Inhalation Exposure Bieniek ( 1994) analyzed the excretion patterns of 1 naphthol in three groups of workers occupationally exposed to naphthalene. The mean excretion rate for these workers was 0.57 mg/ hour, with a calculated excretion half life of approximately 4 hours. The highest urinary levels of 1 naphthol were reported for workers in a naphthalene oil distribution plant. Peak 1 naphthol levels were detected in urine collected one hour after finishing the shift. 7 1 Naphthalene   February 2003 7.0 HAZARD IDENTIFICATION 7.1 Human Effects 7.1.1 Short Term Studies and Case Reports General Population Intentional and Accidental Acute Ingestion The earliest account of acute oral exposure to naphthalene ( Lezenius, 1902) describes the ingestion of impure naphthalene by a man over the course of 13 hours in an attempt to cure an abdominal ailment ( ATSDR, 1995). The dose of naphthalene was not known precisely, but was estimated to be approximately 5 grams. Assuming a body weight of 70 kg for an adult, this amount corresponds to a dose of approximately 71 mg/ kg. Within 8 to 9 hours, vision became severely impaired. No evidence of hematological impacts was reported, but painful urination and urethral swelling were noted. Upon examination 1 month later, bilateral zonular cataracts were seen, visual fields were constricted, and the subject could count fingers up to a distance of only 1.5 meters. The contribution of impurities in the naphthalene to the observed toxic effects is unknown.

Several reports describe cases in which accidental or intentional ingestion of naphthalene resulted in death. Gupta ( 1979) reported a case of a 17 year old male who had ingested an unknown quantity of mothballs. He died after exhibiting symptoms that included vomiting, gastrointestinal bleeding, blood tinged urine, jaundice, and coma. Additional observations included liver enlargement, elevated creatine and blood urea nitrogen, and reduction in urine output. Death occurred 5 days after ingestion. Proximal tubular damage and general tubular necrosis were recorded at autopsy.

Kurz ( 1987) reported the death of a 30 year old woman after she ingested a large number of moth balls. The patient reported consuming at least 40 mothballs, 25 of which were recovered at autopsy. The patient exhibited abdominal pain, blood in the urine, and vomiting of blood. Neurological signs included malaise, loss of response to painful stimuli, and muscular twitching or convulsions. Hemolytic anemia was diagnosed prior to death, and the increased plasma level of liver enzymes indicated potential hepatic injury. Death occurred 5 days after ingestion. Limited areas of mucosal hemorrhage in the small bowel and colon were seen at autopsy.

Two cases describe the death of a child following the ingestion of naphthalene. In the first case, a Japanese child died after ingesting approximately 5 grams of mothballs ( Ijiri et al., 1987). Assuming a body weight of 10 kg ( the age of the child is unknown), this amount corresponds to a dose of approximately 500 mg/ kg. The blood level of naphthalene was reported to be 0.55 mg/ L. Pulmonary edema, congestion, and hemorrhage of the lungs were found to be present at autopsy. Liver pathology included fatty changes, and leucocyte and lymphocyte infiltration. In the second case, a 6 year old child died after ingesting an estimated 2 grams of 7 2 Naphthalene   February 2003 naphthalene over approximately 2 days ( Gerarde, 1960). Assuming a body weight of 21 kg ( U. S. EPA, 1996c), this amount corresponds to an estimated dose of 95 mg/ kg.

Gidron and Leurer ( 1956) reported sublethal acute effects in the case of a 16 year old girl who consumed 6 g of naphthalene in a suicide attempt. Assuming a body weight of 55 kg, this dose corresponds to 109 mg/ kg. Symptoms and treatment were recorded during 18 days of hospitalization. Indications of hemolytic anemia ( low hemoglobin concentration, low erythrocyte count, discolored urine), fever, and pain in the kidney region were observed.

Dreisbach and Robertson ( 1987) reported a fatal dose from oral exposure to be approximately 2 grams, although this information was not well documented. This dose is equivalent to about 28 mg/ kg for a 70 kg reference human.

Additional reports of sublethal acute naphthalene poisoning have been summarized in ATSDR ( 1995) and U. S. EPA ( 1998a). Most of these cases involved naphthalene ingestion by children. Case reports document hemolytic anemia characterized by methemoglobinemia, the occurrence of Heinz bodies, reduced hemoglobin levels, reduced hematocrit, increased reticulocyte counts, and increased serum bilirubin levels. None of these cases provides estimates of the dose levels associated with the development of hemolytic anemia.

Acute and Short Term Inhalation Exposure Household inhalation exposures to naphthalene have also been associated with adverse effects. Eight adults and one child reported gastrointestinal ( nausea, vomiting, abdominal pain) and neurological ( headache, malaise, confusion) symptoms after exposure to large numbers of mothballs in their homes ( Linick, 1983). The duration of exposure was not specified, and a single measurement of the level of naphthalene in indoor air ( 20 ppb) was taken at a time when exposures were thought to be lower because the mothballs were not " fresh." Symptoms were relieved when the mothballs were removed ( U. S. EPA, 1998a).

Short Term Exposure by Other Pathways Dermal exposure to naphthalene has occasionally been associated with adverse effects in humans. Valaes et al. ( 1963) reported adverse health effects in an infant exposed to naphthalene by wearing diapers that had been stored with mothballs. The infant developed severe hemolytic anemia accompanied by jaundice, enlarged liver, methemoglobinemia, and cyanosis. A similar case was reported by Schafer ( 1951). In the latter case, symptoms persisted after cessation of exposure, and death resulted. Levels of exposure were not estimated in either case.

Three reports ( Zinkham and Childs, 1958; Anziulewicz et al., 1959; Athanasiou et al., 1997) describe apparent transplacental exposure of a fetus during pregnancy, which resulted in neonatal hemolysis. In the two older cases, unspecified amounts of naphthalene had been ingested by the mother during pregnancy. The more recent report by Athanasiou et al. ( 1997) documented the occurrence of hemolytic anemia in a neonate whose mother had inhaled naphthalene during the 28th week of pregnancy. 7 3 Naphthalene   February 2003 Sensitive Populations Short term inhalation exposures to naphthalene have been associated with hemolytic anemia, and occasionally, death. Valaes et al. ( 1963) reported adverse effects in 21 Greek infants exposed to naphthalene from clothing, diapers, blankets, and other items that had been stored in contact with mothballs. Durations of exposure ranged from 1 to 7 days. Inhalation was identified as the primary route of exposure because 19 of the 21 infants did not have dermal contact with the naphthalene contaminated materials. A total of 21 infants developed hemolytic anemia and two infants died from kernicterus, a severe neurological condition that was thought to be a consequence of massive hemolysis. Ten of the 21 anemic children and 1 of the 2 infants that died from naphthalene exposure had a genetic polymorphism that resulted in a deficiency in glucose 6 phosphate dehydrogenase ( G6PD). This enzyme helps to protect red blood cells from oxidative damage, and G6PD deficiency makes the cells more sensitive to a wide variety of toxicants, including naphthalene.

7.1.2 Long Term and Epidemiological Studies General Populations Ghetti and Mariani ( 1956) reported the development of pin point lens opacities in 8 of 21 individuals employed for five years at a dye manufacturing plant. The individuals were involved in the heating of large amounts of naphthalene in open vats. Exposure of these workers likely occurred primarily via inhalation and dermal contact, but exposure levels were not estimated. Although cataracts may develop spontaneously with age, seven of the affected individuals were younger than 50 years old. The probability of spontaneous cataract development in these individuals was therefore considered to be low. The lesions, which did not affect visual acuity, were attributed to naphthalene exposure because no correlation existed between incidence and age, and because they occurred in the crystalline lens ( ATSDR, 1995).

Two epidemiological studies addressed a potential relationship between occupational exposure to naphthalene and cancer in German workers. An abstract of a case control study by Kup ( 1978) described 12 cases of laryngeal carcinomas, 2 cases of epipharyngeal cancer, and one case of nasal carcinoma. All but three workers were smokers. Four of the patients with laryngeal cancer also had a history of occupational exposure to naphthalene. Limitations to this study include the small number of patients studied, uncertainty about how naphthalene exposures were identified, and known exposures to other potential carcinogens. Consequently, this study does not provide strong evidence for an association between naphthalene exposure and pharyngeal cancer. The author suggested that most of the observed cancers were probably due to nonoccupational causes ( U. S. EPA, 1998a).

The second epidemiological study reported the finding of 6 cases of cancer among 15 workers exposed to naphthalene vapors at a coal tar and naphthalene production facility ( Wolf, 1976). The duration of exposure ranged from 7 to 32 years. Four workers developed carcinomas of the larynx. Two workers developed cancer of the stomach and cecum. All of the subjects were smokers. Limitations to this study include lack of a control population, the small numbers 7 4 Naphthalene   February 2003 of workers involved, lack of quantitative exposure data, and the presence of both occupational and nonoccupational exposures to other potential carcinogens. Therefore, this study does not provide strong evidence for a relationship between naphthalene exposure and cancer incidence ( U. S. EPA, 1998a).

Sensitive Populations No long term studies conducted in sensitive populations were identified in the materials reviewed for this document.

7.2 Animal Studies This section presents the results of toxicity studies of naphthalene in animals. The first four subsections provide study results by duration of exposure. Acute studies are those which address exposure durations of 24 hours or less. Short term studies are those in which the exposure duration is greater than 24 hours but less than approximately 90 days. The exposure duration of subchronic studies is typically 90 days, and chronic studies are those in which exposure lasts one year or more. Some studies fall into more than one category because they measure impacts over several exposure periods. The discussion of acute, short term, subchronic, and chronic studies summarizes observed toxicological effects on all body systems.

The last four subsections of Section 7.2 provide toxicological data related to specific organ systems and types of endpoints: ocular toxicity, neurotoxicity, developmental/ reproductive toxicity, and carcinogenicity.

7.2.1 Acute Toxicity Oral Exposure Acute lethality data have been reported for rats and mice. LD 50 values in various strains of rats typically range between 1,780 mg/ kg and 2,800 mg/ kg ( Gaines, 1969; NIOSH, 1977; Papciak and Mallory, 1990), although LD 50 values as high as 9,430 mg/ kg have been reported in one study ( Union Carbide Corp., 1968). Shopp et al. ( 1984) reported LD 50 values of 533 mg/ kg for male mice and 710 mg/ kg for female mice.

Zuelzer and Apt ( 1949) administered single dietary doses of 410 mg/ kg or 1,530 mg/ kg naphthalene to two dogs. Both dogs developed signs of hemolytic anemia including a 29   33% reduction in hemoglobin concentrations, decreased hematocrit, presence of Heinz bodies, and reticulocytosis.

Shopp et al. ( 1984) administered 0, 200, 400, 600, 800, or 1,000 mg/ kg naphthalene via oral gavage ( in a corn oil vehicle) to CD 1 mice ( 8 animals/ sex/ dose). Mice displayed ptosis ( drooping of eyelids) and red discharge soon after receiving doses of 400 mg/ kg or higher ( males) or 600 mg/ kg or higher ( females). These findings suggest NOAEL and LOAEL values 7 5 Naphthalene   February 2003 for this study of 200 and 400 mg/ kg, respectively, based on the occurrence of ptosis ( drooping of the eyelids) in male mice.

Naphthalene related cataract formation has been reported in animals following acute oral exposure. Van Heyningen and Pirie ( 1976) administered naphthalene by gavage at 1,000 mg/ kgday to rabbits and observed cataract formation in some animals after a single dose. Ikemoto and Iwata ( 1978) observed that oral administration of 1,000 mg/ kg to albino rabbits of both sexes for two consecutive days resulted in cataract formation. The ocular toxicity of naphthalene is further discussed in Section 7.3.2.

Dermal Exposure Acute toxicity testing in rabbits revealed that 2,000 mg/ kg of naphthalene causes moderate dermal irritation ( erythema, edema, and/ or fissuring that resolved within 7 days) when applied directly to intact or abraded skin ( Papciak and Mallory, 1990). Application of 500 mg/ kg to intact or abraded skin resulted in slight irritation ( some reversible erythema at 24 and 72 hours after application) ( Papciak and Mallory, 1990). In a separate study, application of 500 mg to intact shaved skin ( area not specified) resulted in mild to well defined erythema and some fissuring ( PRI, 1985). Mild ocular irritation occurred following the instillation of 100 mg of naphthalene into the eye ( PRI, 1985; Papciak and Mallory, 1990). These effects were reversed within 7 days, and they occurred only when naphthalene was left on the eye surface rather than rinsed off after application ( ATSDR, 1995).

Inhalation Exposure U. S. EPA ( 1987b) has previously summarized acute inhalation data for naphthalene. Union Carbide ( 1968) reported that the 8 hour LC 50 value for naphthalene was 100 ppm. Buckpitt ( 1985) suggested that this value may be too low, on the basis of calculated body burdens. Buckpitt ( 1985) calculated that following 8 hours of inhalation exposure at 100 ppm, the body burden would be less than 30 mg/ rat, or approximately 150 to 200 mg/ kg. This value is considerably less than the oral or intraperitoneal LD 50 values for rats. Fait and Nachreiner ( 1985) reported that exposure of male and female Wistar rats to 78 ppm naphthalene for 4 hours did not result in mortalities or any abnormalities in the lung, liver, kidney, or nasal passages. Buckpitt ( 1985) conducted an inhalation study with male Swiss Webster mice. No deaths were observed after exposure to 90 ppm naphthalene for 4 hours, but lung lesions were reported to be prominent.

No new data for acute inhalation toxicity were identified in the materials reviewed for this document. 7 6 Naphthalene   February 2003 7.2.2 Short Term Studies Oral Exposure Zuelzer and Apt ( 1949) administered seven consecutive daily doses of naphthalene in the diet to a single dog. The daily doses ranged from 74 to 441 mg/ kg, with an average daily dose of 262 mg/ kg day. The dog developed signs of hemolytic anemia, including decreased hemoglobin concentration, decreased hematocrit, presence of Heinz bodies, extreme leukocytosis, and reticulocytosis. Other signs noted included pronounced lethargy and ataxia.

Shopp et al. ( 1984) administered 0, 27, 53, or 267 mg/ kg day naphthalene in corn oil via oral gavage to CD 1 mice ( 76   112 males/ dose, 40   76 females/ dose) for 14 days. Gross pathology was performed, but a histopathological examination was not conducted. No adverse effects were noted at doses of 53 mg/ kg day or less. Adverse effects observed in animals exposed to 267 mg/ kg day included increased mortality and decreased terminal body weights ( 4   10%) in males and females, decreased absolute thymus weights ( 30%) in males, increased bilirubin in females, and increased spleen and lung weights ( relative and absolute) in females. Serum enzyme activities ( lactic dehydrogenase and serum glutamic oxaloacetic transamidase) and electrolyte levels were not altered in a dose dependent pattern. There were no effects on hexobarbital sleeping time or on various immunological screening parameters ( with the exception of decreased lymphocyte response to concanavalin A in high dose females). Values for hematological and coagulation parameters were similar to controls, with the exception of decreased prothrombin time in high dose females, and a small but significant increase in eosinophils in high dose males. Neither red cell hemolysis nor cataract formation was observed in the naphthalene exposed mice, and the authors suggested that this mouse strain appears to be resistant to these toxic effects. This study identified a NOAEL of 53 mg/ kg day and a LOAEL of 267 mg/ kg day.

