NAC: 02/ 2004 18 FIGURE 2: CONCENTRATION RESPONSE CURVE FOR ACUTE LETHALITY FOLLOWING INHALATION OF STYRENE IN RATS ( Figure from Bonnet et al. 1982a)

Shugaev ( 1969) exposed rats ( strain, sex and number of animals not reported) to analytically ( gas chromatography) controlled styrene vapor concentrations for 4 hours in dynamic flow exposure chambers. A LC50 of 11.8 mg/ l ( 95 % confidence interval 10.3   13.5 mg/ l) ( 2761 ppm; 95 % confidence interval 2410   3159 ppm) was calculated. It was also reported that animals " often" died after the exposure. It is further reported that these animals were not used for the determination of lethal brain styrene concentrations but it is not clear if data for animals dying after the end of exposure were included in the determination of the LC values.

Groups of ten female Sprague Dawley rats were exposed to analytically ( infrared analysis) confirmed styrene vapor concentrations for 4 or 8 hours, respectively ( Lundberg et al. 1986). Deaths were counted 24 hours after the start of exposure. At 33,200 mg/ m ³ ( 7769 ppm), a concentration that corresponded to approximately saturated styrene vapor in air at 25 ° C ( see footnote 1), no deaths were seen after a 4 hour exposure but four of ten animals died within 24 hours after an 8 hour exposure. No LC50 could be determined.

TABLE 3: SUMMARY OF LETHAL EFFECTS IN ANIMALS AFTER ACUTE INHALATION EXPOSURE TO STYRENE Species, sex, strain Concentration in ppm Exposure Duration Effect/ Remarks Reference Rat, f, m, S D 6410 6310 6480 4 hours LC50, female and male LC50, females only LC50, males only BASF 1979b Styrene NAC: 02/ 2004 19 TABLE 3: SUMMARY OF LETHAL EFFECTS IN ANIMALS AFTER ACUTE INHALATION EXPOSURE TO STYRENE Species, sex, strain Concentration in ppm Exposure Duration Effect/ Remarks Reference Rat, f, m, S D 8407 7218 6621 5911 4814 3766 2983 4 hours 18/ 20 f, 20/ 20 m, 38/ 40 m + f died 5/ 10 f, 8/ 10 m, 13/ 20 m + f died 6/ 10 f, 3/ 10 m, 9/ 20 m + f died 6/ 10 f, 1/ 10 m, 7/ 20 m + f died 1/ 10 f, 2/ 10 m, 3/ 20 m + f died 0/ 10 f, 0/ 10 m, 0/ 20 m + f died 0/ 10 f, 0/ 10 m, 0/ 20 m + f died BASF 1979b Rat, nd, nd 1300 2000 2500 5000 10,000 30 hours > 40 hours 16 hours > 30 hours 8 hours 21 hours 2 hours 8 hours 1 hour 3 hours LC0 LC100 LC0 LC100 LC0 LC100 LC0 LC100 LC0 LC100 Spencer et al. 1942 Rat ( 18 f; 15 m) 5100 1 hour No death during exposure Niklasson et al. 1993 Rat, nd, nd 2270 ( 9.7 mg/ l) 2761 ( 11.8 mg/ l) 3276 ( 14.0 mg/ l) 4 hours 4 hours 4 hours LC16 LC50 LC84 Shugaev 1969 Rat, nd, nd 2700 4 hours LC50; abstract only Jaeger et al. 1974 Rat, f, m, CD 1500 6 hours 0/ 20 died after repeated ( subchronic) exposure Cruzan et al. 1997b Rat, f, m, CD 1000 6 hours 0/ 70 died after repeated ( chronic) exposure Cruzan et al. 1998 Rat, m, S D ~ 5000 4618 ~ 3300 6 hours 6 hours 6 hours LC90 estimated from figure LC50 LC10 estimated from figure Bonnet et al. 1982a Rat, f, S D 7769 ( 33.2 mg/ l) 4 hours 8 hours 0/ 10 animals died 4/ 10 animals died Lundberg et al. 1986 Mouse, f, m, NMRI 1600 1840 1370 4 hours LC50, female and male LC50, females only LC50, males only BASF 1979a Mouse, f, m, NMRI 3766 2983 1528 1420 864 680 4 hours 10/ 10 f, 10/ 10 m died 7/ 10 f, 9/ 10 m died 3/ 10 f, 8/ 10 m died 4/ 10 f, 6/ 10 m died 1/ 10 f, 0/ 10 m died 0/ 10 f, 0/ 10 m died BASF 1979a Styrene NAC: 02/ 2004 20 TABLE 3: SUMMARY OF LETHAL EFFECTS IN ANIMALS AFTER ACUTE INHALATION EXPOSURE TO STYRENE Species, sex, strain Concentration in ppm Exposure Duration Effect/ Remarks Reference Mouse, f, OF1 2429 6 hours LC50 Bonnet et al. 1979b; 1982a Mouse, nd, nd 4142 ( 17.7 mg/ l) 4914 ( 21.0 mg/ l) 5873 ( 25.1 mg/ l) 2 hours 2 hours 2 hours LC16 LC50 LC84 Shugaev 1969 Mouse 2223 ( 9.5 mg/ l) 4 hours LC50 ( no details reported) Izmerov et al. 1982 Mouse, B6C3F1, 65 f, 23 27 m 500 250 6 hours 6 hours 5 m, 0 f died after one exposure no death after one exposure Morgan et al. 1993c Mouse, B6C3F1, 5 m 500 250 6 hours 6 hours 4/ 5 found moribund and sacrificed, no death/ moribund after one exposure Morgan et al. 1993c Mouse, B6C3F1, 36 f, 36 m 500 250 6 hours 6 hours 6/ 36 m, 1/ 36 f died after one exposure no death after one exposure Morgan et al. 1993a Mouse, B6 C3F1, 30 m 500 6 hours 2/ 30 died after one exposure Mahler et al. 1999 Mouse, B6 C3F1, 20 f, 20 m 500 6 hours no death after one exposure Cruzan et al. 1997b Mouse, CD 1, 20 f, 20 m 500 250 6 hours 1/ 20 m died after one exposure no death after one exposure Cruzan et al. 1997b Mouse, B6 C3F1, 39 m 250 6 hours 4/ 39 died after one exposure Sumner et al. 1997 Mouse, CD 1, 30 m 250 6 hours 0/ 39 died after one exposure Sumner et al. 1997 Guinea pig, nd, nd 1300 2000 2500 5000 10,000 16 hours 40 hours 7 hours 30 hours 6 hours 14 hours 3 hours 8 hours 1 hour 3 hours LC0 LC100 LC0 LC100 LC0 LC100 LC0 LC100 LC0 LC100 Spencer et al. 1942 f: female; m: male; nd: no data; S D: Sprague Dawley.

