that the proportion found in the controls, 5%, was in agreement with population surveys of sperm density. When oligospermia ( deficiency in number of sperm) is defined as a sperm count per ejaculate< 100 million, 33% ( 24 painters) and 20% ( 18 controls) were oligospermic ( p= 0.20). The rate of oligospermia was analyzed separately for smokers and non smokers. Among the non smokers, the exposed group had a higher rate of oligospermia ( p= 0.05). When smoking was controlled, the odds ratio calculated for a decreased count per ejaculate among the painters was 1.85, with a 95% confidence interval of 0.6  5.6 Because of the regular rotation of painters from one job to another at the shipyard, the painters could not be classified into dose groups. Because of the cyclical nature of spermatogenesis the authors stated that exposure from two to six months prior to semen analysis was likely to have produced an effect at the time of the study, and it was not possible to determine each man's job and exposure at that time. Therefore, the researchers assumed that all the painters had the same exposure Painters were also exposed to two other substances that have been reported in the past to affect semen quality, lead and epichlorohydrin. Lead is known to cause a depression of sperm count The mean lead levels of the 45 men who had been monitored for lead were mostly below 20 ug/ deciliter( dl), and the highest single level in any individual was 40 ug/ dl. The authors stated that this level of lead exposure has not been documented to cause a depressed count. Epichlorohydrin was not detected in air sampling during the study The authors thus concluded that exposure to the ethylene glycol ethers 2 EE and 2 ME lowered sperm count in this group of painters. The authors pointed out that this finding is consistent with the effect seen in animal studies. Studies in several species show that these glycol ethers cause loss of germinal epithelium and testicular atrophy. Cellular studies show that this effect occurs by inhibition of cell division in the early pachytene stage of spermatogenesis, an effect that would be expected to result in a decreased count rather than an effect on motility or morphology Welch and Cullen ( Ex. 5 102) undertook a cross sectional clinical appraisal of a sample of painters and unexposed workers to evaluate the relationship between measurements of peripheral blood of the workers and ethylene glycol ether exposure. The study of hematologic function included : a complete blood count, a manual differential count of 200 cells, and a manual platelet count. In addition, each subject's past medical record from the employer's medical department was requested, including routine blood counts and whole blood levels. Complete records were obtained for two thirds of the subjects The authors reported that the only other compounds known to be toxic to bone marrow or circulating blood cells that painters at the shipyard were exposed to, in addition to ethylene glycol ethers, were lead and benzene. Lead exposure was limited to abrasive blasting operations; the highest lead level detected during brushing or cleaning operations was 10 ug/ m3. Sampled levels during blasting were as high as 11 mg/ m3. Painters engaged in blasting use air supplied respirators and their blood lead was routinely monitored. Forty five of 94 painters were categorized by the employer as " lead exposed" and were participating in the routine blood testing; only nine of the forty five men had a mean lead level greater than 15 ug/ dl, and only two had a mean greater than 20 ug/ dl, with the highest at 30 ug/ dl. The highest single value was 40 ug/ dl Paints or cleaning solutions containing more than 1% benzene have not been used in the shipyard sine 1977. Ten air samples for benzene were obtained by NIOSH during the 1978 survey; levels of 0.08 to 0.53 mg/ m3 were detectable in eight samples. None of the bulk samples of paints or cleaning solutions in the current industrial hygiene survey revealed any benzene. Mean hemoglobin levels did not differ between the painters ( 15.43 g/ dl + 1.09 S. D.) and controls ( 15.67 g/ dl + 0.84); p= 0.14 in a two tailed test. Additionally, there were no statistically significant correlations between hemoglobin and cumulative exposure measured as years painting at the shipyard The hemoglobin data were rank ordered and analyzed by the Wilcoxon rank order test. There was no significant difference in rank for the entire group. However, when only those study subjects in the lowest quartile for hemoglobin were included in the analysis, the majority of low values were in painters ( p= 0.028)

Using an a priori standard for anemia in working age adult males of less than 14 grams hemoglobin/ dl blood, nine of the 147 subjects with adequately coded data were below this cutoff; all nine were painters. The past medical records of the shipyard for the anemic painters were reviewed; complete medical records were available on 7 of the 9. Normal hemoglobins were noted on hire in four of the seven anemic men with available records. In a fifth, the initial hemoglobin of 13.8 g/ dl had dropped steadily to 12 at the time of the study. For the two men for whom there were no preemployment blood counts, their hemoglobins were compared to those of the respondents of the National Health and Nutrition Examination Survey of the same age, sex, and race, and found to be less than the 10th percentile. In the remaining two, hemoglobins were 13.1 and 13.7 g/ dl on hire, comparable to those found during the study. After eliminating these two, whose values did not change since first employed, the rate of anemia is significantly different between painters and controls ( p= 0.04). Two of the anemic painters also had an abnormal semen analysis; one was oligospermic, and one was azoospermic( lack of sperm)

Total polymorphonuclear leukocyte ( PMN) count was calculated by multiplying the total white count by the percentage PMNs in the differential count. The mean values did not differ significantly between the two groups ( painters, 4,602 cell/ ul + 2,041 S. D.; controls, 4,650 + 1,771). A lower limit of 1,800 cells/ ul was used to define " normal" and " abnormal" groups of painters and controls. The lowest total counts were found among painters; five, or 3.4% of the painters had values below 1,800 cell/ ul, whereas none of the controls had such low levels ( p= 0.07)

The authors concluded that the differences in hematologic values seen between the groups of painters and the unexposed controls is significant and that preexisting host factors or rates of participation are not able to explain their results. Welch and Cullen concluded that an analysis of other exposures demonstrates that the difference is attributable to ethylene glycol ethers. They added that the absence of a significant effect on the group as a whole and the inability to detect a dose response pattern, make strong conclusions unwarranted. The authors called for further research on hematologic effects of these compounds in human populations In summary, although limited in part by confounding exposures to other solvents, data among workers exposed to 2 ME and 2 EE have exhibited anemia, reduced white and red blood cell counts, bone marrow injury and reduced sperm counts. In some cases these effects were observed at levels which were reportedly below those of the current permissible exposure limits for 2 ME and 2 EE. These findings support the strong body of experimental animal evidence, which show, in [ page 15544]

several species, that 2 ME and 2 EE induce adverse hematologic, reproductive and developmental effects G. Mutagenicity Studies in general indicate a lack of mutagenic potential for 2 ME and 2 EE. Mutagenicity is the ability to induce genetic mutation, i. e., a change in the genetic material. Mutations may give rise to developmental effects in cases where the genetic material of the egg or the sperm has been changed such as to induce abnormal development in the fetus. ( Mutations may also give rise to cancer. However, there are substances which may be carcinogenic which are not mutagenic. The carcinogenicity of these glycol ethers has not been tested.)

2 ME and 2 EE have been tested in various tests including Ames tests, unscheduled DNA synthesis( UDS) assays in human embryo fibroblasts, sister chromatid exchange ( SCE) tests in hamster ovary cells, cytogenic analyses in rat bone marrow cells, dominant lethal tests in rats, sperm abnormality tests in mice and sex linked recessive ( SLR) tests in fruit flies. ( Exs. 5 022, 5 056 and 5 076)

Neither 2 ME nor 2 EE induced effects in either the Ames test or UDS assays. 2 EE did induce chromosomal abnormalities in SCE tests. The authors stated that the positive findings in 2 EE are in contrast to the general negative findings in most mutagenic assays. Thus the authors concluded that it may be premature to classify these substances as mutagenic. In the SLR assays 2 EE was found to be negative while inconsistent results were observed for 2 ME. Positive results were observed for 2 ME in the sperm abnormality and dominant lethal tests. For example, 2 ME induced abnormal sperm head morphology and a reduction in male rat fertility. While the dominant lethal test showed a decrease in male fertility, the authors raised the possibility that the reduction in fertility could also be attributed to reduced sperm number rather than a dominant mutation Thus, the majority of the available data indicates that 2 ME and 2 EE lack mutagenic potential. However the presence of positive findings raise the possibility that these substances may have some weak mutagenic potential. No mutagenicity testing has been conducted with 2 MEA or 2 EEA, but the metabolic data discussed earlier suggest that all four compounds are metabolized by similar pathways and are thus likely to induce similar effects. Thus the results observed for 2 ME and 2 EE are predictive of mutagenic potential in their respective acetates H. Conclusions Health effects data from experimental animal studies clearly and consistently show that 2 ME, 2 EE and their acetates produce dose related adverse hematologic, reproductive and developmental effects. These effects include testicular damage, reduced fertility, maternal toxicity, early embryonic death, external, skeletal and visceral malformations, delayed development, and adverse effects on the blood Evidence also indicates that both inhalation and dermal exposures are significant routes of exposure for glycol ethers and the induction of adverse effects. In addition persons occupationally exposed to 2 ME and 2 EE through inhalation and dermal exposures have exhibited adverse reproductive and hematologic effects. Although not as extensive, in major part due to methodological limitations, the human data are nevertheless highly consistent with and supportive of the strong body of data in experimental animals showing adverse hematologic, reproductive and developmental effects I. Other Glycol Ethers Past research on the health effects of glycol ether compounds has primarily been concentrated on 2 ME and 2 EE as these two compounds and their acetates have represented a major percentage of the industrial use of glycol ethers Although less extensive there is also research on other glycol ether compounds. Much of the concentration in this area has been on substitutes for 2 ME and 2 EE such as 2 Butoxyethanol and the propylene glycol ethers ( e. g., propylene glycol monomethyl ether and its acetate)

1. 2 Butoxyethanol ( 2 BE)

In a series of experiments Carpenter et al ( Ex. 5 146) exposed various animal species ( e. g., rats, guinea pigs and mice) to 2 BE by inhalation. Groups of rats and guinea pigs were exposed for 7 hours/ day, 5 days/ week for 30 days at doses of 54, 107, 203, 314 or 432 ppm ( rats) and doses of 54, 107, 203 376, or 494 ppm ( guinea pigs). Significant increases in osmotic fragility in red blood cells was observed in rats at doses of 107 ppm 2 BE and higher. Osmotic fragility was also observed at 54 ppm when doses were administered daily for 30 days. No statistically significant evidence of osmotic fragility was observed among the guinea pigs at any of the doses tested. Mice were exposed to exposed 7 hours/ day for 30, 60 or 90 days to 100, 200 or 400 ppm. No controls were included. Osmotic fragility was observed at all doses tested Hematologic analyses were also conducted by Tyl et al.( Ex. 4 152) on pregnant rats and rabbits exposed to 2 BE by inhalation. Rabbits and rats were exposed to 0, 25, 50, 100 or 200 ppm 2 BE on days 6 18 ( rabbits) and days 6 15 ( rats) of gestation. Red blood cell counts, hemoglobin and hematocrit were analyzed in blood samples from both pregnant rats and rabbits. In rats osmotic fragility of red blood cells were not detected at any of the tested doses. However, significant reductions in red blood cell count and mean corpuscular hemoglobin concentration were observed at both 200 and 100 ppm. Mean cell volume and hemoglobin were significantly increased at 200 and 100 ppm. The only significantly treatment related effects observed among rabbits were increases in hemoglobin content and hematocrit at 100 ppm. However these same blood effects were not observed at 200 ppm Dodd et al. ( Ex. 5 050) performed acute, 9 day and 13 week inhalation studies in rats to investigate the toxicity of 2 BE. In the acute study rats were exposed for 4 hours to target concentrations of 200, 500 and 850 ppm 2 BE. The acute 4 hour LC50 was 486 ppm for males and 450 ppm for females. Rats exposed to 500 and 850 ppm exhibited loss of coordination. Post mortem examinations of these animals revealed red stained urine and kidney damage. Rats exposed to 200 ppm appeared normal. In the 9 day study rats were exposed 6 hours/ day to 0, 25, 100 and 250 ppm 2 BE. At 250 ppm rats exhibited significant decreases in red blood cell count and hemoglobin concentration. SSignificant effects in the blood were also observed among rats in the 100 ppm exposure group. However the authors stated that the effects were less profound. No statistically significant adverse hematological effects were observed among the rats exposed to 25 ppm. In the 13 week study, rats were exposed to 0, 5, 25 or 75 ppm 2 BE, 6 hours/ day, 5 days a week for 13 weeks. At 75 ppm female rats exhibited significant decreases in red blood cell count and hemoglobin concentration and increases in mean corpuscular hemaglobin after 6 weeks of exposure. However by the end of the study these decreases had either lessened or returned to controls levels. Male rats exposed at 75 ppm showed a significant decrease only in red blood cell count. No other dose related effects were observed among male or female rats. In particular there were no alterations in testes weight among males exposed to 2 BE, nor were any lesions observed [ page 15545]

which would have been indicative of a testicular effect.