Plasterer et al. ( 1985) administered doses of 0, 125, 250, 500, 1,200, and 2,000 mg/ kgday to non pregnant female CD 1 mice ( 10 per dose group) by gavage in corn oil. A steep doseresponse relationship was observed for lethality in naphthalene exposed mice. After 8 daily gavage doses, an LD 50 value of 354 mg/ kg was determined for male and female mice. This value is based on 100% mortality at 500 mg/ kg day and no deaths at 250 mg/ kg day. In pregnant mice, 15% mortality was observed in the 300 mg/ kg day group. In contrast, no mortality was reported in 2 strains of rabbits that received 1,000 mg/ kg naphthalene via gavage, twice a week for 12 weeks ( Rossa and Pau, 1988), suggesting that there are species differences in response to naphthalene exposure.

Liver changes have been reported in rats exposed to relatively high doses of naphthalene ( approximately 1,000 mg/ kg day or more) when administered for durations of 10 days to 9 weeks ( ATSDR, 1995). Rao and Pandya ( 1981) reported hepatic toxicity in rats administered 1,000 mg/ kg day naphthalene ( LOAEL) for 10 days. Observed effects included a 39% increase in liver weight, increased lipid peroxidation, and a modest increase in aniline hydroxylase activity ( ATSDR, 1995). Increased lipid peroxidation was also reported in rats that received 1,000 mg/ kg day naphthalene for 18 days ( Yamauchi et al., 1986), and in rats that received escalating 7 7 Naphthalene   February 2003 doses of naphthalene up to 750 mg/ kg day over a 9 week period ( Germansky and Jamall, 1988). No effects on liver weight were noted in the 14 day gavage study reported by Shopp et al. ( 1984) at tested doses as high as 267 mg/ kg day.

Dermal Exposure No short term animal studies evaluating the dermal route of exposure were identified in the materials reviewed for this document.

Inhalation Exposure No short term inhalation studies of naphthalene exposure were identified in the materials reviewed for this document.

7.2.3 Subchronic Studies Oral Exposure Naphthalene (> 99% in corn oil) was administered to Fischer 344 rats ( 10/ sex/ dose), 5 days per week for 13 weeks ( BCL, 1980a). Unadjusted daily dose levels were 0, 25, 50, 100, 200, and 400 mg/ kg day. Weekly food consumption and body weights were measured, and rats were examined twice daily for clinical signs of adverse effects. Hematological parameters ( hemoglobin, hematocrit, total and differential white cell count, red blood cell count, mean cell volume and mean cell hemoglobin) were measured in all animals at the end of the study. All rats were necropsied, and detailed histopathological examinations on 27 tissues were performed on all rats in the control and 400 mg/ kg day groups. The tissues examined included eyes, stomach, liver, reproductive organs, thymus, and kidneys. In the 100 mg/ kg day group, male kidneys and female thymus tissues were subject to detailed histopathological examinations.

Male and female rats in the 400 mg/ kg day dose group exhibited diarrhea, lethargy, hunched posture, and rough coats during the study, and one high dose male rat died during the last week of exposure. Food consumption was not affected in any dose group, but body weights were significantly decreased in several of the groups ( Table 7 1). Males in the high dose group experienced a 94% increase in the number of mature neutrophils and a 25.1% decrease in circulating lymphocytes, as compared to control group rats. No other differences were observed in hematological parameters. Histopathological examination of kidney and thymus tissues revealed the following changes: focal cortical lymphocyte infiltration was observed in 1 of 10 males in the 200 mg/ kg day group; focal tubular degeneration was observed in 1 of 10 males in the 200 mg/ kg day group; diffuse renal tubular degeneration was observed in 1 male in the 400 mg/ kg day group; and lymphoid depletion of the thymus was seen in 2 of 10 females in the high dose group. No other tissue abnormalities were seen in any group. The NOAEL and LOAEL values derived from this study were 100 and 200 mg/ kg day, respectively, on the basis of reduced body weight (> 10%) in males ( U. S. EPA, 1998a). These NOAEL and LOAEL values correspond to duration adjusted doses of 71 and 143 mg/ kg day, respectively. 7 8 Naphthalene   February 2003 Table 7 1. Terminal Body Weights in Controls and in Fischer 344 Rats Exposed to Naphthalene by Gavage for 13 Weeks Dose ( mg/ kg day ) Average Terminal Body Weight Males ( g) Average Terminal Body Weight Females ( g) Unadjusted Durationadjusted 0 0 348.9 203.4 25 17.9 353.4 197.8 50 35.7 351.2 203.5 100 71.4 333.4 197.2 200 142.9 306.7* 190.5 400 285.7 250.6* 156.7*

* Decrease > 10% relative to controls Source: U. S. EPA ( 1998a)

In a second study ( BCL, 1980b), the same investigators exposed B6C3F 1 mice to naphthalene in corn oil by gavage. The administered doses were 0, 12.5, 25, 50, 100, and 200 mg/ kg day, 5 days per week for 13 weeks. Seven mice died during the exposure period, all from gavage trauma unrelated to naphthalene dose. In weeks 3 and 5 of the exposure period, transient signs of toxicity ( lethargy, rough coats, decreased food consumption) occurred in the highestdose groups. The average weight gain during the study was higher for all of the exposed male groups than for the control group. Female mice exposed to naphthalene, in contrast, gained less weight than the control group. The reduction in weight gain was dose related, ranging from 2.5% in the 12.5 mg/ kg day females to 24.5% in the 200 mg/ kg day females. The authors of the study indicated that the weight gain reduction " was not large enough to conclusively indicate a toxic effect". Complete histological evaluations were performed on all control and high dose animals at the end of the study. No exposure related lesions were observed in any organ system. Mild focal or multifocal subacute pneumonia was observed in similar proportions for both control and high dose animals. Hematological evaluation indicated an increase in circulating lymphocytes of 18% in the high dose males relative to these in control groups. A decrease of 38.8% in segmented neutrophils was also noted in high dose males. No significant differences were observed in hematological parameters.

The authors of the study indicated that given the " marked indication" of sex differences in body weight responses, the observed weight gain differences did not constitute an adverse effect. If this interpretation is accepted, a LOAEL of 200 mg/ kg day ( adjusted dose: 143 mg/ kgday can be identified from this study based on the occurrence of transient clinical signs of 7 9 Naphthalene   February 2003 toxicity discussed above ( U. S. EPA, 1998a). The corresponding NOAEL would be 100 mg/ kgday ( adjusted dose 71 mg/ kg day).

Shopp et al. ( 1984) employed larger numbers of mice to evaluate the subchronic toxicity of naphthalene. Groups of 76 male and 40 female CD 1 mice were exposed by gavage to daily doses of 5.3 or 53 mg/ kg day naphthalene in corn oil for 90 consecutive days. In addition, a high dose group of 96 male and 60 female mice received a daily dose of 133 mg/ kg day. Toxicological responses were measured against naive control groups of 76 male and 40 female mice, and against vehicle control groups of 112 male and 76 female animals. No differences in survival or terminal body weights were seen between the control and exposed groups. In the high dose females, significant decreases were seen in absolute weights of the brain, liver, and spleen, and in the relative spleen weight. No differences in organ weights were seen in males in any exposure group. Histopathological examinations were not performed, but the authors noted an absence of cataracts in all dose groups. In general, serum chemistry parameters for naphthalene treatment groups did not differ significantly from control groups. However, hematological evaluation showed slight but significant increases in hemoglobin levels in highdose females. All of the exposed female groups had significantly decreased blood urea nitrogen ( BUN) levels. Significant changes in serum globulin levels were observed in females, but a consistent dose response relationship was not evident. Hematological parameters for males were normal. No exposure related impacts on immunological function were observed. Assays for enzyme activity indicated that hepatic benzo( a) pyrene hydroxylase activity was decreased significantly in males and females treated with the 53 and 133 mg/ kg day doses, and in males treated with the 5.3 mg/ kg day dose. Aniline hydroxylase activity was significantly increased in females receiving the 133 mg/ kg day dose. The authors of this study did not report a NOAEL. Because the effects on serum chemistry parameters and hepatic enzymes are not clearly adverse, U. S. EPA ( 1998a) identified a NOAEL of 53 mg/ kg day. The LOAEL of 133 mg/ kg day reflects the observed effects on organ weight and suggestive evidence for impacts on hepatic enzyme function.

Dermal Exposure Frantz et al. ( 1986) applied doses of 0, 100, 300, or 1,000 mg naphthalene/ kg day to the skin of albino rats for 6 hours/ day, 5 days/ week, for 13 weeks. Following exposure, clinical signs, food consumption, body weight, clinical chemistry, hematology, and urinalysis were evaluated. A significantly increased incidence of excoriated skin lesions and papules was reported in the high dose group relative to those in the controls. Similar lesions were observed in the control group and low dose groups. The severity of the lesions appeared to increase with dose.

Inhalation Exposure No studies that addressed subchronic exposures by inhalation were identified in the materials reviewed for this document. 7 10 Naphthalene   February 2003 7.2.4 Neurotoxicity Relatively little information is available regarding the neurological effects of naphthalene exposure in experimental animals. PRI ( 1986) observed treatment related signs of labored breathing, body drop, and decreased activity ( incidence data not provided) in New Zealand White rabbits exposed to 200 or 400 mg/ kg day. In a study of developmental toxicity ( NTP, 1991), pregnant Sprague Dawley rats received daily gavage doses of 0, 50, 150, or 450 mg/ kg day naphthalene for 10 days during organogenesis ( gestation days 6 to 15). Animals in all dose groups showed signs of neurotoxicity, including lethargy, slow respiration ( including periods of apnea), and apparent inability to move after dosing. Incidence of symptoms in the low dose group was 73%, while incidence in the highest dose group was over 90%. These effects were transient, however, and diminished as the animals apparently acclimatized to the treatment. Animals in the low dose group appeared to acclimatize to naphthalene exposures after a few days. Incidence in the higher dose groups declined with continued exposure, but never dropped below 15% ( ATSDR, 1995).

Male mice exposed to 10 or 30 ppm naphthalene in a two year inhalation study exhibited increased huddling behavior during exposure and a reduced inclination to fight ( NTP, 1992a). Although the observed activities may indicate neurological effects, the authors of this study did not speculate on the underlying basis for the behavioral changes, and no additional signs of neurotoxicity were reported.

Other large studies found no evidence of neurotoxicity at naphthalene doses similar to those producing symptoms mentioned in the studies above. No neurological effects were found in Fischer 344 rats ( BCL, 1980a) or B6C3F 1 mice ( BCL, 1980b), at gavage doses up to 400 mg/ kg day and 200 mg/ kg day, respectively.

7.2.5 Developmental/ Reproductive Toxicity Studies of the reproductive and developmental toxicity of naphthalene are summarized in Table 7 2.

PRI ( 1985) conducted a range finding developmental study in New Zealand White rabbits. Gavage doses of 0, 50, 250, 630, or 1,000 mg/ kg day were administered to pregnant rabbits ( 4/ dose) by gavage in 1% methylcellulose on gestation days ( GD) 6 to 18. All does in the high dose group died. At 630 mg/ kg day, 2 of 4 animals died. The surviving animals experienced decreased weight gain and aborted their pregnancies. No exposure related changes were observed in the incidence of early resorption, postimplantation loss, number of corpora lutea, fetal survival, or gross fetal structural development.

In a subsequent study of developmental toxicity, PRI ( 1986) administered doses of 0, 40, 200, or 400 mg/ kg day by gavage in 1% methyl cellulose to pregnant New Zealand White rabbits ( 18/ dose). Dosing occurred on GD 6   18. Caesarean sections were performed on GD 29. 7 11 Naphthalene   February 2003 Table 7 2. Summary of Developmental and Reproductive Data on Naphthalene Study Species ( Strain) Sex n Doses mg/ kg day Route Duration NOAEL LOAEL Effect mg/ kg day PRI ( 1985) Rabbit ( New Zealand White) Female 4 0 50 250 630 1,000 Gavage Methylcellulose GD 6   18 Maternal 250 Fetal 250 Maternal 630 ( FEL)

Fetal 630 ( abortion) Maternal: Mortality and decreased wt. gain at 630 mg/ kg day Fetal: Aborted at 630 mg/

kgday PRI ( 1986) Rabbit ( New Zealand White) Female 18 0 40 200 400 Gavage Methylcellulose GD 6 18 Maternal 400 Fetal 400 Maternal  Fetal  Maternal: survival, body wt.

and body wt. gain unaffected Fetal: No effect on reproduction or development of fetus Plasterer et al.

( 1985) Mouse ( CD 1) Female 33   40 0 300 Gavage oil GD 7   14  300 ( FEL) Maternal: Reduced wt. gain;

reduced survival Fetal: Reduced no. of pups/ litter; no abnormalities in surviving pups NTP ( 1991) Rat ( Sprague

Dawley CD) Female 25   26 0 50 150 450 Gavage GD 6   15 Maternal   Fetal 450 Maternal 50 ( central nervous system depression)

Fetal  Maternal: Two deaths; central nervous system depression manifested as lethargy, slow breathing, prone body posture,

and increased rooting Fetal: no finding of fetoxicity or embryotoxicty Table 7 2 ( continued)

Study Species ( Strain) Sex n Doses mg/ kg day Route Duration NOAEL LOAEL Effect mg/ kg day 7 12 Naphthalene   February 2003 NTP ( 1992b) Rabbit ( New Zealand White) Female 25   27 0 20 80 120 Gavage oil GD 6   19 Maternal 120 Fetal 120 Maternal  Fetal  Maternal: No consistently observed toxicity Fetal: No effect on reproduction or development of fetus Shopp et al.

( 1984) Mouse ( CD 1) Male 76   112 0 27 53 267 Gavage oil 14 267  No effect on testicular weight Male 76   96 53 133 Gavage oil 90 133  No effect on testicular weight BCL ( 1980a) Rat ( F344) Male 10/ dose 0 25 50 100 200 400 Gavage oil 13 weeks 5 days/ week 400  Absence of gross testicular lesions BCL ( 1980b) Mouse ( B6C3F 1) Male 10/ dose 0 12.5 25 50 100 200 Gavage oil 90 days 200  Absence of gross testicular lesions GD = gestation day FEL = fetal effect level 7 13 Naphthalene   February 2003 Maternal survival, body weight, and body weight gain were unaffected by naphthalene treatment. Treatment related signs of labored breathing, cyanosis, body drop, decreased activity and salivation were reportedly noted in animals receiving the 200 and 400 mg/ kg day doses, but incidence data were not provided. No effect of treatment was noted on number of corpora lutea, total implantations, fetal viability, pre or postimplantation loss, fetal body weight, fetal sex distribution, or fetal skeletal or visceral abnormalities.

Plasterer et al. ( 1985) examined the developmental toxicity of naphthalene in CD 1 mice. Doses of 0 or 300 mg/ kg day ( 40 and 33 animals/ group, respectively) were administered by gavage in corn oil on GD 7 to 14. Mortality occurred in 5/ 33 exposed dams, while all control dams survived the treatment. Average weight gain was significantly reduced in exposed dams when compared with controls. The average number of live pups per litter was significantly reduced by naphthalene treatment, but the average body weight of the living pups was not affected by exposure. No treatment related gross structural abnormalities were seen in the surviving pups. The 300 mg/ kg day dose is considered a frank effect level ( FEL) based on maternal ( death and reduced body weight) and fetal ( decreased live pups per litter) effects.