According to an abstract ( Jaeger et al. 1974), a 4 hour LC50 of 2700 ppm was estimated for fed and fasted rats. It is further reported that styrene caused death by pulmonary irritation and edema, but no Styrene NAC: 02/ 2004 21 further details were presented ( strain, no. of animals and dose gropus, treatment with inducers, inhalation exposure conditions, occurrence of CNS effects).

No deaths occurred in CD ( Sprague Dawley) rats exposed 6 hours/ day, 5 days/ week to 1500 ppm for 13 weeks ( Cruzan et al. 2001a) or to 1000 ppm for 104 weeks ( Cruzan et al. 1998). Acute non lethal ( irritation) effects observed in these studies are described in section 3.2.2.

Studies with non inhalation exposure The oral toxicity of styrene was determined in a total number of 57 young adult white rats raised from a stock obtained from the Wistar institute. Styrene was given by gavage, but it is not clear whether the compound was given undiluted or as an olive oil or corn oil solution emulsified with an aqueous soltion of gum arabic. The oral toxicity was low as indicated by an LD50 of 5.0 g/ kg b. w. ( Wolf et al. 1956). In accordance with these data, in another study no death of rats was observed after oral administration of 1600 mg/ kg b. w. but all rats died after treatment with 8000 mg/ kg b. w. ( Spencer et al. 1942).

Groups of six female Sprague Dawley rats were treated i. p. with styrene ( Lundberg et al. 1986). Deaths were counted 24 hours and 14 days after the injection. The reported LD50 of 898 mg/ kg b. w. and the 95 % confidence limits ( 768   1051 mg/ kg b. w.) were identical at both time points indicating that there were no delayed deaths after intraperitoneal administration of styrene.