A similar lack of testicular effect after exposure to 2 BE was noted by Nagano et al. ( Ex. 4 135). In this study mice were orally exposed to 500, 1000, or 2000 mg/ kg body weight of 2 BE, 5 days/ week for 5 weeks. Animals exposed at 2000 mg/ kg died. Decreases in red blood cell count were observed among both the 1000 and 500 mg/ kg exposure groups. However males at 100 and 500 mg/ kg 2 BE did not exhibit any statistically changes in testicular weight. This observation was in contrast to results from this same study which showed marked testicular degeneration after exposure to 2 ME, 2 EE and their acetates Doe ( Ex. 4 112) studied the testicular effects of single high dose exposures to 2 BE, in addition to examining the effects of 2 ME and 2 EE. Rats were exposed for 3 hours to single high doses of 2 BE ( 800 ppm), 2 ME ( 7500 ppm), or 2 EE ( 3500 ppm) and then were followed for 14 days. 2 ME and 2 EE significantly induced testicular atrophy however no significant reduction in testes weight were seen among the 2 BE exposed rats Similar comparative analyses were performed by Foster et al ( Ex. 5 052). However in this study the metabolites of 2 BE, 2 ME and 2 EE were administered rather than the parent glycol ethers. Male rats were exposed by gavage to single oral doses of butoxyacetic acid ( 0, 174, 434 or 868 mg/ kg), methoxyacetic acid ( 0, 118, 296, or 595 mg/ kg) or ethoxyacetic acid ( 0, 137, 342, or 684 mg/ kg). No statistically significant evidence of testicular toxicity was observed at any of the test doses for butoxyacetic acid whereas both methoxy and ethoxyacetic acid were found to significantly decrease testicular weight ( at all doses for 2 ME and at high doses only for 2 EE). As a part of this same study in vitro testicular cell cultures were exposed to the above metabolites to investigate the effects on testicular germ cells. Adverse effects in spermatocytes were observed after administration of methoxy and ethoxyacetic acids. For example, MAA and EAA produced an enhancement of germ cell loss from Sertoli cell cultures. In contrast no specific effects such as those that were observed after administration of butoxyacetic acid The developmental effects of 2 BE were studied by Tyl et al ( Ex. 4 152). Pregnant rats and rabbits were exposed to either 0, 25, 50 100 or 200 ppm 2 BE for 6 hours/ day on days 6 15 ( rats) or days 6 18 ( rabbits) of gestation. Signs of maternal toxicity were observed in rats at 100 and 200 ppm ( e. g., significant reductions in body weight, food consumption and absolute and relative organ weights). A significant increase in the number of resorbed litters, a significant decrease in the number of viable implantation per litter and a significant reduction in skeletal ossification were also observed after exposure to 200 ppm 2 BE. No significant increases in the incidence of malformations were observed at any doses among the rats In rabbits, increases in resorptions and reduced body weight gain were observed at 200 ppm however these effects were not statistically significant. Significant reductions in the number of viable implants were observed at 200 ppm. No evidence of statistically significantly increased incidences of malformations were found among any of the exposed rabbits. The authors concluded that 2 BE induced maternal and fetotoxic effects but not teratogenic effects No significant increases in maternal or developmental effects of 2 BE were observed by Nelson et al ( Ex. 5 091). In this study pregnant rats were exposed to 0, 150, or 200 ppm 2 BE for 7 hours/ day on days 7 15 gestation. These levels were chosen as earlier findings reported death at doses from 250 to 500 ppm 2 BE. The only significant adverse effect observed was " slight" hematuria among the maternal animals after the first day of exposure. Otherwise, no other significant maternal or developmental adverse effects were observed. Effects examined included resorptions, fetal weights and incidence of malformations. These findings are in contrast to results of this same study in which 2 ME and 2 EE were shown to induce adverse maternal and developmental effects Dermal application of 2 BE has also shown a similar lack of maternal or developmental effect. Hardin et al ( Ex. 5 073) exposed pregnant rats by dermal application to 0.35 mL 2 BE, four times daily on days 9 13 of gestation. Deaths occurred through the third and seventh days of exposure. Only one of the 11 rats treated survived. Therefore, tests were repeated at 0.12 mL, four times daily. No significant adverse maternal or developmental effects were observed at this exposure dose In the recent final Air Contaminants standard ( 54 FR 2332) OSHA revised the Permissible Exposure Limit ( PEL) for 2 BE from 50 ppm to 25 ppm. OSHA concluded that, [ T] he former PEL of 50 ppm was insufficiently protective against the risk of 2 butoxyethanol's irritant, hematological, and other potential systemic effects, which constitute material health impairments. The limit of 25 ppm included in the final rule will reduce this significant risk to a level below that at which these toxic effects have been observed in animals and humans. This lower limit will also prevent the discomfort experienced by workers at exposure levels of 40 ppm. ( Air Contaminants Final Rule, 54 FR 2554)

In 1990 NIOSH published a Criteria Document for 2 BE and its acetate, 2 BEA ( Ex. 5 145). NIOSH reported that data from animals indicate that 2 BE and 2 BEA do not cause adverse reproductive or development effects. However they report that the animal evidence shows that these substances do induce marked adverse effects on the blood. Based on the adverse blood effects observed in animals, NIOSH recommended occupational exposure limits of 5 ppm for both 2 BE and 2 BEA 2. Propylene Glycol Ethers The production of propylene glycol ethers is analogous to that of ethylene glycol ethers. Ethylene glycol ethers are made by reacting ethylene oxide and the appropriate alcohol. Propylene glycol ethers are produced by reacting propylene oxide with the appropriate alcohol. As such the propylene and ethylene glycol ethers are structurally analogous. For example, ethylene glycol monomethyl ether ( 2 ME) is structurally very similar to propylene glycol monomethyl ether. However despite some structural similarities, differences in toxicities have been observed between the two general types of compounds For example, in a series of experimental studies Hanley et al. ( Exs. 5 068 and 4 120) compared the developmental effects of 2 ME and propylene glycol monomethyl ether ( 2 PGME). In studies on 2 ME ( Ex. 4 120) rats and rabbits were exposed to 0, 3, 10, or 50 ppm 2 ME, 6 hours/ day on days 6 15 and days 6 18 of gestation respectively. In rabbits, exposure at 50 ppm 2 ME resulted in a significant decrease in maternal body weight gain, a significant increase in resorption rates and significant increases in major malformations. Increased resorption rates were also observed at 10 ppm compared to concurrent controls, but because the resorption rates were not statistically different from historical control values, the authors did not consider the effects to be dose related. Rats did not show any signs of maternal toxicity after exposure to 50 ppm. However fetuses from this exposure group exhibited a significant increase in the incidence in lumbar spurs and delayed ossification. Neither rats nor rabbits had any other significant adverse effects at 10 or 3 ppm. In comparison,

[ page 15546]

Hanley et al.( Ex. 5 068) exposed rats and rabbits to 0, 500, 1500 or 300 ppm 2 PGME to similar periods of gestation for 6 hours/ day. At 3000 ppm both rats and rabbits exhibited maternal effects including central nervous system depression and a significant decrease in body weight gain. No significant maternal effects were observed at 1500 or 500 ppm for either species. Neither rats nor rabbits exhibited any significant increase in resorption rates or major malformations at any of the dose levels tested. It was noted by the authors that a significant increase in malformations among rat fetuses at 3000 ppm was observed compared to concurrent controls. However this increase was similar to historical control values and thus was not considered to be dose related. The only significant effect observed, delayed sternebral ossification, was observed at 3000 ppm in rats. This result was interpreted by the authors to be an indication of slight fetotoxicity Miller et al. ( Ex. 5 088) also compared the toxicities of 2 ME and 2 PGME in rats and rabbits. Rats and rabbits were exposed to 0, 30, 100 or 300 ppm 2 ME or 0, 300, 1000 or 5000 ppm 2 PGME, 6 hours/ day for 13 weeks. Exposure to 300 ppm 2 ME induced testicular degeneration, decreased sperm count, decreased white blood cell counts and decreased hemoglobin concentrations. No significant effects were observed among the 100 or 30 ppm exposure groups. In contrast, no significant effects on testes weight or blood were observed among rats or rabbits exposed to 2 PGME at any dose tested. The authors attributed the difference in toxicity to differences in metabolism. The authors noted that 2 ME is a primary alcohol and has been shown to be metabolized by an alcohol dehydrogenase mediated pathway to methoxyacetic acid. In addition methoxyacetic acid is considered to be the active metabolite in the induction of reproductive and developmental toxicity. In contrast, 2 PGME is a secondary alcohol and is metabolized by microsomal enzymes to propylene glycol. The authors concluded that this difference in metabolism is most likely to be responsible for the differing toxicities of 2 ME and 2 PGME However Miller et al. ( Ex. 5 093) have also noted that there are two isomeric forms of 2 PGME; the alpha isomer ( which is a secondary alcohol) and the beta isomer ( which is a primary alcohol). Because of their differences in structure the two isomers are metabolized differently. The alpha isomer is metabolized by microsomal enzymes to propylene glycol and the beta isomer is metabolized by the alcohol/ aldehyde dehydrogenase pathway to 2 methoxypropionic acid. The beta isomer follows a metabolic pathway similar to that of the ethylene glycol ethers, 2 ME and 2 EE, which are also primary alcohol glycol ethers. Thus it was postulated that the beta isomer may have toxic properties different from its alpha isomer and may possibly be more similar to ethylene glycol ethers. These conclusions are supported by studies by Merkle et al. ( Ex. 5 092) on the pure beta isomer of 2 PGME Acetate. In this study pregnant rats were exposed to 0, 110, 550 or 2700 ppm 2 PGME Acetate and pregnant rabbits were exposed to 0, 36, 145 or 550 ppm 2 PGME Acetate. In rats, exposure to 2700 ppm resulted in a significant increase in the number of litters with skeletal anomalies ( e. g., dumbbell shaped notches of the thoracic vertebrae). A slight, but significant, decrease in fetal body weight was also noted at 2700 ppm. No significant effects were observed at the lower test doses. Rabbits however showed a more sensitive response. At 550 ppm, all fetuses exhibited severe malformations ( e. g., heart defects and anomalies of the paw and sternum). No significant increases in malformations were observed at other tested doses. It was concluded from these results that the beta isomer of the 2 PGME Acetate has teratogenic potential. By analogy, the beta isomer of its parent glycol ether, 2 PGME was also considered to have teratogenic potential While the beta isomers of the propylene glycol ethers appear to have teratogenic potential Miller et al. ( Ex. 5 093) also note in their metabolic study that the commercial product of 2 PGME is usually a mixture of the two isomers, with the alpha isomer accounting for up to 95% of the mixture. In its comment on the ANPR, the ARCO Chemical Company, a primary producer of propylene glycol ethers, has also stated that 2 PGME and its acetate routinely contain less than 2% of the beta isomer ( Ex. 7 19). These types of commercial products were used by Miller et al. ( Ex. 5 088) and Hanley et al. ( Ex. 5 068) in their reproductive and developmental studies and were shown to have a low degree of biological activity in comparison to ethylene glycol ethers 3. Ethylene Glycol Monopropyl Ether ( EGPE)