NTP ( 1991) conducted a developmental study of naphthalene toxicity in pregnant Sprague Dawley CD rats ( 25   26/ dose). Doses of 0, 50, 150, or 450 mg/ kg naphthalene were administered by gavage in corn oil on gestational days 6 to 15. The dams were examined daily for clinical signs until sacrifice on GD 20. Fetuses were examined on GD 20 for gross, visceral, and skeletal malformations. Maternal mortality was limited to two deaths in the low dose group. Treatment with naphthalene produced clinical signs of toxicity, including lethargy, slow breathing, prone body posture, and increased rooting behavior. The effects subsided in the 50 and 150 mg/ kg day groups before the end of the treatment period, but persisted throughout the treatment period in the 450 mg/ kg day treatment group. Dams exposed to the 150 and 450 mg/ kg doses showed significant decreases in weight gain. The average reductions in weight gain were 31% and 53% respectively. No unequivocal treatment related effects on fetal development were noted. The study authors identified the highest dose of 450 mg/ kg day as a NOAEL for fetal effects. U. S. EPA ( 1998a) identified 50 mg/ kg day as the LOAEL for maternal toxicity in this study.

Mild developmental abnormalities were noted in some offspring of New Zealand rabbits that were administered 0, 20, 80, or 120 mg/ kg day naphthalene on gestation days 6   19 ( NTP, 1992b). Slight increases in the incidence of fused sternebrae were seen in the female pups in 2 of 20 litters of animals given 80 mg/ kg day, and in 3 of 20 litters of animals given 120 mg/ kg day. However, these increases were not statistically significant. No significant differences were observed for average litter size, average fetal body weight, or incidence of other malformations on a per fetus or per litter basis. The highest dose in this study was the NOAEL for maternal and developmental toxicity.

Naphthalene does not cause testicular lesions in rats or mice. Testicular weight was unaffected in B6C3F 1 mice given 267 mg/ kg naphthalene for 14 days or 133 mg/ kg for 90 days ( Shopp et al., 1984). Two other subchronic studies also found no gross histopathological testicular lesions in Fischer 344 rats receiving up to 400 mg/ kg day naphthalene ( BCL, 1980a) or in B6C3F 1 mice receiving up to 200 mg/ kg day ( BCL, 1980b) for 13 weeks. 7 14 Naphthalene   February 2003 7.2.6 Chronic Toxicity The few chronic animal studies that are available for naphthalene were conducted primarily to characterize its carcinogenic potential effects. Noncancer endpoints reported in these studies are summarized below.

Oral Exposure Schmähl ( 1955) examined the long term ( 300   700 day) exposure of rats ( in house strain BDI or BDII) to naphthalene in food. High purity naphthalene ( as judged by absorption spectra) was dissolved in oil, mixed in the diet, and administered to 28 rats, 6 times a week. Estimated daily doses were between 10 and 20 mg/ rat. Assuming that the body weight of the test strain was similar to the reference weight of 0.36 kg for a male Fischer 344 rat ( U. S. EPA, 1988), the average daily dose was approximately 42 mg/ kg day. Dosing was stopped at 700 days when the total dose for each animal reached 10 grams. Animals were then observed until spontaneous death, usually between the 700th and 800th experimental day. Survival of the exposed animals was similar to that of the control group. Autopsy and histological results did not identify signs of adverse noncancer effects in any organ system, including the eye. This study is not considered adequate to support the development of a NOAEL or LOAEL value ( U. S. EPA, 1998a), on the basis of the administration of a single dose level, inadequate reporting of results, incomplete histopathological examinations, lack of hematological examinations, and examination of some animals at time points up to 300 days following termination of exposure.

Dermal Exposure No studies evaluating chronic naphthalene exposure by the dermal route were identified.

Inhalation Exposure Adkins et al. ( 1986) investigated the impacts of less than lifetime inhalation exposures to naphthalene on rats. Groups of 30 female A/ J strain rats were exposed to 0, 10, or 30 ppm naphthalene vapors for 6 hours per day, 5 days per week, for 6 months. All animals were sacrificed at the end of the exposure period and their lungs excised and examined for tumors. No adverse noncancer effects on the lung were reported ( U. S. EPA, 1998a). Other organs were not examined in study.

A chronic inhalation study of naphthalene toxicity was conducted in B6C3F 1 mice by NTP ( 1992a). Naphthalene exposure concentrations in air were 0, 10 ppm, or 30 ppm. The concentration of 10 ppm was chosen because it was equal to the ACGIH TLV ® for naphthalene, while the 30 ppm concentration was chosen because it was one half the air saturation concentration. The control and low exposure groups consisted of 75 mice of each sex, while the high exposure group consisted of 150 mice of each sex. Exposure was for 6 hours per day, 5 days per week, for 2 years. Comprehensive histopathological evaluations were performed on all control and high exposure mice, and on all low exposure mice that died or were sacrificed during the first 21 months of exposure. The original study plan called for 50 animals per sex to be exposed for 2 years, and 5 animals per sex to be sacrificed for hematological evaluations at 14 7 15 Naphthalene   February 2003 days, 3, 6, 12, and 18 months. However, as a result of excessive mortality in the control males, only the 14 day hematological evaluation was conducted. All of the remaining animals were incorporated into the two year study.

A statistically significant decrease in survival was noted in the male control group ( Table 7 3). This phenomenon was attributed to the frequent fighting that occurred among the control group mice. In contrast, the exposed groups tended to huddle together during exposure periods, and fought less. Statistically significant increases were seen in several types of noncancer respiratory tract lesions in both exposed groups ( Table 7 3). The observed responses included chronic lung inflammation, chronic nasal irritation with hyperplasia of the nasal epithelium, and metaplasia of the olfactory epithelium. The authors of the study described the lung lesions as a chronic inflammatory response with granuloma. These lesions consisted of " focal intra alveolar mixed inflammatory cell exudates and interstitial fibrosis." The more advanced lesions consisted primarily of " large foamy macrophages sometimes accompanied by giant cells."

No changes in hematological parameters were seen among the exposed animals at 14 days. No cataract formation was observed after 2 years of exposure. Histopathological examination did not reveal treatment related effects on the liver, gastrointestinal system, reproductive system, brain, or any other organs. The results of this study have been interpreted by U. S. EPA ( 1998a) to support a chronic LOAEL for nasal and respiratory irritation of 10 ppm.

Table 7 3. Survival and Incidence of Non neoplastic Lesions in B6C3F 1 Mice Exposed to Naphthalene by Inhalation for Their Lifetime Dose ( ppm) Survival Chronic Lung Inflammation Chronic Nasal Inflammation, Hyperplasia of Nasal Epithelium Metaplasia of Olfactory Epithelium Male Female Male Female Male Female Male Female 0 26/ 70 59/ 69 0/ 70 3/ 69 0/ 70 0/ 69 0/ 70 1/ 69 10 52/ 69* 57/ 65 21/ 69* 13/ 65* 66/ 69* 65/ 65* 67/ 69* 65/ 65*

30 118/ 133* 102/ 135 56/ 135* 52/ 135* 134/ 135* 135/ 135* 133/ 135* 135/ 135*

Source: NTP ( 1992a) * Significantly different from control by logistic regression ( p# 0.001). 7 16 Naphthalene   February 2003 NTP ( 2000) conducted a chronic inhalation study in F344/ N rats. Male and female rats ( 49/ sex/ dose) were exposed to naphthalene vapor concentrations of 0, 10, 30, and 60 ppm for 6 hours per day plus T 90 ( the theoretical time to achieve 90% of the target concentration in the vapor chamber: 12 minutes), 5 days per week for 105 weeks. Additional groups of rats were similarly exposed for up to 18 months for evaluation of toxicokinetic parameters. Dose calculations were based upon model estimates of the amount of naphthalene inhaled by rats at the exposure concentrations used in the two year study, the total amount of naphthalene metabolized following a six hour exposure period ( 21% to 31% of inhaled naphthalene), and average weights of 125 grams ( male rats) and 100 grams ( female rats). Because essentially all of the naphthalene that is absorbed into the bloodstream is metabolized, the total amount of naphthalene metabolized was assumed to represent the internalized dose to rats from the exposure concentrations used in this two year study. The estimated daily doses determined by this method were 0, 3.6, 10.7, 20.1 mg/ kg day for males, and 0, 3.9, 11.4, and 20.6 mg/ kg day for females.

Rats were clinically examined twice daily and findings were recorded every four weeks beginning at week 4 and every two weeks beginning at week 92. Body weights were recorded at study initiation, every four weeks beginning at week 4, and every two weeks beginning at week 92. Full necropsies and complete histopathologies were performed on all core study animals.

There were no clinical findings related to naphthalene exposure from the two year inhalation study. The mean body weights all exposed groups of male and female rats were similar to those observed in the appropriate control chamber group. No significant difference in survival rate was observed for any exposed group when compared to the chamber control. The mean body weights of female rats were generally similar to the body weights of the control group, while the mean body weights of naphthalene exposed male rats were generally less than the chamber control for all exposed groups.

Although naphthalene is a known cataractogen and ocular irritant ( see Section 7.3.2), no naphthalene related cataractogenic effects or ocular abnormalities were observed in rats during this study. Treatment related non neoplastic lesions were observed in the nose and lungs of male and female rats. The incidence and average severity of nasal lesions ( glands, goblet cells, respiratory epithelium and olfactory epithelium) are summarized in Table 7 4. The incidences of these lesions were significantly greater than those in the chamber controls for all male and female exposed groups, with the exception of squamous metaplasia of glands in male and female rats in the 10 ppm exposure groups ( NTP, 2000). In general, the severities of olfactory epithelial and glandular lesions increased with increasing exposure concentrations.

Two noteworthy type of lesions occurred in the lungs of exposed rats: alveolar epithelial hyperplasia and minimal chronic inflammation. Female rats in all exposure groups had increased incidences of alveolar epithelial hyperplasia when compared to the chamber control ( chamber control: 4/ 49, 10ppm: 11/ 49, 30 ppm: 11/ 49, 60 ppm: 9/ 49). This effect reached statistical significance in the 10 and 30 ppm exposure groups. The incidences of alveolar epithelial hyperplasia in male rats ( chamber control: 23/ 49, 10 ppm: 12/ 49, 30 ppm: 9/ 48, 60 7 17 Naphthalene   February 2003 Table 7 4. Incidence and Severity of Nonneoplastic Lesions in the Noses of Rats in a Two year Naphthalene Inhalation Study Lesion Type Incidence and Severity ( average) of Lesions Chamber Control 10 ppm 30 ppm 60 ppm MALE Atypical Hyperplasia of the Olfactory Epithelium 0/ 49 48/ 49* ( 2.1) 45/ 48* ( 2.5) 46/ 48* ( 3.0)

Atropy of the Olfactory Epithelium 3/ 49 ( 1.3) 49/ 49* ( 2.1) 48/ 48* ( 2.8) 47/ 48* ( 3.5)

Chronic Inflammation of the Olfactory Epithelium 0/ 49 49/ 49* ( 2.0) 48/ 48* ( 2.2) 48/ 48* ( 3.0)

Hyaline Degeneration of the Olfactory Epithelium 3/ 49 ( 1.3) 45/ 49* ( 1.7) 40/ 48* ( 1.7) 38/ 48* ( 1.5)

Hyperplasia of the Respiratory Epithelium 3/ 49 ( 1.0) 21/ 49* ( 2.2) 29/ 48* ( 2.0) 29/ 48* ( 2.2)

Squamous Metaplasia of the Respiratory Epithelium 0/ 49 15/ 49* ( 2.1) 23/ 48* ( 2.0) 18/ 48* ( 1.8)

Hyaline Degeneration of the Respiratory Epithelium 0/ 49 20/ 49* ( 1.2) 19/ 48* ( 1.4) 19/ 48* ( 1.2)

Hyperplasia of the Respiratory Epithelium Goblet Cells 0/ 49 25/ 49* ( 1.3) 29/ 48* ( 1.2) 26/ 48* ( 1.2)

Hyperplasia of Glands 1/ 49 ( 1.0) 49/ 49* ( 2.2) 48/ 48* ( 2.9) 48/ 48* ( 3.5)

Squamous Metaplasia of Glands 0/ 49 3/ 49 ( 3.0) 14/ 48* ( 2.1) 26/ 48* ( 2.5)

FEMALE Atypical Hyperplasia of the Olfactory Epithelium 0/ 49 48/ 49* ( 2.0) 48/ 49* ( 2.4) 43/ 49* ( 2.9)

Atropy of the Olfactory Epithelium 0/ 49 49/ 49* ( 1.9) 49/ 49* ( 2.7) 47/ 49* ( 3.2)

Chronic Inflammation of the Olfactory Epithelium 0/ 49 47/ 49* ( 1.9) 47/ 49* ( 2.6) 45/ 49* ( 3.4)

Hyaline Degeneration of the Olfactory Epithelium 13/ 49 ( 1.1) 46/ 49* ( 1.8) 49/ 49* ( 2.1) 45/ 49* ( 2.1)

Hyperplasia of the Respiratory Epithelium 0/ 49 18/ 49* ( 1.6) 22/ 49* ( 1.9) 23/ 49* ( 1.7)

Squamous Metaplasia of the Respiratory Epithelium 0/ 49 21/ 49* ( 1.6) 17/ 49* ( 1.5) 15/ 49* ( 1.8)

Hyaline Degeneration of the Respiratory Epithelium 8/ 49 ( 1.0) 33/ 49* ( 1.2) 34/ 49* ( 1.4) 28/ 49* ( 1.2)

Hyperplasia of the Respiratory Epithelium Goblet Cells 0/ 49 16/ 49* ( 1.0) 29/ 49* ( 1.2) 20/ 49* ( 1.0)

Hyperplasia of Glands 0/ 49 48/ 49* ( 1.9) 48/ 49* ( 3.1) 42/ 49* ( 3.3)

Squamous Metaplasia of Glands 0/ 49 2/ 49 ( 2.0) 20/ 49* ( 2.5) 20/ 49* ( 2.8)

Source: Adapted from NTP Technical Report on the Toxicology and Carcinogenesis Studies of Naphthalene in Rats ( Inhalation Studies), Table 6 ( NTP, 2000). * Significantly different ( P< 0.01) from chamber control using the Poly 3 test 7 18 Naphthalene   February 2003 ppm: 16/ 49) were significantly decreased in the 10 and 30 ppm exposure groups. The incidences of minimal chronic inflammation of the lung were increased in males and females exposed to naphthalene. This lesion is characterized by small focal interstitial and intra alveolar collections of macrophages, neutrophils, and lymphocytes and minimal interstitial fibrosis. As noted by the NTP study authors, foci of minimal inflammation are common in chamber control rats ( as evident in this study). Therefore, this change could not be confidently related to naphthalene exposure.

The study conducted by NTP ( 2000) identified an estimated inhalation LOAEL of 3.6 mg/ kg day based on the occurrence of nasal lesions in male rats in the 10 ppm exposure group. A NOAEL was not identified in this study. The 10 ppm concentration associated with the LOAEL corresponds to the threshold limit value for naphthalene ( ACGIH, 2000).