3.1.2 Mice Bonnet et al. ( 1979) studied the toxicity of styrene in female OF1 mice. Groups of at least 20 mice were exposed by whole body exposure in 170 L dynamic exposure chambers ( as more precisely described in Gradiski et al. ( 1978) to analytically ( gas chromatography) confirmed vapor concentrations of styrene for 6 hours. Animals were observed for 14 days after the end of exposure. The 6 hour LC50 for styrene was 2429 ppm ( 95 % confidence intervals 2352   2530 ppm). The authors reported the occurrence of delayed deaths on the 5th to 10th day after exposure but no detailed data were presented.

Shugaev ( 1969) exposed mice ( strain, sex and number of animals not reported) to analytically controlled styrene vapor concentrations for 2 hours in dynamic flow exposure chambers. A LC50 of 21.0 mg/ l ( 95 % confidence interval 17.8   24.8 mg/ l) ( 2761 ppm; 95 % confidence interval 4165   5803 ppm) was calculated. It was also reported that animals " often" did not die during, but after the exposure.

BASF ( 1979) conducted an acute inhalation toxicity study with NMRI mice. 10 female and 10 male mice per dose group were exposed " whole body" to analytically ( gas chromatography) determined concentrations of 680, 864, 1420, 1528, 2983 or 3766 ppm styrene for 4 hours in a 180 L dynamic exposure chamber. Survival of animals was followed for 14 days after the exposure. No deaths were observed at 680 ppm. At the other conentrations, deaths were observed 1   4 days after the exposure. The concentration response curve was steeper for male mice, and males seemed more sensitive than females. LC50 of 1370 ppm ( 95 % conf. limit 1087   1653 ppm) for male mice and of 1840 ppm ( 1486   2359 ppm) for female mice were determined. Necroscopy revealed acute dilation and congestive hyperemia in the heart, enlarged lung, and centrilobular liver changes. Other, non lethal effects are described in section 3.2.3.

Without presenting further details, a 4 hour LC50 of 9500 mg/ m ³ ( 2223 ppm) for mice is reported by Izmerov et al. ( 1982). Styrene NAC: 02/ 2004 22 In a study to evaluate toxic effects of short term exposure to B6C3F1 mice, 23   27 male and 65 female animals ( 8 weeks old) per group were exposed to analytically confirmed concentrations of 0, 125, 250, or 500 ppm styrene ( 99.9 % pure) for 6 hours/ day ( starting at 7 AM) for up to 14 days ( Morgan et al. 1993c). Each animal was exposed individually in Hazleton 2000 chambers and the styrene concentration was measured every minute by infrared spectophotometry. After one exposure day, 5 males exposed to 500 ppm died. Mortality and morbidity were delayed after exposure and typically animals were found dead or moribund the morning after the exposure day. In an additional experiment in the same study conducted only with male mice, 4 of 5 animals died after one 6 hour exposure at 500 ppm. No deaths were observed after one exposure in male mice at lower concentrations or at any concentration in female mice.

In a further study of the same group ( Mahler et al. 1999), 2/ 30 male, 8 week old B6C3F1 mice were found dead one day after a single 6 hour exposure to 500 ppm styrene. Death was attributed to massive hepatic necrosis. In another study, death of 4/ 39 male B6C3F1 mice was observed following one exposure to 250 ppm for 6 hours ( Sumner et al. 1997).

Sex differences in susceptibility of B6C3F1 mice were further investigated ( Morgan et al. 1993a). 36 animals ( 8 weeks old) per sex and dose were exposed as described above to 0, 125, 250, or 500 ppm styrene. At 500 ppm, six male and one female mice were found dead or were terminated moribund after one exposure. No deaths occurred after one exposure to 250 ppm or 125 ppm. Necropsy of dead or moribund mice revealed that the liver of these animals was engorged with blood, and microscopic examination showed severe congestion and necrosis in the liver of these animals.

In a subacute toxicity study with CD 1 and B6C3F1 mice, one of 20 male CD 1 mice exposed to 500 ppm died after one 6 hour exposure ( Cruzan et al. 1997b).