Katz et al. ( Ex. 5 085) conducted a series of acute and subchronic toxicity tests on EGPE and its acetate EGPEA in rats. In single dose oral studies rats were exposed to 1090, 2180, 4360, 8720 mg/ kg ( EGPE& EGPEA) or 17,470 mg/ kg ( EGPEA only). The LD50 of EGPE and EGPEA were observed to be 3089 and 9456 mg/ kg, respectively. Prior to death animals exhibited weakness, anorexia and hemoglobinuria. In single inhalation dose studies rats were exposed to target concentrations of 0, 250, 100 or 200 ppm EGPE and 0, 250, 500 or 100 pp, EGPEA. No lethality was observed at any dose, therefore the LC50 was concluded to be greater than 2132 ppm for EGPE and greater than 934 ppm for EGPEA. In six week oral studies male rats were exposed to 0, 195, 390, 780 or 1560 mg/ kg body weight EGPE or 0, 1097, 2193, or 4386 mg/ kg EGPEA. Adverse blood effects ( e. g., significant decreases in hemoglobin concentration and significant increases in platelet counts and nucleated red blood cells) were observed for both compounds at all dose levels. However, only rats exposed to EGPEA at 4386 mg/ kg exhibited significant decreases in testicular weight. Pathological examinations revealed atrophy of the seminiferous tubules and degenerated sperm. In the two week inhalation studies both male and female rats were exposed for 6 hours/ day to either 0, 100, 200, 400, 800 ppm EGPE or 0, 100, 200, 400 or 800 ppm EGPEA. Slight, but significant changes in red blood cells ( e. g., decreased count, and increased corpuscular volume) were observed at 800 and 400 ppm for both compounds. Hemoglobinuria was observed in males and females at 800 ppm EGPE and males only at 400 ppm. Both males( 4 out of 5) and females( 5 out of 5) exhibited hemoglobinuria after exposure to 400 and 200 ppm EGPEA. A significant increase in spleen weights were observed at 800 and 400 ppm for both compounds. No significant changes in testicular weight were observed for either compound. Based on these results the authors concluded that the NOELs in this study were 200 ppm for EGPE and 100 EGPEA Krasavage and Katz ( Ex. 5 070) studied the developmental toxicity of EGPEA. In this study pregnant rats were exposed to 100, 200, 400 or 800 ppm EGPEA, 6 hours/ day on days 6 15 gestation. Exposure to 800 and 400 ppm resulted in decreases in mean maternal body weight, feed intake, and red blood cell counts. Exposure at 800 ppm also resulted in a significant increase in the incidence in resorptions and a significant reduction in mean fetal body weight. No significant increases in theincidence of major malformations were observed among fetuses exposed up to 800 ppm. The authors stated that a significant increases in the incidence of minor skeletal effects ( e. g., wavy,

[ page 15547]

knobby, fused and partially ossified ribs and decreased ossification of the skull) were observed at 800 and 400 ppm. A significant increase in rudimentary ribs was observed in the 200, 400 and 800 ppm exposure groups. The authors concluded that adverse fetal effects occur after exposure to EGPEA. However they stated that these effects occurred only after doses which were overtly toxic to the maternal animal ( i. e., 800 and 400 ppm)

4. Di Ethylene Glycol Monomethyl Ether ( DEGME)

In inhalation studies by Miller et al. ( Ex. 5 058) male and female rats were exposed to 0, 30, 100 or 216 ppm DEGME, 6 hours/ day, five days/ week for 13 weeks. No dose related significant effects were observed among the male or female animals for any of the doses tested. Based on the lack of effects the authors concluded that DEGME is unlikely to present the same degree of hazard as its structural homolog 2 ME The teratogenic potential of DEGME was examined by Scortichini et al. ( Ex. 5 060). In this study pregnant rabbits were exposed by dermal application to 0, 50, 250 or 750 mg/ kg day of DEGME on days 6 18 gestation. Rabbits exposed at 750 mg/ kg exhibited a significant decrease in maternal weight gain and red blood cell counts. No statistically significant maternal effects were observed at 250 or 50 mg/ kg/ day. The authors noted an increase in resorptions at 750 mg/ kg/ day, although this effect was not statistically significantly different from controls. In addition no statistically significant increases in major malformations were observed at any of the doses tested. A significant increase in minor skeletal defects such as forelimb flexure, fused ribs, delayed ossification, forked ribs and cervical spurs were observed among litters from rabbits exposed to 250 and 750 mg/ kg DEGME. The authors considered these to be significant signs of fetotoxicity rather than teratogenicity and suggested that these types of fetal defects might be associated with maternal toxicity 5. Ethylene Glycol Monophenyl Ether ( 2 Phenoxyethanol)

Scortichini et al ( Ex. 5 059) have also examined the teratogenic potential of 2 Phenoxyethanol. Pregnant rabbits were dermally exposed to 0, 300, 600 or 1000 mg/ kg/ day of 2 Phenoxyethanol on days 6 18 gestation. Nine of 25 rabbits died after exposure to 1000 mg/ kg/ day and 5 of 25 rabbits died after exposure to 600 mg/ kg/ day. Death was attributed to intravascular hemolysis. The animals surviving in these groups showed no statistically significant treatment related effects. In addition no statistically significant signs of maternal toxicity were observed after exposure to 300 mg/ kg/ day. Among fetuses examined, there were no statically significant increases in the incidence of external, visceral or skeletal malformations at 300 or 600 mg/ kg/ day. ( Fetal observations were not available at 1000 mg/ kg/ day due to the high lethality at 1000 mg/ kg. Animals were sacrificed with no further observations. In addition, no other reproductive parameters such as resorptions or fetal body measurements were adversely affected at 600 or 300 mg/ kg/ day. Based on these results the authors concluded that doses up to 600 mg/ kg/ day produced no significant signs of developmental toxicity 6. Conclusions The available data for other glycol ether compounds suggests that there are differential toxicities between the longer chain glycol ethers and shorter chain glycol ethers such as 2 ME, 2 EE and their acetates. For example, in the case of 2 butoxyethanol, there were observations of adverse hematological effects but no observations of adverse reproductive or developmental effects. Similarly for propylene glycol ethers there was little evidence of any reproductive or developmental toxicity except in the case of the beta isomeric forms of these compounds. There are scattered reports on other ethylene glycol ether compounds showing adverse hematological effects and, in some cases, slight evidence of testicular effects and minor skeletal defects. In some studies the authors have suggested that defects observed in some of the fetuses may be due to maternal toxicity rather than a direct effect on the conceptus. However, as discussed earlier, developmental effects observed at maternally toxic doses do not necessarily imply that the developmental effects are secondary to maternal effects In general, the toxicities of these compounds appear less potent than those of shorter chain glycol ethers. The results from toxicity tests on other glycol ethers strongly contrast with the evidence observed after exposures to 2 ME and 2 EE. The evidence on 2 ME and 2 EE clearly and consistently show reduced sperm count, decreased fertility, testicular degeneration, early fetal death, major external, visceral and skeletal malformations, delayed development and functional deficiency. These effects have been observed in several species and through various routes of exposure. The totality and consistency of the evidence on 2 ME, 2 EE and their acetates in experimental animals, clearly indicate that these agents are potential reproductive and developmental toxins in humans. However, OSHA reiterates that past research primarily concentrated on 2 ME, 2 EE and their acetates. The lack of evidence on other glycol ethers may be due, in part, because less research has been conducted on these compounds. Thus, OSHA requests data and analyses on other glycol ethers and their potential reproductive and developmental toxicity VI. Preliminary Risk Assessment A. Introduction The United States Supreme Court, in the " benzene" decision, Industrial Union Department, AFL CIO v. American Petroleum Institute, 448 U. S. 607 ( 1980), has ruled that the OSH Act requires that, prior to the issuance of a new standard, a determination must be made, based on substantial evidence in the record considered as a whole, that there is a significant risk of health impairment at existing permissible exposure limits and that issuance of a new standard will significantly reduce or eliminate that risk. The Court stated that " before he can promulgate any permanent health or safety standard, the Secretary is required to make a threshold finding that a place of employment is unsafe in the sense that significant risks are present and can be eliminated or lessened by a change in practices." 448 U. S. 642. The Court also stated " that the Act does limit the Secretary's power to require the elimination of significant risks." 448 U. S. 644 Although the Court in the " cotton dust" case, American Textile Manufacturers Institute v. Donovan, 452 U. S. 490 ( 1981), rejected the use of cost benefit analysis in setting OSHA standards, it reaffirmed its previous position in " benzene" that a risk assessment is not only appropriate, but also required to identify significant health risk to workers and to determine if a proposed standard will achieve a reduction in that risk. Although the Court did not require OSHA to perform a quantitative risk assessment in every case, the Court implied, and OSHA as a matter of policy agrees, that assessments should be put into quantitative terms to the extent possible The extent to which a risk assessment may be put in quantitative terms is limited in the case of glycol ethers. This is not because there are no data suitable for assessing the risk. On the contrary, there are a number of well conducted rodent bioassays which clearly demonstrate the adverse health effects [ page 15548]

associated with exposure to glycol ethers ( see the discussion of health effects above). The problem lies in the fact that there is not a quantitative model for extrapolating the risk of developmental and reproductive effects either from high doses to low doses or across species, that is generally accepted in the scientific community. Therefore, unlike other risk assessments which OSHA has prepared in the past, this risk assessment will be far more qualitative than quantitative and will closely follow the guidelines of the Environmental Protection Agency for assessing the risks of suspect developmental and reproductive toxicants ( Ex. 5 153) to determine those levels of occupational exposure to the glycol ethers below which significant risk of adverse health outcomes is unlikely. This approach, which is described in detail in the following sections, is one that has been generally accepted in both the scientific and regulatory communities and is generally accepted as the best methodology for assessing the risks associated with reproductive and developmental toxins Risk assessment is a process in which scientific judgments are made concerning the potential for toxicity to occur in humans. Because human data are often not available, the risk assessment process often requires the use of models to extrapolate experimental data to humans. These models may be quantitative or qualitative. Quantitative models generally involve mathematical descriptions of dose response relationships which allow one to calculate numerical estimates of potential risk for a given exposure. Qualitative models, on the other hand, rely more on narrative descriptions of dose response relationships to describe the likelihood of an adverse effect for a given exposure. However both approaches are based on scientific judgments and scientifically based assumptions about dose response relationships and the predictive value of experimental data The scientific and regulatory communities have chosen a preference for quantitative models especially in the case of carcinogens. However the scientific and regulatory communities also consider qualitative models as an acceptable means of extrapolating animal data to humans. The No Observed Effect Level Uncertainty Factor ( NOEL UF) approach, described herein, is such a qualitative model As a matter of policy, OSHA has chosen to use the NOEL UF approach in describing the risks associated with exposure to glycol ethers. OSHA has chosen to use this qualitative approach because it is the most generally well accepted approach for assessing the risks from reproductive and developmental toxins. OSHA's decision to use the NOEL UF approach is based on agreement in the scientific community that this approach is the best methodology currently available for assessing reproductive health risks. This approach, in addition to its general acceptance in the scientific community, is also the methodology that has been consistently used by both EPA and FDA to assess reproductive health risks in their rulemaking procedures. As such it represents the best evidence available to OSHA for making its risk determinations. However while this is a policy choice it should be kept in mind that OSHA's decision to use the NOEL UF approach is a scientifically informed choice that is supported by scientific expertise and judgment. The selection of the NOEL UF approach, as well as the steps involved in the process ( e. g., the selection of the size of uncertainty factors to extrapolate from animals to humans) are choices based on underlying scientific data and assumptions to account for certain basic scientific uncertainties and are not choices borne solely from a public health perspective to provide a safe workplace in the face of scientific uncertainty B. Assessing the Risk of Developmental and Reproductive Effects Most OSHA risk assessments have focused on the risk of cancer from occupational exposure to toxic substances. In the case of carcinogen risk assessment, mathematical models are fit to dose response data, and the fitted models are used to make predictions of risk at a variety of doses. Although there are a number of mathematical models available to fit to carcinogen dose response data, within the risk assessment community in general, and the regulatory community in particular, a consensus exists as to which are the " best" models In the case of non carcinogen risk assessment, no such generally accepted mathematical models exist for predicting risks. The traditional approach to assessing the risk of noncancer effects has been first to make a qualitative determination that a toxic substance poses a risk of inducing an adverse effect and then to determine the level of exposure below which that adverse effect is unlikely to be induced in humans using an uncertainty or safety factor approach This approach is relatively simple. It is most often applied to experimental ( animal) data, but it can be applied to epidemiological data when such data are available. The first step in this approach is to determine whether an effect occurs in each exposure group at a rate which is statistically significantly elevated over the rate at which the effect occurs in the unexposed or control group. The highest exposure level which does not induce the effect at a statistically significantly elevated rate is called the no observed effect level or NOEL. In its most recent guidelines ( Ex. 5 153), the EPA uses the term NOAEL or no observed adverse effect level instead of NOEL, to make clear that effects being considered are of toxicological significance. For purposes of this document, NOEL is synonomous with NOAEL. The lowest exposure level which does induce the effect at a statistically significantly elevated rate is called the lowest observed effect level or LOEL. ( EPA also refers to this level as the LOAEL or Lowest Observed Adverse Effect Level. Again, for purposes of this document, LOEL and LOAEL are synonomous.) In this approach the NOEL is usually the value of interest, but a substance may induce an effect at a statistically significantly elevated rate at each exposure level under study. In that case, the LOEL becomes the value of interest. Determination of the NOEL and/ or the LOEL is the purpose of this first step The next step in this approach is to divide the NOEL or, in the absence of a NOEL, the LOEL by an uncertainty factor. Choice of the uncertainty factor will depend, in part, upon whether one uses the NOEL or the LOEL, and this is discussed at greater length below. The value NOEL Uncertainty Factor is termed the " acceptable daily intake" or ADI and is considered to represent the level of exposure at which humans are unlikely to experience an adverse effect. ( OSHA notes that for purposes of this document, the ADI is not to be interpreted as a regulatory limit, but rather as a health based level upon which regulatory considerations can be referenced.)