7.2.7 Carcinogenicity Oral Exposure One study was available that evaluated the carcinogenic potential of naphthalene following oral exposure in experimental animals. Schmähl ( 1955) administered 10 to 20 mg naphthalene/ rat ( dissolved in oil) in the diet for 6 days/ week to a group of 28 rats. Compound administration was continued until a total dose of 10 g/ rat was achieved. A concurrent control group was reported, but the number of animals was not specified. Administration of the diet containing naphthalene was terminated on the 700th day of the study, with animals observed until spontaneous death ( approximately 700   800 days of age). An average daily dose of 42 mg/ kg body weight/ day was estimated by U. S. EPA ( 1998a) for this study, assuming that animals ingested 15 mg naphthalene/ day and had an average default body weight of 0.36 kg ( U. S. EPA, 1988). Gross necropsies were conducted on all animals, with histopathological examinations conducted only on those organs that appeared unusual. Reported results were limited to a statement that indicated that no toxic effects were observed, including eye damage or tumors. This study has inadequacies in study design, implementation, and reporting that limit the conclusions that can be drawn regarding the carcinogenicity of naphthalene. These limitations include administration of only one dose level, inadequate reporting of results, incomplete histopathological examinations, lack of hematological examinations, and examination of some animals at time points up to 300 days following termination of exposure. Based on the absence of toxicity, the dose tested is not considered an adequately high dose for detection of carcinogenic effects ( U. S. EPA, 1998a).

Inhalation Exposure Three studies were identified which evaluated the carcinogenic potential of inhalation exposure to naphthalene in animals. Adkins et al. ( 1986) exposed groups of 30 female A/ J mice to 0, 10, or 30 ppm naphthalene via inhalation for 6 hours/ day, 5 days/ week for 6 months. An additional group of 20 mice served as a positive control, and animals in this group were administered a single intraperitoneal injection of 1g urethane/ kg. At termination of exposure, animals were sacrificed and lungs excised and examined for tumors, with tumors examined histologically. Lung tumors were observed in all positive control mice, with an average of 28.9 7 19 Naphthalene   February 2003 tumors/ animal. An increase in the number of mice with alveolar adenomas was observed in the naphthalene exposed groups ( 6, 10, and 11 in the 0, 10, and 30 ppm groups, respectively). The increases were not statistically significant when compared with the incidence of alveolar adenomas observed in the concurrent control group. Statistically significant increases in the number of adenomas per tumor bearing mouse were reported in the exposed mice; however, there was no increase in response with increasing dose. The average number of tumors per tumor bearing animal ( standard deviation in parentheses) was 1.00 ( 0.00), 1.25 ( 0.07), and 1.25 ( 0.07) for the 0, 10, and 30 ppm groups, respectively. This study is limited for use in evaluating the carcinogenic potential in humans following lifetime exposure due to the less than lifetime exposure and observation period and due to the limited histopathological examinations ( U. S. EPA, 1998a).

NTP ( 1992a) conducted a two year inhalation exposure study in B6C3F 1 mice. Groups of male and female mice were housed 5 to a cage and were exposed ( whole body) to atmospheres containing 0 ( 75 mice/ sex), 10 ( 75 mice/ sex), or 30 ppm ( 150 mice/ sex) naphthalene ( 99% pure) for 6 hours/ day, 5 days/ week for 2 years. The high dose group contained twice as many animals as the low dose group to ensure that a sufficient number of animals lived until termination of the study, and because of the insufficient information on the long term toxicity of naphthalene. Comprehensive histopathological examinations were performed on all control and high dose mice, and on low dose mice that died or were sacrificed before 21 months of exposure. In the remaining low dose animals that survived longer than 21 months of exposure, only the nasal cavity and lung were histologically examined. Initially, 50 animals/ sex/ dose group were designated for the two year study, with 5 animals/ sex/ dose group designated for an interim hematology examination at 14 days, and 3, 6, 12, and 18 months of the study. However, because of high mortality in the male control group ( discussed below), only the 14 day hematology examination was conducted, with the remaining animals incorporated into the two year study.

In the male control group, statistically significant decreases in survival were observed due to wound trauma and secondary lesions resulting from increased fighting in this group, compared to the exposed groups. In the exposed groups, male mice tended to huddle in the cage corners during exposure. At study termination, survival was 37% ( 26/ 70), 75% ( 52/ 69), and 89% ( 118/ 133) for the male mice exposed to 0, 10, or 30 ppm, respectively. Survival percentages in exposed female mice were similar to that of the control group. Survival percentages were 86% ( 59/ 69), 88% ( 57/ 65), and 76% ( 102/ 135) for female mice exposed to 0, 10, or 30 ppm, respectively. The occurrence of nonneoplastic lesions observed in this study is summarized in Table 7 3 ( Section 7.2.6 above).

Apparent dose related increases were noted for the incidence of alveolar/ bronchiolar adenomas in female and male mice. In females, the incidence of this tumor type was 5/ 69, 2/ 65 and 28/ 135 at the 0, 10 and 30 ppm concentrations, respectively. An additional female mouse in the 30 ppm group displayed an alveolar/ bronchiolar carcinoma. The incidence of alveolar/ bronchiolar adenomas reached statistical significance at the 30 ppm concentration and the occurrence of this tumor type was considered compound related. In male mice, the incidence of alveolar/ bronchiolar adenomas was 7/ 70, 15/ 69, and 27/ 135 in the control, 10, and 30 ppm groups, respectively. However, when these incidence data were analyzed using a logistics 7 20 Naphthalene   February 2003 regression test ( a statistical test that adjusts for intercurrent mortality), the incidence of tumors in the 10 and 30 ppm groups did not differ significantly from the control.

Hemangiosarcomas were also reported in 5/ 135 female mice in the 30 ppm group. This tumor type was not observed in male mice or in control or 10 ppm female mice. However, this occurrence of hemangiosarcomas did not reach statistical significance, and the incidence of this tumor type was within the range of historical incidence ( 17/ 467) observed in control animals in multiple NTP inhalation studies ( NTP, 1992a).

NTP ( 2000) exposed F344/ N rats ( 49/ sex/ dose) to naphthalene vapor concentrations of 0, 10, 30, and 60 ppm for 6 hours plus T 90 ( the theoretical time to achieve 90% of the target concentration in the vapor chamber: 12 minutes) per day, 5 days a week for 105 weeks. The 10 ppm concentration corresponded to the threshold limit value for naphthalene ( ACGIH, 2000). Naphthalene concentrations were monitored by an on line gas chromatograph and average chamber concentrations were maintained within 1% of the target concentrations throughout the study. A physiologically based toxicokinetic model was used to estimate the daily doses of naphthalene. Data for modeling were obtained from additional groups of male and female rats exposed to 10, 30, or 60 ppm for up to 18 months. Dose calculations were based upon model estimates of the amount of naphthalene inhaled by rats at the exposure concentrations used in the two year study, the total amount of naphthalene metabolized following a six hour exposure period ( 21% to 31% of inhaled naphthalene), and average body weights of 125 grams ( male rats) and 100 grams ( female rats). Because essentially all of the naphthalene absorbed into the bloodstream is metabolized, the total amount of naphthalene metabolized was assumed to represent the internalized dose to rats from the exposure concentrations used in this study. The estimated daily doses determined by this method were 0, 3.6, 10.7, and 20.1 mg/ kg day for male rats, and 0, 3.9, 11.4, and 20.6 mg/ kg day for female rats.

The study animals were clinically examined twice daily. Body weights were recorded on day 1, every 4 weeks beginning at week 4, and every 2 weeks beginning at week 92. Clinical findings were recorded every 4 weeks beginning at week 4 and every 2 weeks beginning at week 92. Surviving rats were sacrificed at the end of the study and full necropsies and complete histopathological examinations were performed on all core study animals.

There were no treatment related clinical findings. All exposed groups of male and female rats had survival rates similar to those of the chamber controls. Mean body weights of females were generally similar to the body weights of the control group. The mean body weights of male rats in all exposure groups were generally less than those of the control group for most of the study. The mean body weights for the 10, 30, and 60 ppm exposure groups of male rats at 4 and 104 weeks were 9% and 5%, 9% and 5%, and 11% and 6% lower than those of chamber controls, respectively.

Neoplasms were observed in the nose of male and female rats in all exposure groups ( Table 7 5). However, neoplasm incidence in the lungs was not affected by naphthalene exposure of male or female rats in any exposure group ( respective tumor incidences for the chamber control, 10 ppm, 30 ppm and 60 ppm exposure groups were 2/ 49, 3/ 49, 1/ 48, and 0/ 49 for males, and 1/ 49, 0/ 49, 0/ 49, and 0/ 49 for females). The observed nasal neoplasms were 7 21 Naphthalene   February 2003 identified as neuroblastomas of the olfactory epithelium and adenomas of the respiratory epithelium. Neuroblastomas of the olfactory epithelium occurred with positive trends in both male and female exposure groups. The incidence of neuroblastomas for female rats in the control, low, mid and high exposure groups were 0/ 49 ( 0%), 2/ 49 ( 4%), 3/ 49 ( 6%), 12/ 49 ( 24%), respectively. Tumor incidence for female rats in the 60 ppm exposure group was significantly greater ( p< 0.001) than control. Incidences of neuroblastomas in the male rat control, low, mid and high exposure groups were 0/ 49 ( 0%), 0/ 49 ( 0%), 4/ 48 ( 8%), and 3/ 48 ( 6%), respectively. Neuroblastomas of the olfactory epithelium have not been historically observed in chamber control rats in other NTP two year inhalation studies. Positive trends in the incidence of respiratory epithelium adenomas in the nose were also observed for both male and female exposure groups. Tumor incidences were significantly increased ( p# 0.01) in all male rat exposure groups relative to the control group. Male rats in the control, low, mid, and highexposure groups had respiratory epithelium adenoma incidences of 0/ 49 ( 0%), 6/ 49 ( 12%), 8/ 48 ( 17%), and 15/ 48 ( 31%), respectively. Female rats exposed to the same concentrations had incidences of respiratory epithelium adenomas of 0/ 49 ( 0%), 0/ 49 ( 0%), 4/ 49 ( 8%), and 2/ 49 ( 4%), respectively. The increased tumor incidence observed in female rats in the 30 and 60 ppm exposure groups was not statistically significant. No historical incidence ( 0/ 299) of respiratory epithelium adenomas has been observed in chamber control rats utilized in previous NTP studies using the same diet as the current study.

Table 7 5. Incidence of Neoplasms in Male and Female F344/ N Rats in a Two year Naphthalene Inhalation Exposure Study Tumor Type Incidences of Neoplasms Chamber Control 10ppm 30ppm 60ppm MALE Adenoma of the Respiratory Epithelium 0/ 49 ( 0%) 6/ 49* ( 12%) 8/ 48* ( 17%) 15/ 48* ( 31%)

Neuroblastoma of the Olfactory Epithelium 0/ 49 ( 0%) 0/ 49 ( 0%) 4/ 48 ( 8%) 3/ 48 ( 6%)

Alveolar/ bronchiolar Adenoma or Carcinoma 2/ 49 ( 4%) 3/ 49 ( 6%) 1/ 48 ( 2%) 0/ 49 ( 0%)

FEMALE Adenoma of the Respiratory Epithelium 0/ 49 ( 0%) 0/ 49 ( 0%) 4/ 49 ( 8%) 2/ 49 ( 4%)

Neuroblastoma of the Olfactory Epithelium 0/ 49 ( 0%) 2/ 49 ( 4%) 3/ 49 ( 6%) 12/ 49* ( 24%)

Alveolar/ bronchiolar Adenoma 1/ 49 ( 2%) 0/ 49 ( 0%) 0/ 49 ( 0%) 0/ 49 ( 0%)

* Significantly different from chamber control ( P< 0.01) from chamber control using the Poly 3 test Source: NTP ( 2000) 7 22 Naphthalene   February 2003 Based upon the absence of neuroblastomas and adenomas in the chamber control rats of this two year study and historically in NTP two year inhalation studies, the increased incidences of these neoplasms are considered to be related to naphthalene exposure. The increased incidences of respiratory epithelial adenoma and olfactory epithelial neuroblastoma of the nose observed in this study are considered by the study authors to be clear evidence of carcinogenic activity of naphthalene in male and female F344/ N rats.

Other Routes of Exposure In a study conducted by Schmähl ( 1955), groups of 10 rats were given subcutaneous or intraperitoneal injections of naphthalene in oil ( 20 mg/ rat/ injection) once a week, starting at 100 days of age and continuing for 40 weeks, for a total dose of 820 mg/ rat. Rats were observed following the administration of naphthalene until natural death ( 700   900 days). Necropsies were performed on animals at death, and organs that appeared unusual were examined histologically. Results were limited to the statements indicating that no toxic effects or tumors were observed in either treatment group ( U. S. EPA, 1998a).

Boyland et al. ( 1964) implanted naphthalene into the bladders of stock Chester Beatty mice to determine the suitability of naphthalene as a potential vehicle of carcinogenicity testing. Thirty animals received the naphthalene implants, with examinations conducted 30 weeks following implantation. Twenty three of the 30 animals survived the implantation period. One mouse ( 1/ 23 or 4%) implanted with naphthalene developed a bladder carcinoma. No adenomas or papillomas were reported in the naphthalene implanted group. When compared to the paraffin wax or cholesterol implanted groups, tumor incidence in the naphthalene implanted group was as low as the incidence in the paraffin implanted groups ( 2   4%), and lower than in groups implanted with cholesterol ( 12%). The limitations of this study that make it inadequate for assessing the carcinogenic potential of naphthalene include the short exposure and observation periods and the lack of untreated controls ( U. S. EPA, 1998a).

La Voie et al. ( 1988) administered naphthalene dissolved in dimethyl sulfoxide by intraperitoneal injection to a group of 49 male and female newborn CD 1 mice on days 1, 8, and 15 of life. The doses at each injection time were 0.25, 0.5, and 1.0 : mol, for a total dose of 1.75 : mol naphthalene. A separate group of 46 pups served as a vehicle control group and received dimethyl sulfoxide alone. Mice were maintained ( 10 mice/ cage) until moribund, or until study termination at 52 weeks. Histopathological examinations were conducted on all gross lesions and on liver sections. Incidences of liver tumors reported in the mice that lived at least 6 months were 0/ 16 and 2/ 31 for exposed females and males, and 0/ 21 and 4/ 21 for vehicle control females and males. This study is limited for assessing the carcinogenic potential of naphthalene by the short exposure ( 2 weeks) and observation ( 52 weeks) periods, and because complete histopathological examinations were not conducted ( U. S. EPA, 1998a). 7 23 Naphthalene   February 2003 7.3 Other Key Data 7.3.1 Mutagenicity and Genotoxicity Numerous in vitro and in vivo assays have been conducted to evaluate the potential genotoxicity of naphthalene and its metabolites. The results of most studies were negative, suggesting that the genotoxic potential of naphthalene and its metabolites is weak ( U. S. EPA, 1998a), and is probably not an area of concern for exposure to naphthalene ( ATSDR, 1995). The results of naphthalene genotoxicity studies are summarized below.

Negative Results in vitro Naphthalene was not mutagenic in several bacterial/ microsomal assay systems, including Salmonella tester strains TA 97, 98, 100, 199, 667, 1535, and 1537, in the presence or absence of Aroclor 1254 induced hamster or rat liver microsomes ( McCann et al., 1975; Kaden et al., 1979; Florin et al., 1980; Gatehouse, 1980; Seixas et al., 1982; Connor et al., 1985; Godek et al., 1985; Sakai et al., 1985; Mortelmans et al., 1986; Nakamura et al., 1987; Narbonne et al., 1987; Bos et al., 1988; NTP, 1992a, 2000). There was no evidence of naphthalene induced DNA damage in Escherichia coli WP2/ WP100 ( Mamber et al., 1983), PQ37 ( Mersch Sundermann et al., 1992), GY5027/ GY4015 ( Mamber et al., 1984), or Salmonella typhimurium TA 1535/ p5K 1002 ( Nakamura et al., 1987).