Data on lethality following repeated short term exposure in Morgan et al. ( 1993a), Morgan et al. ( 1993c) and Cruzan et al. ( 1997) are summarized below (" studies with repeated exposure").

Studies with repeated inhalation exposure In a developmental toxicity study, 2 of 6 pregnant BMR/ T6T6 mice exposed to 500 ppm 6 hours/ day from the 6th day of gestation on died before the intended end of the exposure phase on day 16. At 750 ppm, 3 of 5 mice died. Surviving dams carried a high number of dead and resorbed fetuses ( Kankaanpää et al. 1980, see 3.3.2).

B6C3F1 mice were exposed up to 14 consecutive days to styrene as described above ( see 3.1.2, Morgan et al. 1993c). No animals died at 0 and 125 ppm styrene. The highest mortality was observed at 250 ppm where 7 males and 2 females died after two exposures and a total of 11 males and 6 females after 14 days. At 500 ppm, no deaths occurred in females, 7 males died after 2 exposures and a total of 8 males died during the 14 day exposure.

In a second study investigating sex related differences in susceptibility of B6C3F1 mice to styrene, animals were exposed up to 3 consecutive days to styrene as described above ( see 3.1.2, Morgan et al. 1993a). No control mice or mice exposed to 125 ppm died. At 250 ppm, 2 males and 3 females died or were terminated moribund after 2 exposures. At 500 ppm, 6 males and one female died after one exposure but no additional deaths occurred after subsequent exposures.

The susceptibility of different strains of mice to styrene inhalation exposure was studied ( Morgan et al. 1993b). 8 week old B6C3F1, C57BL/ 6, Swiss and DBA/ 2 mice ( 20 of each sex and strain at each dose group) were exposed to styrene ( 99.9 % pure) at nominal but analytically confirmed Styrene NAC: 02/ 2004 23 concentrations of 0, 125, 250, or 500 ppm in Hazleton 2000 chambers for 6 hours/ day for 4 consecutive days. No animals of any strain died at 0 and 125 ppm. Both strain and sex differences in mortality ( or sacrifice in moribund condition) were observed. The highest mortality occurred in Swiss mice and both sexes proved similarly susceptible ( male: 10/ 20 died at 500 ppm, female: 3/ 20 at 250 ppm, 8/ 20 at 500 ppm died). Overall mortality in B6C3F1 mice was comparable to that in Swiss mice but there was a clear sexspecific effect with mortality in male B6C3F1 mice ( 14/ 20 at 250 ppm, 3/ 20 at 500 ppm died) being much higher than in females ( 1/ 20 at 250 ppm). Mortality in C57/ BL6 mice also differed between males ( 7/ 20 at 250 ppm, 1/ 20 at 500 ppm) and females ( 1/ 20 at 250 ppm and 500 ppm each). Mortality in male B6C3F1 and C57BL/ 6 mice at 250 ppm was higher than at 500 ppm. No mortality was observed in male and female DBA/ 2 mice.

Sex related differences in mortality were also observed in B6C3F1 and CD 1 mice in another subacute study in which mice ( 20 per sex and dose group) were exposed in 0.75 m ³ inhalation chambers to analytically confirmed concentrations of 0, 15, 60, 250 or 500 ppm styrene for 6 hours/ day, 5 days/ week for 14 days ( Cruzan et al. 1997b). No deaths were observed at 15 and 60 ppm. Remarkably, the concentration response was non linear in female mice of both strains as mortality after two weeks clearly was higher at 250 ppm ( 7 CD 1, 10 B6C3F1) than at 500 ppm ( 2 CD 1, 0 B6C3F1). This was not observed in male mice of both strains where mortality increased with increasing concentration: One male of each strain died at 250 ppm, 7 male CD 1 and 8 B6C3F1 males died at 500 ppm.

In a further study by Morgan et al. ( 1995), 8 week old male and female B6C3F1 and Swiss mice were exposed to 0, 150 or 200 ppm styrene as described above for 6 hours/ day on 4 consecutive days. One female Swiss mouse died after four exposures to 200 ppm, necropsy revealed centrilobular hepatocellular necrosis in this mouse. No deaths were observed in male Swiss or in B6C3F1 mice of both sexes.