Although this approach requires only two steps, each step introduces uncertainty as to whether the final ADI estimate does indeed represent an exposure level below which an adverse effect is unlikely to be induced. Implicit in the uncertainty factor approach is the assumption that there is a threshold level of exposure below which a toxic [ page 15549]

response will not be induced, and the NOEL is an estimate of that threshold. There is debate, however, as to whether non cancer effects, in particular developmental effects, are indeed threshold phenomena. Brent, for example, has argued that teratogenesis " is by and large a threshold phenomena, which means that the vast majority of teratogenic agents have a ' no effect' dose." ( Ex. 5 126). He cites thalidomide as an example of a developmental toxin which if administered at 50 mg during the critical gestation period can effect a majority of embryos but which will have no effect on the development of embryos administered at 0.5 mg during the same period Others, however, maintain that not all developmental toxins have a threshold. Gaylor et al argue that " if a chemical produces a malformation by different mechanism than spontaneous malformations, then there is a possibility for a threshold dose. However, if a chemical produces a malformation by augmenting or accelerating an already existing mechanism that produces spontaneous malformations, then no population threshold can exist" ( Ex. 5 128). Rodricks et al maintain that " in cases in which the mechanisms of toxic or carcinogenic action are not understood, it is not possible to establish or reject the threshold hypothesis or no threshold hypothesis, at least with the degree of certainty usually sought in scientific proof. There are numerous reasons to believe that thresholds must exist . . ., but generalization to all agents and all effects is not possible" ( Ex. 5 130)

In its comments in response to OSHA's Advance Notice of Proposed Rulemaking ( ANPR), the Chemical Manufacturer's Association ( CMA) argues that acceptance of the existence of thresholds is central to evaluating reproductive and developmental risk ( Ex. 7 17). CMA bases its position in part on the " demonstrated regenerative, repair and regulation abilities of an embryo and fetus." In addition, CMA notes that fetuses are protected by maternal placenta and the metabolic processes of the pregnant female that break down, excrete, store, or otherwise inactivate chemicals before they can damage the embryo. CMA concludes that " to make appropriate decisions about potential human reproductive risks, OSHA must focus its attention on studies that determine the threshold below which adverse effects on the adult or the conceptus will not occur" ( Ex. 7 17) While OSHA believes it is likely that most chemically induced developmental effects have a threshold, it would seem that CMA is confusing the finding of a NOEL in an animal bioassay with the certainty a threshold exists. As noted by Rodricks et al, the existence of a NOEL from experimental data is consistent with the hypothesis of a threshold but is not sufficient to prove it ( Ex. 5 130). Furthermore, if a threshold does exist, there is little reason to believe that the NOEL is indeed the threshold as CMA implies. The exposure level at which no effect is observed is not only a function of the potency of the substance under test but also a function of the experimental design of a study. For example, an exposure level which is not tested cannot be a NOEL. If a researcher tests a substance at 10, 25, and 50 ppm, then the NOEL can only be 10, 25, or 50 ppm. As noted by Rodricks et al, "[ f] or practical reasons, only a few doses can be used in experimental studies. While these doses may fall above and below the true threshold doses, it is only by chance that any will precisely match the true threshold doses ( and this chance is very small)."

The exposure level found to be the NOEL in a study, ( and the exposure level found to be the LOEL in a study), will depend not only upon the exposure levels chosen by a researcher but also upon the numbers of animals in each exposure group. This is because exposure group size is an important factor in determining whether an observed excess of an effect is statistically significant. For example, suppose an experiment is run, and an effect is found to occur in 20% of the animals in the unexposed group. If there are 15 animals in each exposure group, then 60% of the animals exposed at some level X, ( 9 out of 15), must experience the effect in order to find that level X is the LOEL ( i. e. 60% is the lowest rate at which the effect can occur in order to be statistically significantly elevated at the p= 0.05 level over the 20% rate in the unexposed animals using a Fisher's Exact Test). If level X induces the effect in only 8 of the 15 exposed animals, then the rate for the effect in this exposure group will not be statistically significant If, in the example above, the number of animals in each exposure group were larger, then the proportion of exposed animals which must experience the effect to achieve statistical significance over the 20% rate in the unexposed group decreases. Thus, if there were 30 animals in each exposure group, then only 43.3% of the animals exposed to some level Y, ( 13 out of 30), must experience the effect in order to find that level Y is the LOEL. If there were 1000 animals in each exposure group, then only 23.2% of the animals exposed to some level Z, ( 232 out of 1000), must experience the effect in order to find that level Z is the LOEL It is clear from this example that the exposure levels determined to be the NOEL and LOEL will depend on study group size. The " true" NOEL may be lower than the NOEL determined for a particular study, but the study may not be sensitive enough to detect it. Few studies employ 1000 animals per group in their study design, and thus the direction of uncertainty due to sample size is towards overestimating the NOEL and LOEL; a response rate which is statistically significant for a small number of study animals will always be statistically significant for any larger number of animals Because small exposure group size and therefore lack of statistical power can lead to the erroneous conclusion that exposure induces no effect, the NOEL is not taken by itself to represent the " acceptable daily intake" ( ADI). Instead, the NOEL is adjusted by an uncertainty factor not only to account for uncertainties associated with the experimental design but also to account for uncertainties associated with extrapolation across species ( i. e. from experimental animals to humans) and to account for the variability of responses within a human population ( i. e. intra species variability)

In their chapter on risk assessment for effects other than cancer, Rodricks et al provide a brief history of the origins of the uncertainty factor ( Ex. 5 130). Referring to uncertainty factors as safety factors, these authors write:

The safety factor approach was originated by Lehman and Fitzhugh of the FDA who indicated that variability in sensitivity to chemicals ( expressed as differences in dose causing similar responses) across several species was usually in the range of two or threefold and did not appear to exceed tenfold. They also indicated that the variability among extensively outbred individuals and individuals of all ages and degrees of susceptibility ( e. g., persons in the general population) appeared also to be less than one order of magnitude. They consequently founded the 100 fold safety factor as a general method of dealing with the uncertainties of extrapolation. This incorporated a factor of 10 when extrapolating from animals to humans and an additional factor of 10 to account for differential sensitivities within the human population. When this 100 fold safety factor is applied to the highest experimental animal NOEL, it is considered to approximate a NOEL for humans in the general population, and becomes the ADI.

[ page 15550]

Since the concept of uncertainty factors was first introduced, it has been modified to derive an ADI from data of varying quality. For example, the FDA has expanded the original 100 fold uncertainty factor approach. When a NOEL is derived from subchronic animal data but that NOEL has been identified in two species, then the FDA recommends an uncertainty factor of 1000. Here, the additional factor of 10 is needed to account for the added uncertainty in estimating a chronic ADI from subchronic data. When a NOEL is derived from subchronic animal data but that NOEL has been identified in only one species, FDA recommends an uncertainty factor of 2000. The additional two fold factor is intended to account for possible interspecies differences ( Ex. 5 130)

If a NOEL can not be identified from study data, that is, if the lowest exposure level used in a study induces an effect at a rate statistically significantly greater than observed among the unexposed group, then the uncertainty factor is applied to the LOEL instead of the NOEL to derive the ADI. As with the NOEL, the uncertainty factor applied to the LOEL is used to account for the uncertainties and variability described above, but EPA recommends that an additional uncertainty factor, usually between one and 10, be used to account for the fact that no NOEL was identified from the data ( Ex. 5 131) Although the selection of uncertainty factors in the multiples of ten may appear to be arbitrary, there is some experimental support for their selection, and this is discussed at some length in an article by Dourson and Stara ( Ex. 4 113). ( The scientific basis underlying the selection and use of uncertainty factors is further discussed in OSHA Exhibit 5 155.) In addition, these choices of uncertainty factors as well as the entire uncertainty factor approach for non cancer health effects have been adopted by a number of governmental agencies and international organizations including the U. S. Environmental Protection Agency ( EPA), the U. S., Food and Drug Administration ( FDA), the Joint Food and Agricultural Organization of the World Health Organization ( FAO/ WHO), and the National Academy of Sciences ( NAS). The uncertainty factor approach for regulating occupational exposure to glycol ethers is supported by many of the commentors responding to OSHA's ANPR including CMA ( Ex. 7 17), DOW Chemical ( Ex. 7 21), and Du Pont ( Ex. 7 28), among others, although not all agree on the value of the uncertainty factor which should be used As CMA points out in its comments, the uncertainty factor approach " has been well established for regulating reproductive risks" ( Ex. 7 17). As noted above, the ADI represents an exposure level below which an adverse effect is unlikely, and confidence that the ADI is an exposure level below which an adverse effect is unlikely will depend, to a large extent, upon the quality of the data from which it is derived. If we know something of the mechanism which induces an effect and if we know that that mechanism is activated when exposure exceeds some threshold level, then our confidence that an adverse effect is unlikely at exposures levels at or below the ADI increases further C. Assessment of the Developmental Risk from Exposures to Glycol Ethers 1. Introduction According to the EPA Guidelines for Developmental Toxicity Risk Assessment ( Ex. 5 153), the major manifestations of developmental toxicity include 1) death of the developing organism; 2) malformations; 3) altered growth, and 4) functional deficiency. The studies used by OSHA for its assessment of developmental risk from glycol ethers employed a protocol exposing fetuses in utero during organogenesis, the phase of gestation during which the major organ systems develop. The pregnant dams were sacrificed at the end of this gestational phase and prior to giving birth. Each of the unborn fetuses was then examined. Under this protocol the endpoints of interest in these studies are the first three of the outcomes listed above The endpoint " death of the developing organism" includes resorptions and intra uterine deaths. Pre implantation loss is not a measure of developmental toxicity in these studies because the pregnant females were not exposed to any glycol ether until after implantation had occurred A malformation is usually defined as a permanent structural change that may adversely affect survival, development, or function. A malformation is different than a variation which is usually defined as a divergence beyond the usual range of structural constitution that may not adversely affect survival or health. It is not always possible, however, to distinguish between variations and malformations because, as noted by EPA in its Guidelines, " there exists a continuum of responses from the normal to extreme deviant." Furthermore, there is no generally accepted classification of malformations. Other terminology which is also used includes anomalies, deformations, and aberrations, but, as EPA points out, these terms are no better defined. Nonetheless, these effects indicate toxicity to the developing organism when associated with exposure to a chemical Altered growth is defined by EPA as an alteration in offspring organ or body weight or size. This endpoint may be reversible or may result in a permanent change As noted by the Interagency Regulatory Liaison Group ( IRLG) in its Workshop on Reproductive Toxicity Risk Assessment, " the developmental toxicity endpoints encountered in experimental animals do not and should not be expected necessarily to mimic those observed in humans exposed to the same toxicant" and " the specific agentrelated endpoints in humans are not always reproduced in experimental animals" ( Ex. 5 018). All substances known to cause developmental effects in humans, however, have also been found to induce developmental effects in animals with the exception of the coumarin anticoagulants which have not been studied extensively in animals ( Ex. 4 147). Schardein has compared the effects of all " known or possible" teratogens in humans with the teratogenic responses observed in laboratory animals exposed to these substances ( Ex. 4 147). Each of the developmental toxicants he looked at induced some developmental effect in at least one animal species, but only one class of substances, androgenic hormones, induced the same effect as observed in humans in each species which experienced an effect. Androgenic hormones have been tested in fourteen species, and only one species tested, sheep, experienced no effect The more common result in cross species testing of developmental toxicants can be found in the case of thalidomide which was found to induce limb defects ( i. e. missing limbs) in humans. In laboratory animals, the drug was found to induce developmental effects in seventeen species, but an effect concordant to the effect observed in humans was observed in only nine species. Furthermore, eight of these nine species, the rhesus monkey, the marmoset, the baboon, the bonnet monkey, the crab eating monkey, the green monkey, the Japanese monkey, and the stump tailed monkey, are not the usual animals used in animal bioassays. The rabbit was the sole rodent species to exhibit an effect concordant to the effect observed in humans ( Ex. 4 147)