The frequency of sister chromatid exchanges ( SCE) was not increased upon incubation of human peripheral lymphocytes in a medium containing naphthalene or in a human liver metabolic activation system, when compared with control frequencies ( Tingle et al., 1993; Wilson et al., 1995). Naphthalene did not induce unscheduled DNA synthesis in cultured rat hepatocytes ( Barfknecht et al., 1985). Naphthalene did not induce transformations of Fischer rat embryo cells ( Freeman et al., 1973) or Swiss mouse embryo cells ( Rhim et al., 1974) in vitro. Sina et al. ( 1983) reported that naphthalene did not induce single strand DNA breaks in cultured rat hepatocytes, as detected by alkaline elution ( U. S. EPA, 1998a).

Negative Results in vivo Several experiments have investigated the genotoxicity of naphthalene in vivo. Naphthalene did not increase the number of micronuclei in bone marrow cells of mice following intraperitoneal injection of a single dose of 250 mg naphthalene/ kg body weight ( Sorg et al., 1985). Harper et al. ( 1984) reported no increase in the frequency of micronucleated erythrocytes in mice exposed to single oral doses of naphthalene as high as 500 mg/ kg when compared to frequencies observed in control mice.

Tsuda et al. ( 1980) reported no evidence of the neoplastic transformation of liver cells in a group of 10 young adult Fischer 344 rats administered single gavage doses of 100 mg naphthalene/ kg in corn oil, when compared with the results from a group of 10 vehicle control animals ( U. S. EPA, 1998a). Rats were administered the doses of naphthalene or corn oil following partial hepatectomy, but prior to dietary treatment with 2 acetylaminofluorene and carbon tetrachloride. Gamma glutamyl transpeptidase foci were used as an indicator of 7 24 Naphthalene   February 2003 neoplastic transformation. These foci were observed in both exposed and control animals following the dietary treatments. In contrast to the results observed with naphthalene, a single gavage dose of 200 mg/ kg benzo[ a] pyrene induced significant increases in the number, area, and size of gamma glutamyl transpeptidase foci ( U. S. EPA, 1998a).

Positive results Four studies were available that reported a positive genotoxic response ( ATSDR, 1995; U. S. EPA, 1998a). NTP ( 1992a, 2000) reported that naphthalene caused sister chromatid exchanges ( concentration range of 27   90 : g/ mL) in Chinese Hamster ovary cells when assayed in the presence or absence of metabolic activation with rat liver S9 fraction. Chromosomal aberrations were observed ( concentration range of 30   67.5 : g/ mL) only in the presence of metabolic activation. Naphthalene was mutagenic in the marine bacterium Vibrio fischeri ( Arfsten et al., 1994) and in the Drosophila melanogaster wing somatic mutation and recombination test ( Delgado Rodriguez et al., 1995). Gollahon et al. ( 1990) observed a 10 fold increase in chromosomal damage in mouse embryos cultured in a medium containing 0.16 mM naphthalene, when compared with untreated culture controls. This response was amplified by the inclusion of a hepatic metabolic activation system in the medium.

Genotoxicity Studies of Naphthalene Metabolites Studies have been conducted with several known or possible metabolites of naphthalene, including 1 naphthol, 2 naphthol, naphthoquinone, and naphthalene 1,2 dione ( U. S. EPA, 1998a). The metabolites 1 naphthol and 2 naphthol were not mutagenic in Salmonella typhimurium with or without metabolic activation ( McCann et al., 1975; Florin et al., 1980; Narbonne et al., 1987). The metabolite 1 naphthol gave negative results in several other genotoxicity assays, including tests for sex linked recessive lethal mutations in Drosophila melanogaster ( Gocke et al., 1981), tests for mutations in mouse L5178Y cells ( Amacher and Turner, 1982), tests for unscheduled DNA synthesis in cultured rat hepatocytes ( Probst and Hill, 1980), and acute in vivo tests for the induction of micronuclei in the bone marrow cells of mice ( Gocke et al., 1981) and rats ( Hossack and Richardson, 1977). Naphthoquinone was not mutagenic in several strains of Salmonella typhimurium, with or without metabolic activation ( Sakai et al., 1985). Flowers Geary et al. ( 1994) reported that naphthalene 1,2 dione was mutagenic in strains of Salmonella typhimurium without metabolic activation ( U. S. EPA, 1998a).

7.3.2 Ocular Toxicity The ocular toxicity of naphthalene has been studied extensively, and the association between naphthalene exposure and the development of cataracts in animals is well established. Table 7 6 summarizes the results of representative ocular toxicity studies of naphthalene in various animal species. 7 25 Naphthalene   February 2003 Table 7 6. Summary of Studies of Naphthalene Ocular Toxicity in Animals Study Species ( Strain) Exposure Route Dose mg/ kg day Duration NOAEL LOAEL Result Van Heyningen and Pirie ( 1976) Rabbits ( Dutch, albino) Gavage 1,000 3   28 consecutive daily doses  1,000 Cataracts in 10/ 16 Dutch and 11/ 12 albino animals)

Srivastava and Nath ( 1969) Rabbits ( NS*) Gavage 2,000 5 days  2,000 Cataracts in 8/ 8 animals Rossa and Pau ( 1988) Rabbits ( Chinchilla Bastard) Oral 1,000 Single dose  1,000 Cataracts Rabbit ( New Zealand) Oral 1,000 4 biweekly doses  1,000 Cataracts Orzalesi et al. ( 1994) Rabbit ( pigmented) Gavage 500 5 weeks  500 Cataracts, retinal degeneration, subretinal neovascularization Fitzhugh and Buschke ( 1949) Weanling rats ( NS) Diet 2,000 ( estimated) 2 months ( approx.)  2000 Mild cataracts Koch et al. ( 1976) Rats ( Sprague Dawley, Wistar, and others) Gavage 1,000 Total duration unknowna; cataracts developed within 16 28 days. Doses administered on alternate days  1,000 Cataracts Rao and Pandya ( 1981) Rat ( NS) Gavage 1,000 10 days 1,000  No effects observed Yamauchi et al. ( 1986) Rat ( Wistar) Oral 1,000 18 days  1,000 Cataracts Rathbun et al. ( 1990) Rat ( Black Hooded) Gavage 5,000 79 days  5,000 Lens opacities Tao et al. ( 1991a, b) Rat ( Brown Norway) Gavage 700 102 days  700 Lens opacities Kojima ( 1992) Rat ( Brown Norway) Gavage 500 ( 1,000 mg/ kg day every second day) 4 weeks  500 Lens opacities Table 7 6 ( continued)

Study Species ( Strain) Exposure Route Dose mg/ kg day Duration NOAEL LOAEL Result 7 26 Naphthalene   February 2003 Xu et al. ( 1992a) Rat ( Long Evans, Brown Norway, Sprague Dawley, Wistar, Lewis) Gavage 1,000 28 days  1,000 Cataracts Ikemoto and Iwata ( 1978) Rabbits ( Albino) Oral 1,000 2 days  1,000 Cataracts Murano et al. ( 1993) Rat ( Sprague Dawley, Brown Norway) Gavage 1,000 6 weeks ( administered every other day)  1000 Cataracts Schmähl ( 1955) Rat ( in house strain BDI, BDIII) Food 41 2 years Study not adequate to support LOAEL OR NOAEL  No cataracts observed BCL ( 1980a) Rat ( Fischer) Gavage 0 25 50 100 200 400 13 weeks 5 days/ week 400  No cataracts observed BCL ( 1980b) Mouse ( B6C3F 1) Gavage 0 12.5 25 50 100 200 90 days 5 days/ week 200  No cataracts observed Shopp et al. ( 1984) Mouse ( CD 1) Gavage 0 53 133 90 days 133  No cataracts observed Shopp et al. ( 1984) Mouse ( CD 1) Gavage corn oil 0 27 53 267 14 days 267  No cataracts observed NTP ( 1992a) Mouse B6CF1 Inhalation 0 ppm* 10ppm 30 ppm 2 years ( 6 hr/ day; 5 days/ wk) 30ppm*  No cataract formation observed NTP ( 2000) Rat ( F344/ N) Inhalation 0 3.6   3.9 10.7   11.4 20.1   20.6 2 years 20.1   20.6  No cataractogenic effects or ocular abnormalities observed. Table 7 6 ( continued)

Study Species ( Strain) Exposure Route Dose mg/ kg day Duration NOAEL LOAEL Result 7 27 Naphthalene   February 2003 Shichi et al. ( 1980) Mouse ( C57BL/ 6N, DBA/ 2N) Diet 0 60 120 ( injected twice weekly with cytochrome P 450 inducer) 60 days C57BL/ 6 N mice:

DBA/ 2N mice: 120 C57BL/ 6N mice: 60 DBA/ 2 N mice:  Cataracts observed in 1/ 15 C57BL/ 6N mice at each dose.

No cataracts observed in DBA/ 2N mice)

Holmen et al. ( 1999) Rat ( Brown Norway) Gavage 0 100 500 1,000 1,500 10 weeks 2 doses/ week 100 adjusted: 29 500 adjusted : 143 First signs of ocular changes occurred within 2.5 weeks after start of treatment. All treated with doses of 500 mg/ kg or more developed cataracts.

* NS = Not specified a Information obtained from secondary source in which the indicated data were not provided Cataract formation has been documented primarily in rabbits and rats. Almost all studies have evaluated oral exposures. In rabbits, Van Heyningen and Pirie ( 1976) noted the formation of cataracts as soon as two days after initiation of daily administration of 1,000 mg/ kg by oil gavage. The incidence of cataract formation was higher in albino rabbits ( 11/ 12) than in the pigmented ( Dutch) strain. Srivastava and Nath ( 1969) reported cataracts in 8/ 8 rabbits ( strain not stated) treated with naphthalene doses of 2,000 mg/ kg day for 5 days via gavage. Rossa and Pau ( 1988) found that cataracts appeared in two different strains of rabbits after administering between one and four 1,000 mg/ kg oral doses of naphthalene ( U. S. EPA 1998a). Orzalesi et al. ( 1994) found that pigmented rabbits developed cataracts after 5 weeks gavage exposure at 500 mg/ kg day and retinal degeneration starting at 3 weeks. Retinal degeneration was extensive by the end of the exposure period, resulting in almost complete obliteration of the pigmented layer and extensive neovascularization.

In rats, Fitzhugh and Buschke ( 1949) reported the development of mild cataracts in five weanling rats ( strain unspecified) consuming two percent naphthalene in their diet for two months. U. S. EPA ( 1998a) estimated that this amount is equivalent to a total dose of approximately 2,000 mg/ kg per animal. Tao et al. ( 1991a, b) reported lense opacities ( unspecified incidence) in a group of female Brown Norway rats exposed by gavage at 700 mg/ kg day for 102 days.

Holmen et al. ( 1999) administered doses of 0, 100, 500, 1,000, or 1,500 mg/ kg twice weekly by gavage to female pigmented Brown Norway rats ( 3 to 15 animals/ dose group). When adjusted for duration, these doses correspond to 0, 29, 143, 285, or 429 mg/ kg day, respectively. Ocular changes were monitored by slit illumination and retro illumination. All rats treated with 7 28 Naphthalene   February 2003 doses of naphthalene equal to or greater than 500 mg/ kg developed cataracts. The first ocular changes were evident after 2.5 weeks of treatment when eyes were examined by retroillumination. In contrast, no evidence of cataractous change was noted in control rats or rats administered the 100 mg/ kg dose.

Strain differences have been reported for the incidence and rate of development of cataracts in rats. Koch et al. ( 1976) administered 1,000 mg/ kg naphthalene per day by gavage to rats of several strains ( Sprague Dawley, Wistar, and others) on alternate days. All of the pigmented rats developed cataracts within 16 to 28 days, whereas cataract incidence was lower in the albino strains. Xu et al. ( 1992a) administered gavage doses of 1,000 mg/ kg naphthalene per day in oil to both pigmented ( Long Evans and Brown Norway) and unpigmented ( Sprague Dawley, Wistar, and Lewis) rats for up to 28 days. Eyes were examined ( by slit lamp with focal and retro illumination techniques) twice a week for the first 2 weeks and weekly thereafter. All rats of both pigmented and unpigmented strains were found to have cataracts at the end of the exposure period. However, the rate of cataract development differed among strains, with the order being Brown Norway > Long Evans = Lewis = Sprague Dawley > Wistar. Murano et al. ( 1993) found that gavage doses of 1,000 mg/ kg naphthalene administered every other day for 6 weeks resulted in the development of cataracts in all exposed male Brown Norway and Sprague Dawley rats. Cataracts developed more rapidly in the Brown Norway than in the Sprague Dawley rats, an observation that is consistent with the findings of Xu et al. ( 1992a), above.

Shichi et al. ( 1980) observed a very low incidence ( 1/ 15) of cataracts in C57BL/ 6N mice following administration of doses of approximately 60 or 120 mg/ kg day in the diet for 60 days. The mice were injected twice weekly with an inducer of cytochrome P 450. No cataracts were observed in DBA/ 2N mice treated under the same regimen.

NOAELs for naphthalene induced cataract formation have been identified in chronic and subchronic exposure studies in rats and mice. Schmähl ( 1955) found no cataracts in rats treated orally with naphthalene at 41 mg/ kg day for 2 years, although the method of examination was not documented. BCL ( 1980a) did not observe cataracts in Fischer rats receiving up to 400 mg/ kgday 5 days per week for 13 weeks. In B6C3F 1 mice, ( BCL, 1980b) identified a NOAEL of 200 mg/ kg day ( administered 5 days per week). Shopp et al. ( 1984) found no cataracts ( method of cataract examination was not indicated) in CD 1 mice treated by gavage at 133 mg/ kg day for 90 days. Cataracts were not observed in B6C3F 1 mice exposed to concentrations of naphthalene as high as 30 ppm by inhalation for two years ( NTP 1992a). Cataracts or other ocular changes were not observed in F344/ N rats exposed to concentrations up to 60 ppm ( estimated dose 20.6 mg/ kgday for males) for two years ( NTP, 2000).

Based on the above findings, the relationship between oral naphthalene exposure and the development of cataracts has been clearly demonstrated in rodents. LOAELs range from 500 mg/ kg day ( Brown Norway rats) to 5,000 mg/ kg day ( black rats) across studies of all durations. NOAELs for naphthalene induced cataractogenesis in subchronic studies ranged from 29 mg/ kg day ( duration adjusted dose administered to Brown Norway rats on a biweekly dosing regimen) and 133 mg/ kg ( CD 1 mice) to 400 mg/ kg ( Fischer rats). 7 29 Naphthalene   February 2003 7.3.3 Hematological Effects Hemolytic anemia has been observed in humans exposed to naphthalene via inhalation, combined inhalation and dermal exposure, and combined oral and inhalation exposure ( see Section 7.3.3). In animals, naphthalene induced hemolytic anemia has been observed only in the dog. Zuelzer and Apt ( 1949) administered naphthalene incorporated into a meat diet to three dogs. One dog ( 7.3 kg body weight) received a single dose of 3 g ( equivalent to a 410 mg/ kg dose). A second dog ( 5.9 kg body weight) received a single 9 g dose ( equivalent to a 1,530 mg/ kg dose). The third dog ( 6.8 kg) was administered seven consecutive daily doses ranging from 0.5 to 3.0 g ( equivalent to 74 to 144 mg/ kg). The total dose in the third dog was 12.5 g, which is equivalent to an average daily dose of 262 mg/ kg day. The blood of the treated animals was characterized by decreased hemoglobin concentration and hematocrit; development of Heinz bodies in erythrocytes, erythrocyte fragmentation, and reticulocytosis. Similar indications of hemolytic anemia were not observed when hematological parameters were examined in F344 rats treated with gavage doses of up to 400 mg/ kg day ( BCL, 1980a), 5 days/ week for 13 weeks; in B6C3F 1 mice treated with gavage doses of up to 200 mg/ kg day, 5 days/ week for 13 weeks ( BCL, 1980b); or in CD 1 mice given gavage doses of up to 133 mg/ kg day for 90 consecutive days ( Shopp et al., 1984).