3.1.3 Guinea pigs A total number of 410 guinea pigs ( sex, strain and number of animals per exposure group not reported) were exposed to styrene concentrations ranging from 1300 ppm to 10,000 ppm for one hour to 40 hours ( Spencer et al. 1942). After the exposure the animals were observed for 2   4 weeks. No LC50 but only LC0 and LC100 were reported in the study. The highest concentration that could be reached without condensation of the chemical out of the atmosphere was 10,000 ppm. At this concentration, no deaths were observed after one hour of exposure, but all guinea pigs exposed for 3 hours died. At 5000 ppm, no animal died after exposure for 3 hours but all animals exposed for 8 hours died. At 2500 ppm, all animals survived a 6 hour exposure and death of all animals only was observed when the exposure lasted 14 hours. All animals survived 7 hours of exposure to 2000 ppm but all animals died when exposure at this concentration was extended to 30 hours. Immediate deaths during or shortly after the end of exposure were due to the effect of styrene on the CNS. However, there were also delayed deaths with pulmonary edema and hemorrhages that were considered to develop as a result of the acute irritating effect of styrene on the lung.

3.1.4 Hamsters Studies with non inhalation exposure Groups of 23 male Syrian hamsters were treated with 0, 450 or 600 mg/ kg b. w. styrene in corn oil by gavage ( Parkki 1978). 3 of the animals that had received 600 mg/ kg b. w. styrene died within 24 hours after administration. No deaths occurred at 450 mg/ kg b. w. Styrene NAC: 02/ 2004 24 3.2 Nonlethal Toxicity 3.2.1 Nonhuman primates Studies with repeated inhalation exposure Spencer et al. ( 1942) exposed 4 monkeys ( two of each sex, species not specified) to 1300 ppm styrene 7   8 hours/ day, 5 days/ week. Male monkeys received 142 exposures during 7 months, females 262   264 exposures over a period of 12 months. Additionally, there were at least 3 control monkeys. No further experimental details were reported. There were no signs of irritation or intoxication. Furthermore, the animals were reported to be in excellent condition and to show no gross or microscopic pathological lesions ( at least lung, liver, kidney, spleen, pancreas, adrenals were examined). Blood examination revealed no differences between the four exposed and three control monkeys.

3.2.2 Rats Female and male Sprague Dawley rats ( 10 of each sex/ group; 20 of each sex at the highest concentration) were exposed to 2983, 3766, 4814, 5911, 6621, 7218 and 8407 ppm styrene for 4 hours ( BASF 1979b, see 3.1.1). Styrene was irritating to eyes and respiratory tract as indicated by closed eyes, eye and nasal secretion, salivation, and dyspnoe. Signs of CNS impairment were staggered or stalking gait, tremors, lying on the side, and narcosis. Symptoms were not differentiated with respect to the individual exposure concentrations except that it was reported that narcosis was " slight" at the lowest concentration.

Shugaev ( 1969) reported that rats ( strain, sex and number of animals not reported) inhaling styrene for one hour at a concentration corresponding to the 4 hour LC50 ( reported to be 2761 ppm) were in a state of deep narcosis at the end of the 1 hour exposure.

Effects on the nervous system ( somnolence, tremors, and muscular seizures) that preceded death were also reported by Bonnet et al. ( 1982). Lacrymation was not observed in styrene exposed animals in this study.

Exposure of rats to 1300 ppm led to immediate irritation of eyes and nose with lacrymation, salivation, and nasal discharge ( Spencer et al. 1942, see 3.1.1 for further information). At this concentration, no other signs of intoxication were noted until 12 hours of exposure when general weakness and unsteadiness became apparent. These effects were more evident at 2000 ppm but more pronounced signs of effects on the CNS with loss of consciousness were only seen in " some" animals after 24   30 hours. At 2500 ppm, rats showed definite CNS depression ( weakness, stupor, incoordination, loss of equilibrium, tremor, finally unconsciousness) after 10   12 hours. Animals were " usually" completely unconscious within one hour exposure to 5000 ppm and even more rapid at 10,000 ppm. Unconsciousness was preceded by loss of quilibrium, falling on the side, running leg movements, tremors and convulsions. In addition to the immediate irritant action and the effects on the CNS, pulmonary changes were observed. These varied from slight congestion to hemorrhages, edema, exudation, and leucocytic infiltration. Generally, the severity of effects varied with the exposure concentration and duration. Marked pulmonary lesions were seen at all concentrations when the exposure time was so long that at least some of the exposed animals died following exposure. Regarding other organs, liver and kidney changes were seen in " comparatively few animals"; these changes were most often recorded at 2500 ppm but less frequently at higher concentrations.