The IRLG has noted that there is " no evidence that any particular species or [ page 15551]

strain more consistently predicts human susceptibility to animal teratogens than any other species or strain "( Ex. 5 018). This is borne out by Schardein ( Ex. 4 147). He found that the rabbit, which experienced an effect from thalidomide concordant to the effect induced humans, experienced no adverse developmental effects from alcohol or diethylstilbestrol ( DES), both known to cause birth defects in humans. The mouse experienced effects concordant to those in humans for a number of substances including alcohol, diethylstilbestrol, and antithyroid compounds, but neither aminopterin nor streptomycin, substances found to induce developmental effects in humans, induced any developmental effects in this species. Rats experienced adverse developmental effects from exposure to most of the toxicants considered by Schardein, ( rats, and mice were the most commonly used animals in tests of the toxicants considered by Schardein), but the effects were concordant with those in humans in only little more than half the substances and at least one substance considered by Schardein, trimethadione, induced no effect in this species The response to a developmental toxicant in animal bioassay can be measured in a number of ways. One of these is the number of fetuses affected per number of fetuses exposed. This shall be referred to as the " fetus measure of response". While this measure gives some indication of the potency of a developmental toxicant, it treats each fetus independently of all other fetuses thereby ignoring the " litter effect". The litter effect is the tendency for littermates to respond more like each other than like animals from different litters. Furthermore, the fetus measure cannot distinguish between the case where all litters have one or two affected fetuses and the case where all affected fetuses are from only one or two litters, although these two scenarios have different implications for the potency of a developmental toxicant An alternative measure of response is number of litters with at least one affected fetus per total number of litters exposed, referred to as the " litter measure of response". This measure treats the litters as the experimental unit because, as noted by EPA in its Guidelines, it is the maternal animal and not the conceptus which is treated during gestation ( Ex. 5 153). This is the measure of response favored by EPA for evaluating the potency of a developmental toxicant. The drawback to this measure, however, is that it gives equal weight to a litter with one affected fetus as it gives to a litter with all affected fetuses In addition to both of these measures, a third measure which OSHA has considered for evaluating response in animals exposed to developmental toxicants is average number of fetuses affected per affected litter. This shall be referred to as the " fetus/ litter measure of response". This measure provides a compliment to the fetus measure of response and the litter measure of response, for, whereas the former indicates only the number of fetuses affected and the latter indicates only the number of litters affected, the fetus/ litter measure provides an indication of how severe an effect may be within an affected litter. For example, a fetus/ litter value of 1.0 would indicate that only one fetus was affected in each of the affected litters. A fetus/ litter value of 2.0 would indicate that on average, two fetuses were affected in each of the litters with affected fetuses. Comparison of fetus/ litter values across exposure groups would allow one to determine whether more fetuses were affected in each affected litter as dose increases. The limitation of this measure, however, is that unlike the other two measures discussed above, the fetus/ litter measure of response has utility only as a descriptive measure and can not be used for statistical inference because the statistical distribution of this measure is unknown 2. Choice of Data a. 2 ME OSHA has identified three well conducted animal bioassays for 2 ME which are suitable for assessing the risk of developmental effects from occupational exposure to this glycol ether and for determining the acceptable daily intake or ADI. ( As noted earlier for purposed of this document, the ADI is not a regulatory limit but rather a health based level which describes the level at which humans are unlikely to exhibit effects similar to those obseved in experimental data.) These studies were chosen because in each of these studies, exposure levels were documented, the routes of exposure were the same as is found in most occupational settings ( i. e. inhalation), concurrent controls were used, two or more exposure levels of the test substance were employed, statistically significant excesses of developmental [ page 15552]

effects were observed in exposed groups, and individual litter data were available Hanley and associates of the Dow Chemical Company conducted three animal inhalation bioassays for 2 ME using female rats, rabbits, and mice ( Exs. 4 042a and 4 106). Groups of 30 to 31 bred Fisher 344 rats and 20 to 30 bred New Zealand white rabbits were exposed to 2 ME at levels of 3, 10, or 50 ppm. Groups of 30 to 32 bred CF 1 mice were exposed to 2 ME at levels of 10 or 50 ppm. The test article was supplied by Dow and was 99.96% pure. Thirty bred rats, 30 bred rabbits, and 31 bred mice served as controls The female rats were bred one to one with male rats of the same strain. The female mice were bred two to one with male mice of the same strain ( two females to one male), and the rabbits were bred through artificial insemination. Animals were randomly assigned to exposure groups. Exposure occurred six hours per day through the organogenesis phase of gestation: from day 6 through day 15 of gestation for rats and mice and from day 6 through day 18 gestation for rabbits. All animals were given food and water ad libitum except during periods of exposure and were observed daily throughout the experimental period for indications of toxicity and adverse effects of treatment Animals found dead or moribund during the course of the study were submitted for gross pathological examination. Surviving mice were sacrificed on day 18 of gestation, surviving rats were sacrificed on day 21 of gestation, and surviving rabbits were sacrificed on day 29 of gestation. Caesarean sections and examinations were preformed on all animals to determine: ( 1) the number and position of fetuses in utero; ( 2) the number of live and dead fetuses; ( 3) the number and position of resorption sites; ( 4) the sex, and body weight, and crown rump length of each fetus; and ( 5) any gross external alternations. In addition, the rats and the rabbits were examined for number of corpora lutea. The uteri of apparently non pregnant animals were stained and examined for evidence of implantation sites to determine whether pregnancy had occurred. One half of each litter was dissected and examined for soft tissue alternations. All fetuses were examined for skeletal alternations b. 2 EE OSHA has identified two well conducted animal inhalation bioassays for 2 EE which are suitable for assessing the risk of developmental effects from occupational exposure to this glycol ether and for determining the acceptable daily intake or ADI. As with 2 ME, both of these studies were chosen because in each, exposure levels were documented, the routes of exposure were the same as is found in most occupational settings ( i. e. inhalation), concurrent controls were used, two or more exposure levels of the test substance were employed, statistically significant excesses of developmental effects were observed in exposed groups, and individual litter data were available Tinston, Doe and associates of Imperial Chemical Industries PLC conducted two animal inhalation studies for 2 EE using rats and rabbits ( Exs. 4 038 and 4 039; see also Ex. 5 071). These studies were sponsored by the Chemical Manufacturer's Association ( CMA) and followed a protocol similar to the one used by Hanley et al. Groups of 24 bred rats of the Alpk/ AP ( Wistar derived) strain were exposed to 2 EE at levels of 10, 50, or 250 ppm. Groups of 24 bred Dutch rabbits were exposed to 2 EE at levels of 10, 50, and 175 ppm. The test article was supplied by Imperial Chemical Industries and was more than 99% pure. Twenty four bred rats and 24 bred rabbits served as controls The female rats were bred one to one with male rats of the same strain, and female rabbits were bred with 2 male rabbits of the same strain. Animals were randomly assigned to exposure groups. Exposure occurred six hours per day throughout the organogenesis phase of gestation: from day 6 through day 15 of gestation for the rats and from day 6 through day 18 of gestation for the rabbits. All animals were given food and water ad libitum except during periods of exposure and were observed daily for their clinical condition Terminal sacrifice of the animals occurred on day 21 of gestation for the rats and day 29 of gestation for the rabbits. After sacrifice, the number of corpora lutea in each animal's ovaries was counted. The uterus of each animal was cut open and the number of implantations as well as the number of early and late intra uterine deaths was determined. An intra uterine death was judged to be late if fetal tissues were distinguishable. Each fetus which had not died in utero was removed from the uterus. These fetuses were weighed and examined for gross defects. Half of the rat fetuses and all of the rabbit fetuses were examined for skeletal defects. All fetuses of both species were examined for external and visceral defects 3. Bioassay Results a. 2 ME In measuring the incidence of effects of 2 ME in fetal rats, rabbits, and mice, Hanley et al grouped the effects into three categories: external alterations, soft tissue alterations, and skeletal alterations. Each of these categories of defects was subdivided further into major defects and minor defects. The study authors provided no explanation as to the criteria used to subdivide these categories, and one must assume it was professional judgement ( Exs. 4 047 and 4 106)

Table VI A presents the incidence of developmental effects in fetal rats exposed to 2 ME. Incidence is reported using each of the measures of response discussed above ( i. e. fetus, litter and fetus/ litter). The only effects presented in this discussion are those which occurred in any exposed group at a rate statistically significantly greater than the rate in the unexposed group at the p= 0.05 level using either the fetus measure of response or the litter measure of response. Statistical significance was determined using Fisher's Exact Test Table VI A Incidence of Developmental Effects Observed in Fisher 344 Rats Exposed to 2 ME Days 6 through 15 of Gestation ( 1)

MINOR SKELETAL ALTERATIONS  Control 3 ppm 10 ppm 50 ppm Fetus ( 2) 4/ 287 3/ 283 6/ 293 19/ 307( 3)

Delayed Ossification Litters ( 4) 4/ 29 3/ 28 5/ 28 13/ 30( 5)

of Centra Fetus/ Litter ( 6) 1.00 1.00 1.20 1.46 Fetus 18/ 287 13/ 283 20/ 293 57/ 307( 3)

Rib Spurs Litters 12/ 29 10/ 28 13/ 28 26/ 30( 3)

Fetus/ Litter 1.50 1.30 1.54 2.19 Fetus 125/ 287 142/ 283 131/ 293 97/ 307( 3)

Delayed Ossification Litters 28/ 29 27/ 28 27/ 28 28/ 30 of Sternebrae Fetus/ Litter 4.46 5.26 4.85 3.40 1  Data from Hanley et al, Ex. 4 042a.

2  Incidence is number of fetuses affected divided by the total number of fetuses 3  Significantly different than controls at the p< .01 level 4  Incidence is number of litters with at least one fetus affected 5  Significantly different than controls at the p< .05 level 6  Average number of affected fetuses per affected litter Delayed ossification of the centra and rib spurs are the two developmental effects which occurred at a rate statistically significantly greater in an exposed group than in the controls. Both of these effects were classified as minor skeletal alterations. Both effects were elevated for the 50 ppm group only, but incidence was significant at the p= 0.012 level or lower regardless of measure of response, and the fetus/ litter measure of response increases with dose. Delayed ossification of the sternebra was significantly reduced for the 50 ppm group when measured using the fetus measure of response, but it was not significant using the litter measure of response and the fetus/ litter measure does not show a dose related trend. The authors attribute the observed deficit of delayed ossification of the sternebra to normal variation and not to exposure to 2 ME Table VI B presents the incidence of developmental effects in fetal rabbits exposed to 2 ME. The same measures of incidence presented for the rats are presented for the rabbits, and the same statistical criteria were used for inclusion of an effect in the table [ page 15553]

Table VI B Incidence of Developmental Effects Observed in New Zealand White Rabbits Exposed to 2 ME Days 6 through 18 of Gestation ( 1)

Control 3 ppm 10 ppm 50 ppm Resorptions: Fetus( 2) 7/ 180 14/ 186 23/ 210( 3) 46/ 191( 3)

Litters( 4) 5/ 23 10/ 24 14/ 24( 5) 16/ 24( 3)

Fetus/ Litter( 6) 1.40 1.40 1.64 2.88 MAJOR EXTERNAL ALTERATIONS( 7)

Arthrogryposis:

Fetus 0/ 173 1/ 172 0/ 187 54/ 145( 3)

Litters 0/ 23 1/ 23 0/ 24 15/ 22( 3)

Fetus/ Litter 0.00 1.00 0.00 3.60 Anonychia:

Fetus 0/ 173 0/ 172 0/ 187 14/ 145( 3)

Litters 0/ 23 0/ 23 0/ 24 6/ 22( 3)

Fetus/ Litter 0.00 0.00 0.00 2.33 Brachydactyly:

Fetus 0/ 173 0/ 172 0/ 187 6/ 145( 3)

Litters 0/ 23 0/ 23 0/ 24 4/ 22( 5)

Fetus/ Litter 0.00 0.00 0.00 1.50 Ectrodactyly:

Fetus 0/ 173 0/ 172 0/ 187 6/ 145( 3)

Litters 0/ 23 0/ 23 0/ 24 4/ 22( 5)