7.3.4 Immunotoxicity A limited number of studies document potential immunotoxic effects of naphthalene exposure. Based on the available data, adverse effects on the immune system do not appear to be a prominent feature of naphthalene toxicity.

An enlarged spleen was reported in one human subject that died as a result of ingesting naphthalene ( Kurz, 1987). Enlarged spleens were also observed in two human subjects that were dermally exposed to naphthalene ( Schafer, 1951; Dawson et al., 1958). However, these effects were believed to be associated with hemolysis, rather than indicative of a direct toxic effect on the spleen.

Shopp et al. ( 1984) reported no effects on humoral immune responses, delayed hypersensitivity responses, bone marrow DNA synthesis, or bone marrow stem cell number in CD 1 mice that received naphthalene at oral doses as high as 267 mg/ kg day for 14 days. Thymic weight decreased approximately 30% in the high dose male mice. In the high dose females, mitogenic responses to concanavalin A were reported. This effect was not observed with lipopolysaccharide or in mice that received naphthalene at 27 or 53 mg/ kg day. In addition, no immune system effects or alterations in thymic weights were observed in male mice that received 133 mg naphthalene/ kg/ day for 13 weeks. An approximately 20% decrease in spleen weight was reported in female mice that received 267 mg naphthalene/ kg/ day for 14 days, while a 25% decrease was observed in female mice that received 133 mg/ kg day for 13 weeks ( Shopp et al., 1984).

In other studies, thymic lymphoid depletion was reported in 2 of 10 female rats that received 400 mg naphthalene/ kg/ day for 13 weeks ( BCL, 1980a). Dermal application of pure naphthalene once weekly for 3 weeks to the skin of rabbits did not result in evidence of a delayed 7 30 Naphthalene   February 2003 hypersensitivity reaction ( PRI, 1985; Papciak and Mallory, 1990). The results of an in vivo study in C57B1/ 6 mice indicated that a single oral dose of naphthalene did not suppress antibody responses ( Silkworth et al., 1995).

An in vitro study conducted by Kawabata and White ( 1990) indicated that naphthalene did not have an immunosuppressive effect in the antibody response of splenic cell cultures to sheep red blood cells.

7.3.5 Hormonal Disruption No studies were located that document disruptive effects on the endocrine system associated with naphthalene exposure.

7.3.6 Physiological or Mechanistic Studies Information on the mode of action of naphthalene is available for three health effects associated with exposure: hemolysis, cataract formation, and pulmonary toxicity ( ATSDR, 1995; U. S. EPA, 1998a).

Hemolysis Humans and dogs are susceptible to naphthalene induced hemolysis following inhalation, oral, or dermal exposures. Naphthalene metabolites are believed to be involved in naphthaleneinduced hemolytic anemia, but the mode of action of naphthalene induced hemolysis is not clearly understood. Individuals deficient in glucose 6 phosphate dehydrogenase ( G6PD) are particularly sensitive to naphthalene hemolysis. G6PD deficient cells have a reduced capacity to generate reduced nicotinamide adenine dinucleotide phosphate ( NADPH), which serves as a cofactor in the reduction of oxidized glutathione. G6PD deficient cells, therefore, cannot quickly replenish reduced glutathione ( Dawson et al., 1958; Gosselin et al., 1984), a compound that plays a key role in defense against oxidative damage, and in the conjugation and excretion of some toxicants. Deficits in reduced glutathione levels are thought to decrease the rate of conjugation and the excretion of naphthalene metabolites, thereby leading to elevated levels of toxic naphthalene metabolic intermediates ( U. S. EPA, 1987b). In the absence of glutathione, the metabolites promote damage to red blood cell membranes and the oxidization of hemoglobin to methemoglobin. Both of these actions likely contribute to cell lysis ( U. S. EPA, 1998a). Other possible causes of hemolysis include inhibition of the enzymes glutathione peroxidase or glutathione reductase by a naphthalene metabolite ( Rathbun et al., 1990; Tao et al., 1991a, b).

Cataract Formation Experimental evidence suggests that naphthalene cataractogenesis requires cytochrome P 450 catalyzed bioactivation to a reactive intermediate. Some evidence suggests that the ocular toxicity of naphthalene is mediated by the production of 1,2 naphthalenediol in situ in the lens ( ATSDR, 1995). Alternatively, Van Heyningen and Pirie ( 1967) proposed that naphthalene was metabolized in the liver to epoxide intermediates and subsequently to stable hydroxy compounds. These hydroxy compounds then enter the circulation and are transported to the lens, where 1,2 7 31 Naphthalene   February 2003 naphthalenediol is subsequently oxidized to 1,2 naphthoquinone and hydrogen peroxide. The quinone binds to lens constituents, thus altering the integrity and transparency of the lens ( Uyama et al., 1955; Rees and Pirie, 1967; Van Heyningen and Pirie, 1967; Van Heyningen and Pirie, 1976; Van Heyningen, 1979; Wells et al., 1989).

Wells et al. ( 1989) assessed cataract formation after administration of naphthalene or naphthalene metabolites. Dose related increases in cataract incidence were observed following administration of 125 to 1,000 mg naphthalene/ kg, 5 to 250 mg 1,2 naphthoquinone/ kg, 56 to 562 mg 1 naphthol/ kg, or 5 to 250 mg 1,4 naphthoquinone/ kg to C57BL/ 6 mice by intraperitoneal injection. In contrast, cataract formation was not observed following the intraperitoneal administration of 56 to 456 mg 2 naphthol/ kg. The potency of the quinones was reported to be about 10 times that of naphthalene. Pretreatment with inducers of cytochrome P 450 and a glutathione depleting compound increased the potency of naphthalene in causing cataracts. Pretreatment with a P 450 inhibitor decreased naphthalene toxicity.

Xu et al. ( 1992a, b) conducted experiments that employed five different strains of albino rats. Naphthalene was administered via gavage at a dose of 500 mg/ kg day for three days, followed by 1,000 mg/ kg day for 25 days. All of the naphthalene treated rats developed cataracts. The concentration of reduced glutathione was decreased in the lens following three weeks of treatment, while increases in protein glutathione mixed disulfides and high molecular weight insoluble proteins were reported. Analyses of the aqueous humor indicated that the only naphthalene metabolite present was 1,2 dihydro 1,2 naphthalenediol. The authors speculated that this compound may have been metabolized to 1,2 naphthoquinone, the metabolite believed to be responsible for the formation of cataracts.

Xu et al. ( 1992a) determined that the only metabolite that resulted in formation of morphologically identical cataracts in vitro and in vivo was 1,2 dihydro 1,2 naphthalenediol. Opacities were also formed by 1,2 naphthalenediol and naphthoquinone. However, these cataracts formed on the cortex rather than the inner surface of the lens.

Xu et al. ( 1992a) investigated the role of the enzyme aldose reductase in cataract formation in naphthalene exposed rats. Aldose reductase is found in the lens, liver, and in peripheral neurons ( McGilvery, 1983) and is thought to oxidize 1,2 naphthalenediol to 1,2 naphthoquinone, the metabolite responsible for cataract formation ( Xu et al. 1992a). If this hypothesis is correct, inhibition of the reaction catalyzed by aldose reductase should result in decreased synthesis of 1,2 naphthoquinone, and decreased cataract formation. Groups of rats were dosed with naphthalene alone, naphthalene plus the aldose reductase inhibitor ALO1576, or ALO1576 alone. All naphthalene treated rats developed cataracts. Consistent with the proposed hypothesis, rats given the aldose inhibitor alone, or naphthalene plus aldose inhibitor, did not develop cataracts. The authors of this study suggested that the mechanism of naphthalene cataract formation may involve the transport of the dihydrodiol metabolite formed in the liver into the lens, where it is converted by aldol reductase into the very reactive 1,2 naphthoquinone. The naphthoquinone then causes oxidative damage to the lens, resulting in opacity.

This mode of action is supported by the results of Xu et al. ( 1992b), who found that the aldose reductase inhibitor ALO1537 also prevented naphthalene related cataract formation. In 7 32 Naphthalene   February 2003 contrast, Tao et al. ( 1991a, b) found that the aldose reductase inhibitor TK344 failed to prevent cataracts in naphthalene treated rats. The researchers hypothesized that the cataract preventive activity of ALO1537 might result from the inhibition of a naphthalene metabolizing enzyme other than aldose reductase.

Several studies have investigated biochemical processes that potentially contribute to naphthalene induced cataract formation. Srivastava and Nath ( 1969) reported markedly decreased lactate dehydrogenase activity and elevated o diphenol oxidase activity in the lens and capsule of rabbits ( strain not stated) treated with naphthalene doses of 2,000 mg/ kg day. Yamauchi et al. ( 1986) detected decreased levels of reduced glutathione in lenses of male Wistar rats treated with 1,000 mg/ kg day doses of naphthalene for 18 days. Rathbun et al. ( 1990) observed reduced total glutathione levels and progressive loss of glutathione peroxidase and glutathione reductase activity in Black Hooded rats administered approximately 5,000 mg/ kg day in the diet for 79 days, suggesting that naphthalene exposure impairs defenses against oxidative damage. However, Rao and Pandya ( 1981) did not detect any significant increase in ocular lipid peroxidation following administration of 1,000 mg/ kg day to male rats ( strain not stated) for 10 days.

Pulmonary Toxicity Pulmonary toxicity has been identified in experimental animals exposed to naphthalene via inhalation and parenteral pathways. As noted below, the pulmonary response to naphthalene varies significantly among species. At present, there is no strong evidence that exposure to naphthalene results in pulmonary toxicity in humans ( ATSDR, 1995).

Increased incidences of alveolar bronchiolar hyperplasia were observed in F344/ N female rats exposed to naphthalene via inhalation for two years ( NTP, 2000). A predominantly benign neoplastic response in the alveolar/ bronchiolar region following chronic inhalation exposure to naphthalene has been observed in male and female mice ( NTP, 1992a). Pulmonary bronchiolar epithelial cells, primarily Clara cells, may be damaged following intraperitoneal administration of naphthalene ( Mahvi et al., 1977; Tong et al., 1982; Warren et al., 1982; O'Brien et al., 1985, 1989; Honda et al., 1990; Chichester et al., 1994; Van Winkle et al., 1999). This toxicity has been associated with the metabolism of naphthalene by the cytochrome P 450 system in the lung ( Warren et al., 1982; O'Brien et al., 1985; Rasmussen et al., 1986; Buckpitt and Franklin, 1989). The ultrastructural changes induced by naphthalene are consistent with the type of damage produced by other P 450 bioactivated toxicants ( Van Winkle et al., 1999).

The identity of the toxic, P 450 activated metabolite is not known with certainty. However, it is believed to be one or more of the enantiomeric epoxides, naphthoquinones, or free radical intermediates ( Buckpitt and Franklin, 1989), which likely bind to the Clara cell proteins or nucleic acids ( Chichester et al., 1994). Local pulmonary metabolic processes are thought to be responsible for the observed toxicity, although there is some evidence that other tissues, such as the liver, may metabolize naphthalene to reactive metabolites that enter the circulation, are transported to the lung, and result in pulmonary cytotoxicity ( Warren et al., 1982; O'Brien et al., 1989; Kanekal et al., 1990). 7 33 Naphthalene   February 2003 Epoxide metabolites are considered strong candidates for causing the pulmonary toxicity observed following exposure to naphthalene. This conclusion is based on the observations that some epoxides are cytotoxic, genotoxic and possibly carcinogenic ( U. S. EPA, 1998a), and that cytotoxicity in isolated, perfused mouse lungs was produced by 1,2 naphthalene epoxide at concentrations 10 fold less than naphthalene ( Kanekal et al., 1991). In addition, the epoxide is capable of covalently binding to cellular macromolecules resulting in cell damage. In contrast, the naphthalene metabolites 1 naphthol, 1,2, and 1,4 naphthoquinone were not apparently cytotoxic in the lung at concentrations equal to concentrations of naphthalene that produced cytotoxicity ( U. S. EPA, 1998a). However, Zheng et al. ( 1997) treated mouse lung Clara cells with naphthalene in vitro and identified 1,2 naphthoquinone as a major adduct covalently bound to cellular protein, suggesting that this metabolite has the potential to contribute to pulmonary toxicity.

Species differences exist in the pulmonary metabolism and toxicity of naphthalene. Mice are more sensitive to the pulmonary effects of naphthalene than hamsters or rats ( Buckpitt and Franklin, 1989; Buckpitt et al., 1992; Plopper et al., 1992a, b). Microsomes prepared from mouse lung metabolized naphthalene approximately 92 times faster than microsomes prepared from Rhesus monkeys ( Buckpitt et al., 1992). The primary metabolites formed by the 2 species were also different, with mice and monkeys forming 1R, 2S naphthalene oxide and 1S, 2Rnaphthalene oxide, respectively. The metabolic rates reported for hamsters and rats were intermediate between those reported for mice and monkeys ( Buckpitt et al., 1992). The metabolic rates of human lung microsomes have been reported to be similar to those of monkeys ( Buckpitt and Bahnson, 1986).

Detailed comparison of naphthalene metabolic potential and naphthalene induced cytotoxicity throughout dissected airways confirms that there is a significant degree of speciesspecificity in metabolism and injury. Clara cells appear to be a primary target cell for naphthalene toxicity in the lung of mice, the most sensitive species among those tested. This is consistent with the putative role of Clara cells as one of the primary sites for cytochrome P 450 mediated xenobiotic metabolism in the lung. Studies by Plopper et al. ( 1992a) and Buckpitt et al. ( 1995) evaluated the association between Clara cell toxicity and metabolism in different areas of the tracheobronchial trees of mice, rats, and hamsters. The rate of metabolism of naphthalene and the extent of 1R, 2S naphthalene oxide enantiomer formation by microsomal preparations from specific areas were reported to correlate with differences in pulmonary cytotoxicity observed in the different species. Metabolism of naphthalene in mouse airways was highly stereoselective, producing the 1R, 2S naphthalene oxide enantiomer; similar stereospecificity was not observed in the airways of rats or hamsters. Non ciliated cells in all airway regions of the mouse were heavily labeled when treated with an antibody to cytochrome P 450 2F2, whereas little labeling was observed in any airway region of rats or hamsters.

Studies of species specific responses to naphthalene toxicity in the nose suggest that factors other than metabolic activation may play a role in cell injury. Plopper et al. ( 1992a) compared the sensitivity of nasal tissues to naphthalene toxicity in rat, mouse, and hamster. The close correlation observed between the metabolism and stereospecificity of the metabolites in the lung was not evident in the nose. Damage in the nasal cavity of the three species was limited to necrosis of the olfactory epithelium. Cells in this portion of the nose contain high concentrations 7 34 Naphthalene   February 2003 of several cytochrome P 450 isoforms. Although the target site for naphthalene induced injury was the same for all three species, the dose that produced necrosis differed among them. The level of total naphthalene metabolizing activity in a given species was not predictive of the dose required to elicit necrosis. This result was interpreted by the study authors as evidence for a role of phase II enzymes ( e. g., epoxide hydrolase and/ or glutathione S transferases) in modulating the intracellular levels of naphthalene oxides and thus toxicity in target cells.