Irritation during exposure also was observed in a subchronic study in which female and male CD ( Sprague Dawley) rats ( 10 females, 10 males per group) were exposed to analytically ( gas Styrene NAC: 02/ 2004 25 chromatography) confirmed concentrations of 200, 500, 1000 and 1500 ppm styrene in 0.75 m ³ inhalation chambers for 6 hours/ day, 5 days/ week for 13 weeks ( Cruzan et al. 1997b). Irritation was observed at all concentrations during exposure. Signs reached from closed eyes at 200 ppm to salivation and rubbing of paws and chin on the cage at higher concentrations. Similar effects ( salivation, restlessness, hunched posture) were also observed in groups of 70 male and female CD rats each during exposure to 500 and 1000 ppm for 6 hours/ day, 5 days/ week for 104 weeks; generally, effects tended to decrease during each week of exposure ( Cruzan et al. 1998).

Salivation and reduced attention were observed in a subacute study in which 10 male Wistar rats/ group were exposed 6 hours/ day on 5 consecutive days to an analytically confirmed concentration of 1500 ppm. At the beginning of the exposure period, signs of sensory irritation were also observed at 500 ppm but not at 150 ppm ( Jarry et al. 2002).

Male Wistar rats were exposed to analytically ( infrared spectophotometry) confirmed concentrations of 0, 100, 300, or 600 ppm styrene 12 hours/ day, 5 days/ week for 4 weeks ( Mäkitie et al. 2003). During the exposures, the animals were mostly recumbent, especially at 600 ppm. The authors saw no clear signs of irritation of skin, eye or mucous membranes at the end of the daily exposures.

TABLE 4: SUMMARY OF ACUTE NON LETHAL EFFECTS IN ANIMALS AFTER INHALATION EXPOSURE TO STYRENE Species ( strain, sex, no./ group) a Concentration ( ppm) Exposure Duration Effect Reference Rat ( Wistar, m) 2000 ppm 5 hours Loss of consciousness in " many of the test animals" Withey and Collins 1979 Rat ( nd; 18 f, 15 m) 1730 ppm 1 hour Inability to suppress nystagmus Niklasson et al. 1993 Rat ( Wistar, 10 m) 1500 ppm 500 ppm 6 hours Reduced attention, sensory irritation Sensory irritation at start of exposure Jarry et al. 2002 Rat ( nd) 4 hour LC50 ( 2760 ppm) 1 hour State of deep narcosis Shugaev 1969 Mouse ( Swiss OF1, 10 m) 549 ppm 4 hours 50 % decrease in immobility time in behavioral " despair swimming" test de Ceaurriz et al. 1983 Mouse ( Swiss Webster, m) 156 ppm 3 minutes RD50 Alarie 1973 Mouse ( Swiss OF1, 6 m) 586 ppm 5 minutes RD50 de Ceaurriz et al. 1981 Mouse ( Swiss Webster, m) 980 ppm 10 minutes RD50 Bos et al. 1992 Mouse ( NMRI, 10 f, 10 m) 1420 ppm 2983 ppm 3766 ppm 4 hours Staggered gait Apathy Narcosis BASF 1979a Styrene NAC: 02/ 2004 26 Effects of styrene on the vestibulo and opto oculo motor system effects were studied in a total number of 18 female and 15 male " pigmented rats" ( Niklasson et al. 1993). Each rat was used for an initial control experiment with no solvent exposure and one or two subsequent experiments with exposure to one or two styrene concentration levels. Exposure for each animal was separated by at least one week. Animals were exposed in a dynamic exposure chamber to nominal concentrations ( fluctuating within about 15 % as confirmed analytically during all exposures) of 3600; 7400; 13,400; 17,300; and 21,800 mg/ m ³ of styrene ( 840, 1730, 3140, 4050, 5100 ppm). 10 minutes after initiation of exposure, a few eye saccades were provoked and registered. Then, during continuous exposure, repeated vestibular stimulations in darkness and combined vestibular and optokinetic stimulations were performed ( lasting 35 minutes) followed by optokinetic stimulations ( lasting 15 minutes). Optokinetic stimulation caused nystagmus with a gain that was reduced by styrene exposure in a concentration dependent manner. Effects could be observed at the lowest concentration of styrene applied. Repeated vestibular stimulations in darkness showed a prolongation of nystagmus at 4050 and 5100 ppm. The combined vestibular and optokinetic stimulation caused no or little nystagmus in control experiments since the visual input suppressed the vestibular reaction. Styrene exposure caused a concentration dependent inability to suppress the nystagmus at concentrations   1730 ppm.