Fetus/ Litter 0.00 0.00 0.00 1.50 Omphalocele:

Fetus 0/ 173 0/ 172 0/ 187 5/ 145( 5)

Litters 0/ 23 0/ 23 0/ 24 2/ 22 Fetus/ Litter 0.00 0.00 0.00 2.50 Thinning of Adominal Wall:

Fetus 0/ 173 0/ 172 0/ 187 6/ 145( 3)

Litters 0/ 23 0/ 23 0/ 24 3/ 22 Fetus/ Litter 0.00 0.00 0.00 2.00 Kinky Tail:

Fetus 0/ 173 0/ 172 0/ 187 4/ 145( 5)

Litters 0/ 23 0/ 23 0/ 24 4/ 22( 5)

Fetus/ Litter 0.00 0.00 0.00 1.00 MINOR EXTERNAL ALTERATIONS Misalignment of Palatine Rugae:

Fetus 0/ 173 0/ 174 0/ 187 27/ 145( 3) Litters 0/ 23 0/ 23 0/ 24 11/ 22( 3)

Fetus/ Litter 0.00 0.00 0.00 2.45 Narrowed tip of Tail:

Fetus 0/ 173 0/ 172 0/ 187 6/ 145( 3)

Litters 0/ 23 0/ 23 0/ 24 6/ 22( 3)

Fetus/ Litter 0.00 0.00 0.00 1.00 MAJOR SOFT TISSUE ALTERATIONS f Coarctation of the Aortic Arch:

Fetus 0/ 95 0/ 93 0/ 101 13/ 80( 3)

Litters 0/ 23 0/ 23 0/ 24 6/ 22( 3)

Fetus/ Litter 0.00 0.00 0.00 2.17 Ventricular Septal Defect:

Fetus 0/ 95 0/ 93 0/ 101 34/ 80( 3)

Litters 0/ 23 0/ 23 0/ 24 15/ 22( 3)

Fetus/ Litter 0.00 0.00 0.00 2.27 Hypoplastic Spleen:

Fetus 0/ 95 0/ 93 0/ 101 25/ 80( 3)

Litters 0/ 23 0/ 23 0/ 24 13/ 22( 3)

Fetus/ Litter 0.00 0.00 0.00 1.92 Dilated Renal Pelvis:

Fetus 0/ 95 1/ 93 1/ 101 28/ 80( 3)

Litters 0/ 23 1/ 23 1/ 24 14/ 22( 3)

Fetus/ Litter 0.00 1.00 1.00 2.00 Patent Ductus Arteriosis:

Fetus 0/ 95 1/ 93 0/ 101 5/ 80( 5)

Litters 0/ 23 1/ 23 0/ 24 4/ 22( 5)

Fetus/ Litter 0.00 1.00 0.00 1.25 Hypoplastic Gall Bladder:

Fetus 0/ 95 0/ 93 0/ 101 4/ 80( 5)

Litters 0/ 23 0/ 23 0/ 24 4/ 22( 5)

Fetus/ Litter 0.00 0.00 0.00 1.00 Pale Spleen:

Fetus 4/ 95 2/ 93 1/ 101 30/ 80( 3)

[ page 15554] Litters 1/ 23 1/ 23 1/ 24 16/ 22( 3)

Fetus/ Litter 4.00 2.00 1.00 1.88 Dilated Ureter:

Fetus 0/ 95 0/ 93 0/ 101 7/ 80( 3)

Litters 0/ 23 0/ 23 0/ 24 6/ 22( 3)

Fetus/ Litter 0.00 0.00 0.00 1.17 Convoluted Ureter:

Fetus 3/ 95 2/ 93 0/ 101 12/ 80( 3)

Litters 2/ 23 2/ 23 0/ 24 8/ 22( 5)

Fetus/ Litter 1.50 1.00 0.00 1.50 Paroavarian Cyst:

Fetus g 0/ 48 1/ 47 1/ 53 9/ 40( 3)

Litters h 0/ 23 1/ 21 1/ 24 8/ 22( 3)

Fetus/ Litter 0.00 1.00 1.00 1.13 Testicular Cyst:

Fetus g 0/ 47 0/ 46 0/ 48 5/ 40( 5)

Litters i 0/ 22 0/ 23 0/ 23 3/ 22 Fetus/ Litter 0.00 1.00 1.00 1.13 Shortened Nasals, Maxillae and Mandibles:

Fetus 0/ 173 0/ 172 0/ 187 6/ 145( 3) Litters 0/ 23 0/ 23 0/ 24 1/ 22 Fetus/ Litter 0.00 0.00 0.00 6.00 MAJOR SKELETAL ALTERATIONS e Missing Palange( s):

Fetus 0/ 173 0/ 172 0/ 187 7/ 145( 3)

Litters 0/ 23 0/ 23 0/ 24 4/ 22( 5)

Fetus/ Litter 0.00 0.00 0.00 1.75 Missing Metacarpal( s):

Fetus 0/ 173 0/ 172 0/ 187 4/ 145( 5)

Litters 0/ 23 0/ 23 0/ 24 2/ 22 Fetus/ Litter 0.00 0.00 0.00 2.00 Missing Metatarsal( s):

Fetus 0/ 173 0/ 172 0/ 187 9/ 145( 3)

Litters 0/ 23 0/ 23 0/ 24 4/ 22( 5)

Fetus/ Litter 0.00 0.00 0.00 2.25 Shortened Ribs:

Fetus 0/ 173 0/ 172 0/ 187 4/ 145( 5)

Litters 0/ 23 0/ 23 0/ 24 2/ 22 Fetus/ Litter 0.00 0.00 0.00 2.00 Enlarged Interparietals:

Fetus 0/ 173 0/ 172 0/ 187 5/ 145( 5)

Litters 0/ 23 0/ 23 0/ 24 1/ 22 Fetus/ Litter 0.00 0.00 0.00 5.00 Delayed Ossification of the Hyoid:

Fetus 1/ 173 2/ 172 2/ 187 14/ 145( 3)

Litters 1/ 23 2/ 23 2/ 24 8/ 22( 3)

Fetus/ Litter 1.00 1.00 1.00 1.75 Delayed Ossification of the Tarsals( s):

Fetus 0/ 173 3/ 172 1/ 187 17/ 145( 3)

Litters 0/ 23 3/ 23 1/ 24 8/ 22( 3)

Fetus/ Litter 0.00 1.00 1.00 2.13 Extra Lumbar Ribs:

Fetus 0/ 173 0/ 172 0/ 187 13/ 145( 3)

Litters 0/ 23 0/ 23 0/ 24 7/ 22( 3)

Fetus/ Litter 0.00 0.00 0.00 1.86 Shortened Lumbar Ribs:

Fetus 0/ 173 0/ 172 0/ 187 4/ 145( 5)

Litters 0/ 23 0/ 23 0/ 24 2/ 22 Fetus/ Litter 0.00 0.00 0.00 1.86 Delayed Ossification of the Centra:

Fetus 4/ 173 2/ 172 0/ 187 11/ 145( 5)

Litters 4/ 23 2/ 23 0/ 24 ( 5) 8/ 22 Fetus/ Litter 1.00 1.00 0.00 1.38 Delayed Ossification of the Sternebrae:

Fetus 82/ 173 93/ 172 123/ 187( 3) 127/ 145( 3)

Litters 23/ 23 23/ 23 24/ 24 22/ 22 Fetus/ Litter 3.57 4.04 5.13 5.77 Fused Sternebrae:

Fetus 3/ 173 2/ 172 0/ 187 11/ 145( 5)

Litters 3/ 23 2/ 23 0/ 24 8/ 22 [ page 15555] Fetus/ Litter 3.57 4.04 5.13 1.33 Sternebrae  Extra Site of Ossification: Fetus 0/ 173 0/ 172 0/ 187 4/ 145( 5)

Litters 0/ 23 0/ 23 0/ 24 3/ 22 Fetus/ Litter 3.57 4.04 5.13 1.33 1  Data from Hanley et al, Ex. 4 042a 2  Incidence is number of fetuses affected divided by the total number of fetuses 3  Significantly different than controls at the p< .01 level. 4  Incidence is number of litters with at least one fetus affected 5  Significantly different than controls at the p< .05 level. 6  Average number of affected fetuses per affected litter. 7  Denominator ( i. e. the number of animals at risk) is adjusted for resorptions 8  Only a portion of fetuses in each exposure group were examined for soft tissue alterations. 9  Denominator not specified by study authors. Number of fetuses at risk estimated by applying male/ female ratio for each exposure group to number of fetuses examined for soft tissue alterations 10  Denominator is number of litters with at least one female fetus. 11  Denominator is number of litters with at least on male fetus.

Incidence of resorptions was statistically significantly increased using both the fetus and the litter measures of response for the 10 ppm and the 50 ppm group. The fetus/ litter measure of incidence shows a dose related increase. The study authors note that although resorptions are significantly elevated for the 10 ppm group, the observed rate for fetuses ( 11%) and for litters ( 58%) are comparable to the historical incidence of resorptions observed in other studies in the same laboratory ( 7% to 15% for fetuses and 38% to 74% for litters). They attribute the finding of statistical significance of resorptions in this exposure group to the unusually low control group incidence of resorptions ( 4% for fetuses and 22% for litters) and not to exposure Major external alterations in rabbits occurred at a significant excess in the 50 ppm group only. Incidence of arthrogryposis ( abnormal flexure of the forelimbs), anonychia ( absence of nails), brachydactyly ( short digits), ectrodactyly ( absence of part or all of a digit), and kinky tail occurred at a statistically significantly elevated rate for both the fetus measure of response and the litter measure of response. Incidence of omphalocele ( protrusion of the intestines through the abdominal wall), and thinning of the abdominal wall was significant for fetuses but not for litters As with the major external alterations, the minor external alterations, misalignment of the palatine rugae and narrowed tip of tail, occurred at a significant excess only in the 50 ppm group. Both were statistically significant using both the fetus and the litter measure of response What is most striking about these data is that every external alteration observed, major or minor, was observed only in the 50 ppm group except for one fetus in the 3 ppm group observed with arthrogryposis. The almost total absence of background incidence of these effects reduces the uncertainty as to whether the response in the 50 ppm group could be attributed to chance variation rather than exposure to 2 ME While all the fetal rabbits were examined for external alterations, only half were examined for soft tissue alterations. Thus, the study had less power to detect this type of developmental effect. Nonetheless, a large number of soft tissue alterations were observed at a significant excess in 50 ppm group. Incidence of these effects was not significantly elevated over controls in any other exposure group. The major soft tissue alterations were coarctation of the aortic arch, ventricular septal defect, hypoplastic spleen, and dilated renal pelvis, and incidence of these effects in the 50 ppm group were statistically significant using both the fetus and the litter measure of response at the p= 0.009 level or lower. All but one minor soft tissue alteration occurred at a statistically significant rate in the 50 ppm group using both measures of response These alterations were patent ductus arteriosis, hypoplastic gall bladder, pale spleen, dilated ureter, convoluted ureter, and parovarian cysts. Testicular cysts were statistically significant in male fetal rabbits in the 50 ppm group using the fetus measure but not using the litter measure. Here again, the almost total absence of any of these effects in the control group, the 3 ppm group, or the 10 ppm group lends further support to 2 ME as the cause for these effects in the 50 ppm group All fetuses were examined for skeletal alterations, yet incidence of all but two of these alterations was significantly elevated in the 50 ppm group only. The major skeletal alterations which occurred at a significantly elevated rate in this group using both measures of response were missing phalanges and missing metatarsal; the minor skeletal alterations were delayed ossification of the hyoid, delayed ossification of the tarsals, and extra lumbar ribs. Incidence of shortened nasals, maxillae and mandibles and shortened ribs, both major external alterations, were significantly elevated in the 50 ppm group also but only when measured in fetuses. Likewise, incidence of enlarged interparietals, shortened lumbar ribs, delayed ossification of the centra, fused sternebra, and extra site of sternebra ossification, all minor external alterations, were significantly elevated in the 50 ppm group but again, only for the fetus measure of response. There was a statistically significant deficit of delayed ossification of the centra in litters in the 10 ppm group, this most likely is attributable to chance variation and not to exposure. Delayed ossification of the sternebra was significantly elevated over controls in fetuses in the 10 ppm and the 50 ppm exposure groups, but the incidence of this effect measured in litters at both exposure levels was the same ( 100%) as in the control and 3 ppm exposure group Exposure to 2 ME did not have as strong an effect in fetal mice as it did in fetal rabbits. Table VI C presents the results of the mouse bioassay. Criteria for inclusion of an effect in the table in the same as was used for both rats and rabbits. Only one effect, extra lumbar ribs, a minor skeletal alteration, was significantly elevated using both the fetus and the litter measure of response, and this was in the 50 ppm group. [ page 15556]