Kanekal et al. ( 1990) reported that Clara cell numbers decreased substantially following a 4 hour exposure to 0.13 mg naphthalene when tested using a perfused rat lung system. It was also noted that the Clara cells exfoliated and were found in the airway lumens. As noted above, non ciliated Clara cells contain higher levels of mixed function oxidases and thus are believed to be more sensitive to damage from naphthalene. Chichester et al. ( 1994) reported that Clara cell viability decreased by 39 and 88%, when exposed to 64 or 128 mg/ L naphthalene, respectively. No effect was seen in cells exposed to 1.3 or 6.4 mg/ L naphthalene for 120 or 340 minutes. Exposure to equivalent molar concentrations of naphthalene oxide resulted in effects similar to those produced by naphthalene. The addition of glutathione and glutathione transferase decreased Clara cell damage.

Relatively little is known about repair of naphthalene induced pulmonary injury. However, the number of pulmonary neuroendocrine cells and the surface area covered per cell increased markedly within five days of a single intraperitoneal injection of naphthalene administered to male FVB/ n mice ( Peake et al., 2000). These alterations were interpreted as evidence for a key role of this cell type in epithelial cell renewal after airway injury.

Germansky and Jamall ( 1988) investigated the organ specific effects of naphthalene ( 169 mg/ kg day, time weighted average) on tissue peroxidation, glutathione peroxidases, and superoxide dismutase in lung tissue of male Blue Spruce pigmented rats. In contrast to results obtained in the liver, no effect of naphthalene exposure was evident on levels of peroxidation or activity of the two enzymes.

7.3.7 Structure Activity Relationship There are few studies that systematically examine the toxicological structure activity relationships among naphthalene and its close structural analogues. U. S. EPA ( 1998a) has summarized information related to the metabolism and pulmonary toxicity of the naphthalene structural analogues 1 and 2 methylnaphthalene.

The methylation of naphthalene to form 1 and 2 methylnaphthalene presents opportunities for metabolism via additional oxidative pathways. Due to the lack of a functional group to serve as a site for conjugation, naphthalene metabolism proceeds via P 450 catalyzed ring oxidation. Presence of the methyl groups in 1 and 2 methylnaphthalene enables the formation of potentially toxic aldehydes via side chain oxidation. The potential toxicity of the aldehydes raises the possibility that there are distinct differences between the effects of naphthalene and its methylated derivatives that result from differences in metabolism ( U. S. EPA, 1998a). 7 35 Naphthalene   February 2003 Buckpitt and Franklin ( 1989) reviewed the comparative pulmonary toxicity of naphthalene and the related compound 2 methylnaphthalene. The researchers noted that, while 2 methylnaphthalene is less acutely toxic than naphthalene, the dose response characteristics for subchronic pulmonary toxicity ( alveolar proteinosis, Clara cell damage, bronchiolar necrosis) of naphthalene and the 2 methyl derivative are quite similar. They suggested that metabolism by cytochrome P 450 was more clearly implicated in the toxicity of 2 methylnaphthalene than in the case of naphthalene.

Chronic dietary exposure ( 0.075% and 0.15% in feed) of B6C3F 1 mice to either 1 methylnaphthalene and 2 methylnaphthalene for 81 weeks results in an increased incidence of pulmonary alveolar proteinosis ( Murata et al., 1993; Murata et al., 1997). Exposure to 1 methylnaphthalene also induced a small but statistically significant increase in the incidence of bronchiolar/ alveolar adenomas in the lungs of male, but not female mice ( Murata et al, 1993). Dietary exposures to 2 methylnaphthalene were not associated with an increased tumor incidence.

Additional research is required to determine if and how the pulmonary effects of naphthalene and 1 and 2 methylnaphthalene are mechanistically related ( U. S. EPA, 1998a).

7.4 Hazard Characterization 7.4.1 Synthesis and Evaluation of Major Noncancer Effects As discussed in Section 7.1, data concerning the adverse effects of naphthalene exposure in humans are limited. A number of case reports describe acute accidental and intentional naphthalene ingestion ( Lezenius, 1902; Gerarde, 1960; Gupta et al., 1979; Ijiri, 1987; Kurz, 1987). The utility of these data for the evaluation of health effects associated with occurrence of naphthalene in drinking water is potentially limited by several factors. Quantitative exposure data are not provided in these incident reports. The extent of naphthalene uptake and the toxic endpoints resulting from a single, large dose may differ from those that would occur from exposure in drinking water. In addition, the low aqueous solubility of naphthalene may prevent the occurrence of concentrations in drinking water that are acutely toxic to the general population. An additional important source of uncertainty in these considerations is the potentially greater sensitivity of certain subpopulations to naphthalene toxicity, including infants and children, neonates, fetuses, and individuals deficient in G6PD. At present, little information is available to define acutely toxic levels of exposure for these groups.

Case reports of individuals ( primarily infants) exposed to naphthalene by inhalation or through dermal contact with mothballs or with items stored with mothballs ( Schafer, 1951; Valaes, 1963; Owa, 1989) are more informative. While none of these studies provides information on the exposure levels that are associated with adverse effects, they provide information that establishes hemolytic anemia and its sequelae as the most important toxic effect in humans exposed to naphthalene at levels that might be encountered in the environment. Case reports also indicate that humans with G6PD deficiency are especially susceptible to naphthalene toxicity, particularly infants and the fetus ( Valaes, 1963; U. S. EPA, 1987b; Owa, 1989). 7 36 Naphthalene   February 2003 Studies of occupational exposure to naphthalene are limited to a single report of possible naphthalene related cataracts in chemical workers ( Ghetti and Mariani, 1956) and to two limited epidemiological studies ( Wolf, 1976; Kup, 1978) that provide ambiguous evidence of associations between occupational naphthalene exposure and cancer. Owing to their numerous limitations ( see Section 4.2), neither of these studies is useful in characterizing the potential risks associated with human exposures to naphthalene ( U. S. EPA, 1998a).

Because there are no reliable human studies to establish dose response relationships for specific health effects, most dose response information is derived from animal studies. The results of key toxicological studies are categorized by toxic effect in Table 7 7. An important feature of the data in this table is that hemolytic anemia, which appears to be the critical toxic effect in humans, is not seen in the majority of the animal studies. Thus, mice, rats, and rabbits are less sensitive to naphthalene induced hematotoxicity than humans. This is consistent with the general observation that dogs and humans are generally more sensitive to chemically induced hemolytic anemia than are other species ( ATSDR, 1995). The physiological and biochemical mechanisms responsible for this difference in sensitivity are not known ( U. S. EPA, 1998a). Dogs are apparently more sensitive to naphthalene exposure than other experimental animals, but the single available study in dogs ( Zuelzer and Apt, 1949) is quite old, and it used only a very small number of animals. Thus, it cannot be used to estimate a dose response relationship for naphthalene induced hemolysis.

In contrast to hemolytic anemia, naphthalene induced cataract formation is well studied in experimental animals. Acute, short term, and subchronic studies of cataractogenesis ( see Table 7 6) have established the general features of dose response relationships in different species and dosing regimens. In addition, these studies have helped to elucidate the biochemical basis of naphthalene induced cataractogenesis. The general mechanism for cataract formation, like that for hemolysis, appears to involve oxidative damage of cell components. However, greater progress has been made in identifying the specific metabolic pathways, enzymes, and toxic metabolites that are involved in cataract formation.

Quinone derivatives of naphthalene appear to be the proximate toxic metabolites involved in cataract formation ( U. S. EPA, 1998a). Naphthalene is first oxidized by cytochrome P 450 monooxygenases to the 1,2 epoxide. The epoxide is then converted into naphthalene dihydrodiol by one or more pathways. These metabolic steps probably occur in the liver, but it is known that naphthalene metabolism also occurs in other organs, notably the lung. It is thought that the dihydrodiol diffuses into the crystalline lens where it is converted into 1,2 naphthoquinone. The naphthoquinone then reacts with lens components to cause damage and opacity. The key enzyme in the conversion of the dihydrodiol to the quinone is aldose reductase, as judged by studies that show reduced cataract formation when reductase inhibitors are administered along with naphthalene to experimental animals. Glutathione depletion may also enhance the development of cataracts ( ATSDR, 1995) by preventing detoxifying conjugation reactions.

Species differences in sensitivity to naphthalene induced cataracts have been attributed to differences in enzyme activity levels. These results have not yet been extrapolated to human toxicity, however. The relatively low severity of the cataracts observed in the single epidemiologic study ( Ghetti and Mariani, 1956) of highly exposed subjects suggests that humans 7 37 Naphthalene   February 2003 are not extremely sensitive to naphthalene induced cataract formation after combined inhalation and dermal exposures.

The second specific toxic effect that has been linked to naphthalene exposure in experimental animals is the development of non neoplastic lesions in the nose and lung ( potential carcinogenic responses are discussed in Section 7.4.2 below). Mice ( NTP, 1992a) and rats ( NTP, 2000) had increased incidences of multiple nasal lesions after inhalation exposure to naphthalene for two years. Exposure related increases in the incidences of alveolar bronchiolar hyperplasia were observed in female rats ( NTP, 2000) and in the incidences of chronic inflammation in the lung of male and female B6C3F 1 mice ( NTP, 1992a). In addition, respiratory tract lesions have been observed in mice after parenteral administration of naphthalene ( summarized in U. S. EPA, 1998a). The occurrence of lung lesions after noninhalation exposure suggests that lung tissue may be especially sensitive to naphthalene or its metabolites, or that particular metabolic pathways are acting in the lung to produce high concentrations of toxic intermediates.

Several studies have found that the pattern of naphthalene induced lesions in a mouse lung closely correlates with cytochrome P 450 activity ( Warren et al., 1982; Buckpitt and Franklin, 1989). In vitro studies suggest the epoxides may be the key cytotoxic metabolites in mouse lung, although down stream metabolites ( the dihydrodiol and quinones) cannot be conclusively ruled out. Buckpitt et al. ( 1992) found that mouse lung microsomes metabolize naphthalene approximately 92 times faster than lung microsomes from Rhesus monkeys, and that the enantiomeric composition of the metabolic products was different in mice than in monkeys ( U. S. EPA, 1998a). The study authors suggested that these differences at least partially explain the differences in sensitivity to lung toxicity of mice and primates. A more recent study ( Buckpitt et al., 1995) identified the rate of conversion of naphthalene to naphthalene 1R, 2S oxide by cytochrome P 450 2F2 as the most important determinant of naphthalene toxicity in mouse lung.

The mode( s) of action for the other toxic effects reported in Table 7 7 are not wellunderstood The decreased body weights seen in several of the subchronic studies do not appear to be related to reduced food intake, but may indicate generally depressed metabolic function. Changes in organ weights have only been observed sporadically, with different organs affected in different studies, and no specific patterns of histopathological changes in the affected organs ( other than the lung). Numerous studies suggest that naphthalene is a very weak reproductive and developmental toxicant, with detectable effects occurring only at doses associated with substantial maternal toxicity or even mortality. Finally, no biochemical explanation has been put forward for the neurological effects seen in pregnant rats ( BCL 1980a; NTP, 1991). However, the available studies support a clearly defined NOAEL and LOAEL for this effect.

7.4.2 Synthesis and Evaluation of Carcinogenic Effects The available human data are inadequate to evaluate any association between naphthalene and cancer occurrence. The available epidemiological studies ( Wolf, 1976; Kup, 1978) are limited due to the size of the populations examined ( n= 15) and co exposure to other potential carcinogens, such as tobacco smoke or other polycyclic aromatic hydrocarbons, such as 7 38 Naphthalene   February 2003 benzo[ a] pyrene. No large scale epidemiological study has been conducted to examine the possible association between naphthalene exposure and cancer ( U. S. EPA, 1998a).

Data available from animal studies are also limited. Only two inhalation studies were adequately designed to examine the carcinogenicity of lifetime naphthalene exposure. NTP ( 1992a) examined the carcinogenicity in mice exposed to naphthalene for 2 years by inhalation. A statistically significant increase in the incidence of alveolar/ bronchiolar adenomas and carcinomas combined was reported for female B6C3F 1 mice, but not male mice, exposed via inhalation to 30 ppm naphthalene for 6 hours/ day, 5 day/ week for 2 years ( NTP, 1992a). However, NTP ( 1992a) concluded that the study provided " some evidence" only of carcinogenicity in female mice, but not " clear evidence," because only one carcinoma was observed ( U. S. EPA, 1998a). In a similar study, NTP ( 2000) examined tumor occurrence in F344/ N rats exposed to naphthalene vapor for 2 years. Increased incidences of two types of nasal tumors were noted in naphthalene treated animals. The incidences of adenoma of the respiratory epithelium were increased in male rats exposed to 10, 30, and 60 ppm ( approximately 3.6, 10.7, and 20.1 mg/ kg day, respectively). The incidence of neuroblastoma of the olfactory epithelium was significantly increased in female rats exposed to 60 ppm ( approximately 20.6 mg/ kg day). Because these tumors did not occur in control animals and because the historical incidence in NTP chamber control rats is low, the increased incidence of these tumors in naphthalene exposed animals was considered by the study authors to be " clear evidence" of carcinogenic activity.

In the Adkins et al. ( 1986) study, A/ J strain mice were exposed to 10 or 30 ppm naphthalene vapors for 6 months. Following the exposure period, excised lungs were examined for pulmonary adenomas. Histopathological study of lung tissue was limited to the examination of the tumors. Increased numbers of adenomas were found in the lungs of naphthalene exposed mice when compared to the control group, but the differences were not statistically significant. A significant increase in the number of alveolar adenomas per tumor bearing lung was reported in both dose groups. However, the response did not increase with increasing dose. Limitations of this study include the less than lifetime exposure duration and the restricted histopathology.

Several studies have been conducted in which naphthalene was administered by routes of exposure other than inhalation or diet ( Schmähl, 1955; Boyland et al., 1964; La Voie et al., 1988). However, no carcinogenic responses were observed in these studies, and each has at least one limitation that makes it inadequate for assessing the potential for lifetime naphthalene exposure to produce cancer ( U. S. EPA, 1998a). 7 39 Naphthalene   February 2003 Table 7 7. Summary of Key Studies of Noncancer Toxic Effects of Naphthalene Study Species ( Strain) Sex n Doses mg/ kg day Route Duration NOAEL LOAEL Effect mg/ kg day Hemolytic anemia Zuelzer and Apt ( 1949) Dog 3 410 1,530 262 ( average of 7 daily doses ranging from 74 to 441 mg/ kg) Diet single dose single dose 7 days  262 Hemolytic anemia observed ( decreased hemoglobin and hematocrit concentrations,

development of Heinz bodies in erythrocytes,

erythrocyte fragmentation and reticulocytosis).

BCL ( 1980a) Rat ( F344) Male and Female 10/ sex/ dose 0 25 50 100 200 400 Gavage corn oil 13 weeks 5 days/ week 400  No indications of hemolytic anemia observed BCL ( 1980b) Mouse ( B6CF 1) Male and Female 10/ sex/ dose 0 12.5 25 50 100 200 Gavage corn oil 90 days 200  No indications of hemolytic anemia observed Shopp et al.