Pulmonary toxicity was studied in Sprague Dawley rats ( Green et al. 2001b). Groups of 5 female and 5 male animals were exposed " whole body" in 3.4 m ³ chambers to analytically ( gas chromatography) controlled concentrations of 0 or 500 ppm styrene for 6 hours. No treatment related effects were observed in the lung of animals exposed to styrene for 1, 5, 6, or 10 days.

The serum activity of sorbitol dehydrogenase ( SDH) was used as an indicator of liver damage in a study with female Sprague Dawley rats ( Lundberg et al. 1986). Animals were exposed from 1/ 32 to ½ of the saturation concentration of styrene in air ( 33,200 mg/ m ³ ; 7769 ppm) for 4 hours and sacrificed 20 hours after the end of exposure. No increase in serum SDH activity was observed at any concentration.

Studies with non inhalation exposure In the study of Lundberg et al. ( 1986), female Sprague Dawley rats were also treated by i. p. injections of styrene in peanut oil at doses of 1/ 8, 1/ 16, and 1/ 32 of the LD50 ( 898 mg/ kg b. w.). The serum activity of sorbitol dehydrogenase ( SDH) was used as an indicator of liver damage; no increase was observed at any of the concentrations.

Studies with repeated inhalation exposure The degeneration and regeneration of respiratory mucosa of the trachea and the nose following subacute exposure of male Sprague Dawley rats was studied by Ohashi et al. ( 1986). Groups of 10 animals each were exposed to analytically ( gas chromatography) determined concentrations of 171 ± 21.8 ppm or 1108 ± 73.8 ppm of styrene or air in dynamic exposure chambers for 4 hours/ day, 5 days/ week for 3 weeks. On the first day of the post exposure period, the ciliary activity of the tracheal mucosa showed some deterioration in the 171 ppm group ( 80 % of control value). There was also an increased number of dense bodies and small vacuoles in the epithelial cells and small compound cilia were observed but there was no severe degeneration of epithelial cells. In the nasal mucosa, ciliary activity was reduced ( 41 % of control) and morphological alterations ( ballooning of cells, fewer ciliated cells, increase of dense bodies) were seen. 12 weeks after the last exposure, the ciliar activity of the tracheal and nasal mucosa was normal and cells with an increased number of dense bodies were only sporadically found in the trachea. At 1200 ppm, ciliar activity of the tracheal mucosa was poor ( 18 % of control) the first day after the last exposure, and cells showed morphological changes ( vacuolization, increased number of dense bodies, cytoplasm Styrene NAC: 02/ 2004 27 protuberances). In the nasal mucosa, ciliary activity was disabled the first post exposure day, there were few ciliated cells and severe degeneration of epithelial cells. The effects in the trachea largely resolved within the following 12 weeks but in the nasal mucosa reduced ciliary activity and morphological alterations were still detectable.

In the nasal olfactory epithelium of female and male CD ( Sprague Dawley) rats, histopathological changes were seen in a subchronic study after exposure to 500, 1000 and 1500 ppm for 6 hours/ day, 5 days/ week for 13 weeks. At 200 ppm, no effects were seen. In the same study, no styrenerelated effects were observed in the lungs at any concentration ( Cruzan et al. 1997b).