( 1984) Mouse ( CD 1) Male and Female 40   112 0 27 53 267 Gavage corn oil 14 days 267  Red cell hemolysis not observed Table 7 7 ( continued)

Study Species ( Strain) Sex n Doses mg/ kg day Route Duration NOAEL LOAEL Effect mg/ kg day 7 40 Naphthalene   February 2003 Shopp et al.

( 1984) Mouse ( CD 1) Male and Female 40   76 0 53 133 Gavage corn oil 90 days 53  No indications of hemolytic anemia observed NTP ( 1992a) Mouse ( B6CF 1) Male and Female 75   150 0 10 30 Inhalation 2 years ( 6 hr/ day;

5 days/ wk) 30  No changes in hematological parameters observed after 14 days Cataracts Van Heyningen and Pirie ( 1976) Rabbits ( Dutch,

albino) Sex not stated 39 0 1,000 Gavage oil 3   28 consecutive daily doses  1,000 Cataracts in 10/ 16 Dutch and 11/ 12 albino animals)

Rossa and Pau ( 1988) Rabbits ( Chinchilla Bastard) Sex not stated 4 0 1,000 Oral Single dose  1,000 Cataracts Rabbit ( New Zealand) Sex not stated 4 0 1,000 Oral 4 biweekly doses  1,000 Cataracts Orzalesi et al.

( 1994) Rabbit ( pigmented Male 31 0 1,000 durationadjusted 500 Gavage 5 weeks  500 Cataracts, retinal degeneration, subretinal neovascularization Fitzhugh and Buschke ( 1949) Weanling rats ( NS*)   a 2,000 ( estimated) Diet 2 months ( approx.)  2,000 Mild cataracts Table 7 7 ( continued)

Study Species ( Strain) Sex n Doses mg/ kg day Route Duration NOAEL LOAEL Effect mg/ kg day 7 41 Naphthalene   February 2003 Tao et al. ( 1991a,

b) Rat ( Brown Norway) Female 80 0 700 Gavage 102 days  700 Lens opacities Koch et al.

( 1976) Rats ( Sprague

Dawley,

Wistar,

albino)   a 0 1,000 Gavage total duration not specified;

cataracts appeared in 16 to 28 days.

Doses administered on alternate days  1,000 Cataracts Xu et al. ( 1992a,

b) Rats ( Sprague

Dawley,

Wistar,

Lewis,

Long

Evans and Brown Norway) Male 6   10 0 1,000 Gavage oil 28 days  1,000 Cataracts Murano et al.

( 1993) Rats ( Brown Norway, Sprague

Dawley) Male 6 1,000 Gavage 6 weeks ( administered every other day)  1,000 Cataracts Table 7 7 ( continued)

Study Species ( Strain) Sex n Doses mg/ kg day Route Duration NOAEL LOAEL Effect mg/ kg day 7 42 Naphthalene   February 2003 Shichi et al.

( 1980) Mouse ( C57BL/ 6N and DBA/ 2N) Male and Female 15/ group 60 120 Diet 60 days C57BL/ 6N mice:

 DBA/ 2N mice:

120 C57BL/ 6N mice:

60 DBA/ 2N mice:

 Cataracts observed in C57BL/ 6N mice ( 1/ 15) at each dose No cataracts observed in DBA/ 2N mice Schmähl ( 1955) Rat ( in house strain BDI,

BDIII) Male and Female 28 41 Food 2 years Study not adequate to develop LOAEL or NOAEL  No cataracts observed BCL ( 1980a) Rat ( Fisher 344) Male and Female 10/ sex/ dose 0 25 50 100 200 400 Gavage corn oil 13 weeks ( 5 days/ week) 400  No cataracts observed BCL ( 1980b) Mouse ( B6CF 1) Male and Female 10/ sex/ dose 0 12.5 25 50 100 200 Gavage corn oil 13 weeks ( 5 days/ week) 200 Adjusted 143  No cataracts observed Shopp et al.

( 1984) Mouse ( CD 1) Male and Female 40   76 0 53 133 Gavage 90 days 133  No cataracts observed Table 7 7 ( continued)

Study Species ( Strain) Sex n Doses mg/ kg day Route Duration NOAEL LOAEL Effect mg/ kg day 7 43 Naphthalene   February 2003 Shopp et al.

( 1984) Mouse ( CD 1) Male and Female 40   112 0 27 53 267 Gavage corn oil 14 days 267  No cataracts observed NTP ( 1992a) Mouse ( B6CF 1) Male and Female 75   150 0 ppm 10 ppm 30 ppm Inhalation 2 years ( 6 hr/ day;

5 days/ wk) 30 ppm*  No cataract formation observed NTP ( 2000) Rat ( F344/ N) Male and Female 49/ sex/ dose 0 3.6   3.9 10.7   11.4 20.1   20.6 Inhalation 2 years 20.1 20.6  No cataracts observed.

Srivastava and Nath ( 1969) Rabbits ( NS*) NS 6   8 0 2,000 Gavage 5 days  2,000 Cataracts in 8/ 8 animals Yamauchi et al.

( 1986) Rat ( Wistar) Male 4   5 0 1,000 Oral 18 days  1,000 Cataracts Rathbun et al.

( 1990) Rat ( Black Hooded) NS 0 5,000 Gavage 79 days  5,000 Lens opacities Rao and Pandya ( 1981) Rat ( NS) Male 6 0 1,000 Gavage 10 days 1,000  No effects observed Ikemoto and Iwata ( 1978) Rabbits ( Albino) Male and Female NS 100 Oral 2 days  1,000 Cataracts Table 7 7 ( continued)

Study Species ( Strain) Sex n Doses mg/ kg day Route Duration NOAEL LOAEL Effect mg/ kg day 7 44 Naphthalene   February 2003 Holmen et al.

( 1999) Rat ( Brown Norway) Female 3   15 0 100 500 1,000 1,500 Gavage 10 weeks 2 doses/ week 100 adjusted:

29 500 adjusted:

143 First signs of ocular changes occurred within 2.5 weeks after start of treatment, leading to cataract formation Kojima ( 1992) Rat ( Brown Norway) Female 3   12 0 1,000 every second day Gavage 4 weeks  1,000 adjusted: 500 Lens opacities Nasal Pulmonary Lesions Plasterer et al.

( 1985) Mouse Male and Female 33   40 250 500 Gavage 8 days  500 No exposure related lesions observed in any organ system Germansky and Jamall ( 1988) Rats, weanling ( Blue Spuce) Male 24 100   750 169 mg/

kgday ( TWA) 9 weeks 169  No effect observed on peroxidation in lung BCL ( 1980b) Mouse ( B6CF 1) Male and Female 10/ sex/ dose 0 12.5 25 50 100 200 Gavage corn oil 13 weeks ( 5days/ week) 200 No exposure related leisons observed Table 7 7 ( continued)

Study Species ( Strain) Sex n Doses mg/ kg day Route Duration NOAEL LOAEL Effect mg/ kg day 7 45 Naphthalene   February 2003 Adkins et al.

( 1986) Rats ( A/ J) Female 30 0 ppm*

10 ppm 30 ppm Inhalation 2 years ( 6 hr/ day; 5 days/ wk) 30 ppm No adverse non cancer effects reported on the lung NTP ( 1992a) Mouse ( B6CF 1) Male and Female 75   150 0 ppm*

10 ppm 30 ppm Inhalation 2 years ( 6 hr/ day;

5 days/ wk)  10 ppm*

( for chronic nasal and respiratory irritaiton) Respiratory tract lesions ( chronic lung inflammation, chronic nasal irritation with hyperplasia of the respiratory epithelium,

metaplasia of the nasal epithelium)

NTP ( 2000) Rat ( F344/ N) Male and Female 49/ sex/ dose 0 3.6   3.9 10.7   11.4 20.1   20.6 Inhalation 2 years  3.6   3.9 Non neoplastic lesions of the nose were observed.

Body Weight BCL ( 1980a) Rat ( Fisher 344) Male and Female 10/ sex/ dose 0 25 50 100 200 400 Gavage corn oil 13 weeks ( 5 days/ wk) 100 200 Reduced body weight (> 10% males)

BCL ( 1980b) Mouse ( B6CF 1) Male and Female 10/ sex/ dose 0 12.5 25 50 100 200 Gavage corn oil 13 weeks ( 5 days/ wk) 200  No effect observed Table 7 7 ( continued)

Study Species ( Strain) Sex n Doses mg/ kg day Route Duration NOAEL LOAEL Effect mg/ kg day 7 46 Naphthalene   February 2003 NTP ( 1991) Rat Pregnant Female 25   26 0 50 150 450 GD 6   15 50 ( for maternal toxicity) 150 Significant decrease in weight gain ( 150 and 450 dose groups)

NTP ( 1992b) Rabbit ( New Zealand,

White) Pregnant Female 25   27 0 20 80 120 Gavage corn oil GD 6   19 120  Maternal: No consistently observed toxicity No effect on fetal body weight NTP ( 2000) Rat ( F344/ N) Male and Female 49/ sex/ dose 0 3.6   3.9 10.7   11.4 20.1   20.6 Inhalation 2 years 20.1   20.6  No difference in mean body weights observed.

Shopp et al.

( 1984) Mouse ( CD 1) Male and Female ( 40   112) 0 27 53 267 Gavage corn oil 14 days 53 267 Decreased body weight ( males and females)

Germansky and Jamall ( 1988) Rats, weanling ( Blue Spuce) Male 24 100   750 169 ( TWA) 9 weeks  169 Decreased body weight ( 20%)

NTP ( 1992a) Mouse ( B6CF 1) Male and Female 75   150 0 ppm*

10 ppm 30 ppm Inhalation 2 years ( 6hr/ day; 5 days/ week) 30  No significant change in mean body weight Table 7 7 ( continued)

Study Species ( Strain) Sex n Doses mg/ kg day Route Duration NOAEL LOAEL Effect mg/ kg day 7 47 Naphthalene   February 2003 Holmen et al.

( 1999) Rat ( Brown Norway) Female 3   15 0 100 500 1,000 1,500 Gavage 10 weeks 2 doses/ week 500 adjusted:

143 1,000 adjusted:

285 Decreased mean body weights observed in rats administered 1000 and 1500 mg/ kg.

Shopp et al.

( 1984) Mouse ( CD 1) Male and Female ( 40   76) 0 53 133 Gavage corn oil 90 days 133  No effects observed on body weight Organ Weight Shopp et al.

( 1984) Mouse ( CD 1) Male and Female ( 40   112) 0 27 53 267 Gavage corn oil 14 days 53 267 Decreased thymus weight ( male)

Increased spleen and lung weights ( female)

Shopp et al.

( 1984) Mouse ( CD 1) Male and Female ( 40   76) 0 53 133 Gavage corn oil 90 days 53 133 Decreased brain, liver,

and spleen wts. ( Female only)

No effects observed on organ weights from all exposure groups ( male)

Rao and Pandya ( 1981) Rats ( NS) Males 6 0 1,000 10 days  1,000 Increased liver weight ( 39%)

Nervous System Depression Table 7 7 ( continued)

Study Species ( Strain) Sex n Doses mg/ kg day Route Duration NOAEL LOAEL Effect mg/ kg day 7 48 Naphthalene   February 2003 BCL ( 1980a) Rat ( Fisher 344) Male and Female 10/ sex/ dose 0 25 50 100 200 400 Gavage corn oil 13 weeks ( 5 days/ wk)  400 Lethargy observed in highest dose group ( 400 mg/ kg day) for males and females BCL ( 1980b) Mouse ( B6CF 1) Male and Female 10/ sex/ dose 0 12.5 25 50 100 200 Gavage corn oil 13 weeks ( 5 days/ wk)  200 Transient signs of lethargy observed in highest dose groups during weeks 3 and 5 PRI ( 1986) Rabbit ( New Zealand White) Female 18 0 40 200 400 Gavage Methylcellulose GD 6   18 40 200 Treatment related signs of labored breathing,

body drop, decreased activity and salivation were observed.

NTP ( 1991) Rat ( Sprague

Dawley) Pregnant Females 25   26 0 50 150 450 Gavage 10 days ( GD 6   15)  50 Neurotoxic effects observed in all dose groups ( lethargy, slow respiration, periods of apnea, apparent inability to move after dosing).

Effects were transient,

and diminished with continued exposure. Table 7 7 ( continued)

Study Species ( Strain) Sex n Doses mg/ kg day Route Duration NOAEL LOAEL Effect mg/ kg day 7 49 Naphthalene   February 2003 NTP ( 1992a) Mice Male 0ppm*

10 ppm 30 ppm Inhalation 2 years   Increased huddling behavior during exposure and reduced inclination to fight ( may indicate neurological effects,

although basis for behavioral changes were not speculated on by authors).

No additional signs of neurotoxicity reported.

BCL ( 1980a) Rats ( Fisher 344) Male and Female 10/ sex/ dose 0 25 50 100 200 400 Gavage corn oil 13 weeks ( 5 days/ wk) 400  No neurological effects found.

BCL ( 1980b) Mice ( B6C3F 1) Male and Female 10/ sex/ dose 0 12.5 25 50 100 200 Gavage corn oil 13 weeks ( 5 days/ wk) 200  No neurological effects found. Table 7 7 ( continued)

Study Species ( Strain) Sex n Doses mg/ kg day Route Duration NOAEL LOAEL Effect mg/ kg day 7 50 Naphthalene   February 2003 Developmental Toxicity Plasterer et al.

( 1985) Mouse ( CD 1) Female 33   40 0 300 Gavage corn oil GD 7   14  300 ( FEL) Maternal: Reduced wt.

gain; reduced survival Fetal: Reduced no. of pups/ litter; no abnormalities in surviving pups PRI ( 1985) Rabbit ( New Zealand White) Female 4 0 50 250 630 1,000 Gavage Methylcellulose GD 6   18 Maternal 250 Fetal 250 Maternal 630 ( FEL)

Fetal 630 ( abortion) Maternal: Mortality and decreased wt. gain at 630 mg/ kg day Fetal: Aborted at 630 mg/ kg day PRI ( 1986) Rabbit ( New Zealand White) Female 18 0 40 200 400 Gavage Methylcellulose GD 6   18 Maternal 400 Fetal 400 Maternal  Fetal  Maternal: survival, body wt. and body wt. gain unaffected Fetal: No effect on reproduction or development of fetus Table 7 7 ( continued)

Study Species ( Strain) Sex n Doses mg/ kg day Route Duration NOAEL LOAEL Effect mg/ kg day 7 51 Naphthalene   February 2003 NTP ( 1991) Rat ( Sprague

Dawley CD) Female 25   26 0 50 150 450 Gavage GD 6   15 Maternal   Fetal 450 Maternal 50 ( central nervous system depression)

Fetal  Maternal: Central nervous system depression manifested as lethargy, slow breathing,

prone body posture, and increased rooting Decreased weight gain ( 150 and 450 mg/ kg day)

Fetal: no finding of fetal toxicity or embryo toxicity NTP ( 1992b) Rabbit ( New Zealand,

White) Female 25   27 0 20 80 120 Gavage oil GD 6   19 Maternal 120 Fetal 120 Maternal  Fetal  Maternal: Two deaths in low dose group Fetal: No effect on reproduction or development of fetus Shopp et al.

( 1984) Mouse ( CD 1) Male 76   112 0 27 53 267 Gavage oil 14 267  No effect on testicular weight Male 76   96 53 133 Gavage oil 90 133  No effect on testicular weight Table 7 7 ( continued)