Effects on serum prolactin and dopamine levels and on hypothalamic and striatal catecholamie concentrations were studied in male Wistar rats ( Jarry et al. 2002). Groups of 10 animals each were exposed to analytically ( gas chromatography) confirmed concentrations of 0, 150, 500, and 1500 ppm styrene for 6 hours/ day on 5 consecutive days. Parameters were measured immediately after the end of exposure and after a recovery period of 24 hours. No significant changes in dopamine, dihydroxyphenylacetic acid, noradrenaline, and homovanillic acid levels were observed in hypothalamus and striatum of styrene exposed rats compared to controls. Also, no change in prolactin level in serum was observed. The dopamine level in peripheral blood was higher at 150 ppm and at 1500 ppm in the 24 hour recovery group at 150 ppm, but no concentration response was obvious; no change was seen immediate after cessation of exposure.

Ototoxicity of styrene was investigated in several studies. When male Long Evans rats were exposed to concentrations between 500 ppm and 1500 ppm 6 hours/ day, 5 days/ week, for 4 weeks, a permanent increase in auditory threshold was observed at mid frequency ranges in animals exposed to 850 and 1000 ppm. At higher concentrations, the threshold was increased in the mid low, mid and high frequency ( Loquet et al. 1999). In a further study, Long Evans rats were exposed to 1000 ppm, 6 hours/ day, for 5 days. Immediately after the end of exposure, cochlear function as tested by DPOAE ( distortion product otoacoustic emissions) showed no decrease in DPOAE amplitude compared to preexposure However, 2 and 4 weeks after the end of exposure, DPOAE indicated a disruption of auditory function ( Lataye et al. 2003). Histological lesions of the cochlea and worsening of the electrophysiological results ( evoked potentials from the inferior colliculus of the cochlea) after the end of exposure was also described in another publication of the same group in which Long Evans rats were exposed to 1000 ppm, 6 hours/ day, 5 days/ week, for up to 4 weeks ( Campo et al. 2001). In male Wistrar rats exposed to 100, 300, or 600 ppm styrene for 12 hours/ day, 5 days/ week, for 4 weeks, 100 and 300 ppm caused no hearing impairment as measured by auditory brain response ( ABR). At 600 ppm, a slight hearing loss (~ 3 dB) was observed only at the highest test frequeny of 8 kHz; cytocochleograms showed a substantial loss of the outer hair cells. A synergism with exposure to noise ( 100 dB) was observed only when styrene was applied in concentrations that were ototoxic without noise ( Mäkitie et al. 2003).

3.2.3 Mice 10 female and 10 male NMRI mice per dose group were exposed to 680, 864, 1420, 1528, 2983 or 3766 ppm styrene for 4 hours ( BASF 1979a, see 3.1.2). Symptoms ( not reported separately for both sexes) included hunched position at exposures exceeding 1420 ppm and rough fur at all concentrations. Styrene was irritating to the respiratory tract and to the eyes ( intermittent breathing at all concentrations, rubbing of nose and mouth, secretion from nose and eyes, eyes closed at 2983 and 3766 ppm). Signs of CNS impairment ( staggered or stalking gait) were noted from 1420 ppm upwards and were more severe at higher concentrations with apathy and narcosis occurring at 2983 and 3766 ppm. Styrene NAC: 02/ 2004 28 In a subacute study, B6C3F1 and CD 1 mice ( 20 per sex and dose group) were exposed in 0.75 m ³ inhalation chambers to analytically confirmed concentrations of 0, 14, 58, 250 or 519 ppm styrene for 6 hours/ day, 5 days/ week for 14 days ( Cruzan et al. 1997b). Mice exposed to all concentrations of styrene showed signs of irritation. At 500 ppm, animals adopted a prone position during exposure. Treatment related signs between exposures occurred in mice exposed to 250 or 519 ppm. These signs included lethargy, shallow breathing, and unsteady gait. 250 and 500 ppm caused liver lesions with centrilobular hepatocyte necrosis and associated changes. B6C3F1 mice were more susceptible than CD 1 mice. Mortality also occurred at these two highest concentrations ( see 3.1.2).

Sensory irritation Male Swiss OF1 mice ( six at each concentration) were exposed " head only" to at least 4 different analytically ( gas chromatography) controlled concentrations of styrene in dynamic 200 L inhalation test chambers for 5 minutes. For the determination of the reflex decrease in respiratory rate that served as an index of sensory irritation, the animals were secured in individual body plethysmographs. An RD50 of 586 ppm ( no confidence limits given) was determined ( de Ceaurriz et al. 1981).