Incidences of resorption, hypoplastic testicle, and extra site of sternebra ossification in the 50 ppm group were significantly elevated over controls but only in fetuses and not in litters. Incidence of testicular hemorrhage and delayed ossification of the sternebra was significantly elevated over controls in the 10 ppm group and the 50 ppm using the fetus measure of response, but incidence of these effects was not significant when response was measured in litters Table VI C Incidence of Developmental Effects Observed in CF 1 Mice Exposed to 2 ME days 6 through 15 of Gestation ( 1)

Control 10 ppm 50 ppm Resorptions:

Fetus b 25/ 342 25/ 285 35/ 286( 3)

Litters c 16/ 26 14/ 23 18/ 24 Fetus/ Litter d 1.56 1.79 1.94 MINOR SOFT TISSUE ALTERATIONS e Hypoplastic Testicle:

Fetus f 2/ 86 3/ 64 8/ 66( 3)

Litters g 2/ 26 3/ 23 6/ 22 Fetus/ Litter 1.00 1.00 1.33 Testicular Hemorrhage:

Fetus f 2/ 86 3/ 64 8/ 66( 3)

Litters g 2/ 26 3/ 23 6/ 22 Fetus/ Litter 1.00 1.00 1.33 MINOR SKELETAL ALTERATIONS h Extra Lumbar Rib:

Fetus 48/ 317 49/ 260 82/ 251( 10)

Litters 14/ 26 14/ 23 21/ 24( 3)

Fetus/ Litter 3.43 3.50 3.90 Delayed Ossification of the Sternebrae:

Fetus 76/ 317 43/ 260 ( 3) 77/ 251( 3) Litters 18/ 26 13/ 23 21/ 24 Fetus/ Litter 3.43 3.50 3.90 Sternebrae  Extra Site of Ossification:

Fetus 21/ 317 9/ 260 8/ 251( 3)

Litters 9/ 26 6/ 23 4/ 24 Fetus/ Litter 3.43 3.50 3.90 ________________________________________________________________________ ___

1  Data from Hanley et al, Ex. 4 106. 2  Incidence is number of fetuses affected divided by the total number of fetuses 3  Significantly different than controls at the p< .05 level. 4  Incidence is number of litters with at least one fetus affected 5  Average number of affected fetuses per affected litter. 6  Only a portion of fetuses in each exposure group were examined for soft tissue alterations 7  Denominator not specified by study authors. Number of fetuses at risk estimated by applying male/ female ratio for each exposure group to number of fetuses examined for soft tissue alterations 8  Denominator is number of litters with at least on male fetus. 9  Denominator ( i. e. the number of animals at risk) is adjusted for resorptions 10  Significantly different than controls at the p< .01 level b. 2 EE In their studies of the developmental effects of 2 EE on fetal rats and fetal rabbits, Tinston, Doe et al, ( Exs. 4 038 and 4 039; see also Ex. 5 071), classified effects differently than Hanley et al. Abnormalities which were deemed either rare or lethal or both were classified as major external and visceral defects or major skeletal defects while those defects which were judged to be small changes that would not normally impair survival and that occur at a moderate to low frequency in the strain were classified as minor external and visceral defects or minor skeletal defects. A third classification, variant, was used to describe those defects which are common in the species and are not normally deleterious Another difference between the 2 EE bioassays and the 2 ME bioassays is that the investigators in the 2 EE bioassays considered much more specific effects than did the investigators in the 2 ME bioassays. For example, Tinston et al looked at the degree of ossification ( i. e. partially ossified or not ossified) of each centrum and sternebra in each fetus examined. Thus, the study authors report the incidence of partial ossification of the first sternebra, partial ossification of the second sternebra, and so forth. In the 2 ME bioassays, on the other hand, Hanley et al grouped any ossification defect of any sternebra into the category " delayed ossification of the sternebra" and any ossification defect of any centrum into the category " delayed ossification of the centra." The different approaches for measuring the incidence of effects have implications for the inferences which can be drawn from analysis of the study results, and these implications will be discussed in the next section Table VI D presents the incidence of developmental effects in fetal rats exposed to 2 EE. As with the results from the 2 ME bioassays, incidence is reported using the fetus, litter, and fetus/ litter measures of response, and only those effects which occurred in any exposed group at a statistically significantly greater rate than in control using either the fetus or the litter measure of response are included.

[ page 15557]

Again, statistical significance was determined using Fisher's Exact Test with a critical value of p= 0.05.

Table VI D Incidence of Developmental Effects Observed in Wistar Rats Exposed to 2 EE days 6 through 15 of Gestation ( a)

Control 10 ppm 50 ppm 250 ppm All Intra uterine Deaths:

Fetus ( b) 16/ 297 22/ 277 12/ 261 32/ 266 **

Litters ( c) 12/ 23 15/ 24 10/ 22 12/ 21 Fetus/ Litter ( d) 1.33 1.47 1.20 2.67 Late Intra uterine Deaths:

Fetus ( e) 3/ 284 3/ 258 2/ 251 17/ 251 **

Litters 2/ 23 2/ 24 2/ 22 7/ 21 *

Fetus/ Litter 1.50 1.50 1.00 2.43 MINOR SKELETAL ANOMALIES ( f) Skull  Partially Ossified Frontals:

Fetus 1/ 147 0/ 131 4/ 129 14/ 122 **

Litters 1/ 23 0/ 24 4/ 22 7/ 21 *

Fetus/ Litter 1.00 0.00 1.00 2.00 Skull  Partially Ossified Parietal:

Fetus 10/ 147 1/ 131 ** 15/ 129 35/ 122 **

Litters 6/ 23 1/ 24 * 8/ 22 17/ 21 **

Fetus/ Litter 1.67 1.00 1.88 2.06 Skull  Partially Ossified Interparietal:

Fetus 26/ 147 3/ 131 ** 24/ 129 40/ 122 **

Litters 10/ 23 3/ 24 * 12/ 22 17/ 21 *

Fetus/ Litter 2.60 1.00 2.00 2.35 Skull  Odontoid Not Ossified:

Fetus 26/ 147 19/ 131 32/ 129 93/ 122 **

Litters 13/ 23 10/ 24 16/ 22 20/ 21 **

Fetus/ Litter 2.00 1.90 2.00 4.65 Cervical centrum # 3 Not Ossified:

Fetus 14/ 147 21/ 131 25/ 129 * 115/ 122 **

Litters 6/ 23 9/ 24 12/ 22 * 21/ 21 **

Fetus/ Litter 2.33 2.33 2.08 5.48 Cervical Centrum # 4 Not Ossified:

Fetus 10/ 147 7/ 131 16/ 129 109/ 122 **

Litters 5/ 23 4/ 24 11/ 22 * 21/ 21 **

Fetus/ Litter 2.00 1.75 1.45 5.19 Cervical Centrum # 5 Not Ossified:

Fetus 2/ 147 5/ 131 9/ 129 * 98/ 122 **

Litters 1/ 23 3/ 24 6/ 22 * 21/ 21 ** Fetus/ Litter 2.00 1.67 1.50 4.67 Cervical Centrum # 6 Not Ossified:

Fetus 1/ 147 2/ 131 6/ 129 * 84/ 122 **

Litters 1/ 23 2/ 24 4/ 22 20/ 21 **

Fetus/ Litter 1.00 1.00 1.50 4.20 Cervical Centrum # 7 Not Ossified:

Fetus 0/ 147 0/ 131 2/ 129 26/ 122 **

Litters 0/ 23 0/ 24 2/ 22 10/ 21 **

Fetus/ Litter 0.00 0.00 1.00 2.60 Thoracic Centrum # 8 Partially Ossified:

Fetus 0/ 147 0/ 131 0/ 129 6/ 122 **

Litters 0/ 23 0/ 24 0/ 22 6/ 21 **

Fetus/ Litter 0.00 0.00 0.00 1.00 Thoracic Centrum # 9 Partially Ossified:

Fetus 0/ 147 1/ 131 0/ 129 7/ 122 **

Litters 0/ 23 1/ 24 0/ 22 5/ 21 *

Fetus/ Litter 0.00 1.00 0.00 1.40 Thoracic Centrum # 10 Partially Ossified:

Fetus 0/ 147 0/ 131 0/ 129 18/ 122 **

Litters 0/ 23 0/ 24 0/ 22 12/ 21 **

Fetus/ Litter 0.00 0.00 0.00 1.50 Thoracic Centrum # 11 Partially Ossified:

Fetus 5/ 147 0/ 131 1/ 129 19/ 122 **

Litters 5/ 23 0/ 24 1/ 22 10/ 21 Fetus/ Litter 1.00 0.00 1.00 1.90 Thoracic Centrum # 12 Partially Ossified:

Fetus 2/ 147 1/ 131 4/ 129 17/ 122 ** Litters 2/ 23 1/ 24 4/ 22 10/ 21 **

Fetus/ Litter 1.00 1.00 1.00 1.70 [ page 15558]

Thoracic Centrum # 13 Partially Ossified:

Fetus 1/ 147 2/ 131 3/ 129 12/ 122 **

Litters 1/ 23 2/ 24 3/ 22 8/ 21 **

Fetus/ Litter 1.00 1.00 1.00 1.50 Lumbar Centrum # 1 Partially Ossified:

Fetus 0/ 147 0/ 131 1/ 129 9/ 122 **

Litters 0/ 23 0/ 24 1/ 22 6/ 21 **

Fetus/ Litter 0.00 0.00 1.00 1.50 Lumbar Traverse Process Partially Ossified  4th Right:

Fetus 0/ 147 5/ 131 1/ 129 6/ 122 **

Litters 0/ 23 5/ 24 1/ 22 5/ 21 *

Fetus/ Litter 0.00 1.00 1.00 1.20 Lumbar Traverse Process Partially Ossified  4th Both:

Fetus 0/ 147 3/ 131 0/ 129 4/ 122 *

Litters 0/ 23 2/ 24 0/ 22 3/ 21 Fetus/ Litter 0.00 1.50 0.00 1.33 Sternebra # 1 Partially Ossified:

Fetus 4/ 147 2/ 131 4/ 129 35/ 122 **

Litters 3/ 23 2/ 24 4/ 22 14/ 21 **

Fetus/ Litter 1.33 1.00 1.00 2.50 Sternebra # 2 Partially Ossified:

Fetus 1/ 147 2/ 131 6/ 129 * 10/ 122 **

Litters 1/ 23 2/ 24 5/ 22 7/ 21 *

Fetus/ Litter 1.00 1.00 1.20 1.43 SKELETAL VARIANTS Sternebra # 6 Partially Ossified:

Fetus 0/ 147 0/ 131 1/ 129 8/ 122 **

Litters 0/ 23 0/ 24 1/ 22 3/ 21 Fetus/ Litter 0.00 0.00 1.00 2.67 Sternebra # 4 Misaligned:

Fetus 1/ 147 0/ 131 4/ 129 6/ 122 *

Litters 1/ 23 0/ 24 4/ 22 5/ 21 Fetus/ Litter 1.00 0.00 1.00 1.20 Sternebra # 5 Misaligned:

Fetus 1/ 147 0/ 131 2/ 129 6/ 122 *

Litters 1/ 23 0/ 24 2/ 22 4/ 21 Fetus/ Litter 1.00 0.00 1.00 1.50 Sternebra # 1 Bipartite:

Fetus 0/ 147 0/ 131 0/ 129 9/ 122 **

Litters 0/ 23 0/ 24 0/ 22 8/ 21 **

Fetus/ Litter 0.00 0.00 0.00 1.13 Skull  Partially Ossified Occipitals:

Fetus 141/ 147 113/ 131 124/ 129 122/ 122 *

Litters 23/ 23 23/ 24 22/ 22 21/ 21 Fetus/ Litter 6.13 4.91 5.64 5.81 cervical Centrum # 1 Not Ossified:

Fetus 28/ 147 40/ 131 * 48/ 129 ** 94/ 122 **

Litters 13/ 23 15/ 24 17/ 22 21/ 21 **

Fetus/ Litter 2.15 2.67 2.82 4.48 Cervical Centrum # 2 Not Ossified:

Fetus 52/ 147 63/ 131 * 80/ 129 ** 118/ 122 ** Litters 17/ 23 22/ 24 21/ 22 21/ 21 *

Fetus/ Litter 3.06 2.86 3.81 5.62 Extra ( 14th) Rib  Unilateral Left Short:

Fetus 5/ 147 7/ 131 4/ 129 15/ 122 **

Litters 5/ 23 7/ 24 3/ 22 8/ 21 Fetus/ Litter 1.00 1.00 1.33 1.88 Extra ( 14th) Bilateral Short:

Fetus 14/ 147 12/ 131 33/ 129 ** 75/ 122**

Litters 10/ 23 6/ 24 15/ 22 18/ 21 **

Fetus/ Litter 1.40 2.00 2.22 4.17 Pelvic Girdle Moved Posteriorly  27 Pre Sacral Vertebrae ( g):

Fetus 1/ 147 3/ 131 2/ 129 15/ 122 **

Litters 1/ 23 2/ 24 2/ 22 7/ 21 *

Fetus/ Litter 1.00 1.50 1.00 2.14 Both Calcaneum Not Ossified:

Fetus 136/ 147 115/ 131 119/ 129 122/ 122 **

Litters 23/ 23 24/ 24 22/ 22 21/ 21 Fetus/ Litter 5.91 4.79 5.41 5.81 [ page 15559]

MINOR EXTERNAL AND VISCERAL DEFECTS 10 Renal Pelvic Dilation:

Fetus 19/ 281 25/ 255 22/ 249 30/ 234 *

Litters 12/ 23 14/ 24 12/ 22 18/ 21 *

Fetus/ Litter 1.58 1.79 1.83 1.67 Hydroureter:

Fetus 13/ 281 6/ 255 4/ 249 * 10/ 234 Litters 5/ 23 6/ 24 3/ 22 5/ 21 Fetus/ Litter 2.60 1.00 1.33 2.00 Limb Malrotation:

Fetus 0/ 281 9/ 255 ** 2/ 249 3/ 234 Litters 0/ 23 4/ 24 1/ 22 2/ 21 Fetus/ Litter 0.00 2.25 2.00 1.50 a  Data from Tinston, Doe et al, Exs. 4 038. See also Ex. 5 071 b  Incidence is number of fetuses affected divided by the total number of fetuses c  Incidence is number of litters with at least one fetus affected d  Average number of affected fetuses per affected litter e  Denominator ( i. e. the number of animals at risk) is adjusted for early intra uterine deaths f  Only a portion of fetuses in each exposure group were examined for soft tissue alterations g  This skeletal defect was not classified as either a minor skeltal anomalie or a skeletal variant h  Denominator ( i. e. the number of animals at risk) is adjusted for all intra uterine deaths *  Significantly different than controls at the p <. 05 level **  Significantly different than controls at the p <. 01 level A significant excess of late intra uterine deaths occurred in the 250 ppm group using both the fetus and the litter measures of response, but when combined with early intra uterine deaths ( i. e. all intra uterine deaths) the effect is significant for the 250 ppm group in fetuses only. Early intra uterine deaths considered separately were not found to be related to exposure Most of the minor skeletal defects which occurred at a significantly elevated rate were also in the 250 ppm group. The effects which are significant using both measures of response were partially ossified frontals, partially ossified parietals, partially ossified interparietals, odontoid not ossified, third, fourth, fifth, sixth, and seventh cervical centra not ossified, eighth ninth, tenth, eleventh, twelfth, and thirteenth thoracic centra partially ossified, first lumbar centrum partially ossified, fourth right lumbar traverse process partially ossified, and first and second sternebrae partially ossified. One minor skeletal defect, fourth right lumbar traverse process partially ossified, was significant for fetuses in the 250 ppm group but not for litters Two minor skeletal defects were significant for the 50 ppm group using both measures of response: third cervical centrum not ossified and fifth cervical centrum not ossified. The minor skeletal defects of unossified sixth cervical centrum and partially ossified second sternebra shows a significant excess in this exposure group but only for fetuses. The incidence of unossified fourth cervical centrum was significantly elevated over controls in the 50 ppm group for litters but not for fetuses Two minor skeletal defects showed a significant deficit of occurrence in the 10 ppm group. Using both the fetus and the litter measure of response, study results show that fetal rats exposed to 10 ppm of 2 EE were significantly less likely to experience partially ossified parietals or partially ossified interparietals than were controls. The study authors offer no explanation for this, but given the large number of effects for which each fetus was examined, the statistical significance of this deficit can easily be attributed to chance variation A number of skeletal variants were observed to be associated with exposure in fetal rats. Those which were found to be statistically significantly elevated over controls using both the fetus and the litter measures of response, bipartite first sternebra, unossified first cervical centrum, and extra ( 14th) rib  bilateral short, were found only in the 250 ppm group. Interestingly, incidence of unossified first and second cervical centra were also significantly elevated in the 10 ppm group and the 50 ppm group when measured in fetuses but not when measured in litters. The fetus/ litter measure shows a dose related trend for ossified first cervical centrum but not for unossified second cervical centrum. The effect extra ( 14th) rib  bilateral short showed a showed a significant excess in 50 ppm fetuses but not in 50 ppm litters There were an additional number of skeletal variants which were statistically significant in the 250 ppm group but only when response was measured in fetuses. These effects were partially ossified sixth sternebra, misaligned fifth sternebra, partially ossified occipital and extra ( 14th) rib  unilateral ( left) short, and although these effects were not significant when measured in litters, when measured in fetuses these effects were significant at the P= 0.035 level or lower Three minor external and visceral defects were found to be statistically significant. Renal pelvic dilation occurred at a significantly elevated rate in fetuses and in litters in the 250 ppm group. Incidence of hydroureter was significantly reduced in the 50 ppm fetuses but in no other group of fetuses and in no group of litters. Incidence of limb malrotation was significantly elevated in the 10 ppm group of fetuses but in no other group of fetuses and in no groups of litters There were two skeletal defects which occurred at a statistically significant rate in the 250 ppm group which were not classified. " Pelvic girdle moved posteriorly ( 27 pre sacral vertebrae)" was not categorized as either a major or minor skeletal defect or as a variant. This effect was significant in both fetuses and litters. Likewise, " both calcaneum not ossified" was not classified as either a major or minor skeletal defect or as a variant. This effect was significant only in fetuses in the 250 ppm group. [ page 15560]

The incidence of developmental effects in fetal rabbits exposed to 2 EE are presented in Table VI E. As for fetal rats, three measures of response are presented, and the same statistical criteria were used for inclusion of an effect in the table Table VI E Incidence of Developmental Effects Observed in Dutch Rabbits Exposed to 2 EE days 6 through 18 of Gestation ( a)

MINOR SKELETAL DEFECTS ( b)

Control 10 ppm 50 ppm 175 ppm Skull  Partially Ossified Hyoid:

Fetus ( c) 15/ 136 32/ 138 ** 12/ 96 28/ 134 *

Litters ( d) 8/ 21 9/ 20 5/ 16 11/ 22 Fetus/ Litter ( e) 1.88 3.56 2.40 2.55 27 Pre sacral Vertebrae:

Fetus 3/ 136 7/ 138 5/ 96 31/ 134 **

Litters 3/ 21 4/ 20 2/ 16 10/ 22 *

Fetus/ Litter 1.00 1.75 2.50 3.10 6th Sternebra Partially Ossified:

Fetus 2/ 136 1/ 138 2/ 96 8/ 134 *

Litters 2/ 21 1/ 20 2/ 16 6/ 22 Fetus/ Litter 1.00 1.00 1.00 1.33 5th Sternebra Not Ossified:

Fetus 9/ 136 20/ 138 * 9/ 96 13/ 134 Litters 5/ 21 7/ 20 6/ 16 5/ 22 Fetus/ Litter 1.80 2.86 1.00 2.00 Pelvic Girdle  Pubes Not Ossified:

Fetus 0/ 136 1/ 138 1/ 96 7/ 134 *

Litters 0/ 21 1/ 20 1/ 16 4/ 22 Fetus/ Litter 0.00 1.00 1.00 1.75 Extra ( 13th) Rib  Unilateral Short:

Fetus 3/ 136 7/ 138 7/ 96 15/ 134 **

Litters 3/ 21 6/ 20 7/ 16 10/ 22 *

Fetus/ Litter 1.00 1.17 1.00 1.50 Extra ( 13th Rib  Bilateral Normal:

Fetus 10/ 136 17/ 138 7/ 96 38/ 134 **

Litters 5/ 21 7/ 20 3/ 16 13/ 22 *

Fetus/ Litter 2.00 2.43 2.33 2.92 Extra ( 13th) Rib  Bilateral One Normal One Short:

Fetus 4/ 136 5/ 138 6/ 96 16/ 134 **

Litters 4/ 21 4/ 20 4/ 16 9/ 22 Fetus/ Litter 1.00 1.25 1.50 1.78 5th Sternebra Partially Ossified:

Fetus 53/ 136 61/ 138 45/ 96 53/ 134 Litters 14/ 21 20/ 20 ** 14/ 16 18/ 22 Fetus/ Litter 3.79 3.05 3.21 2.94 a  Data from Tinston, Doe et al, Exs. 4 039. See also Ex. 5 071 b  Only a portion of fetuses in each exposure group were examined for soft tissue alterations c  Incidence is number of fetuses affected divided by the total number of fetuses d  Incidence is number of litters with at least one fetus affected e  Average number of affected fetuses per affected litter *  Significantly different than controls at the p< .05 level **  Significantly different than controls at the p< .01 level Only one skeletal defect, 27 pre sacral vertebrae, which Tinston et al classified as minor in rabbits, was statistically significant in fetuses and litters and this was in the 175 ppm group. Three minor skeletal defects, partially ossified hyoid, partially ossified sixth sternebra, and unossified pubes, were significant in fetuses in the 175 ppm group but not in litters. The defect partially ossified hyoid occurred at a significantly elevated rate in 10 ppm fetuses but was not significant in this group when measured in litters. The same result was seen for the defect unossified fifth sternebra which was significant in fetuses in the 10 ppm group but not in litters and not in any other exposure group The 175 ppm group had statistically significant excess of two skeletal variants when measured in fetuses and in litters: extra ( 13th) rib  unilateral short and extra ( 13th) rib  bilateral normal. The skeletal variant extra ( 13th) rib  one normal and one short was significantly elevated in fetuses in the 175 ppm group but was not significant in litters One skeletal variant was significantly elevated in the 10 ppm group. This variant was partially ossified fifth sternebra. Incidence was significant only when measured in litters but not when measured in fetuses and was not significant for any other exposure group using any measure of response. Incidence of partially ossified fifth sternebra does not show a dose related trend using the fetus/ litter measure of response, and the study authors attribute the observed excess to coincidence because similar increases in this variant were not observed in the 50 and 175 ppm groups.

[ page 15561]

4. Derivation of the No Observed Effect Level a. 2 ME In reviewing Table VI A, VI B, and VI C, it is clear that almost all effects which occurred in an exposed group at rates statistically significantly elevated over controls occurred in the highest dose group, the 50 ppm 2 ME dose group, in each species. Only one effect, resorptions, was statistically significant in litters at a dose below 50 ppm, but, as noted by the study authors, the rate of resorptions in exposed rabbits fell well within the range of historical controls It is apparent from these data that 10 ppm is the no observed effect level ( NOEL) in each of the species studied by Hanley et al despite the varying sensitivity of each of the species to 2 ME Still, the question arises as to how the incidence of effects can or should be combined to arrive at a measure of overall response Intuitively, one would have greater confidence in a NOEL derived from an overall measure of response rather than one derived from specific effects which may be statistically significant due to chance alone OSHA proposes that an overall measure of the incidence of developmental effects be arrived at by pooling the incidence of effects which occurred at a statistically significant excess in any exposed group when measured in litters. The Agency has chosen litters as the most appropriate unit of measure for a number of reasons. First, as noted above, it is the pregnant female that is exposed to the test substance, therefore it is her litter which is the affected unit. Because dose is administered to the fetus through the pregnant dam, the dose each fetus receives is unknown and may depend upon the individual sensitivity of the mother animal to the test substance. Furthermore, the dose the fetus does receive may be affected by the number of littermates in utereo Another reason for prefering the litter as the unit of measure is that fetuses in a litter are more likely to respond like each other than like fetuses from other litter ( i. e. the litter effect). Therefore, if an effect is observed in some number of fetuses, but all affected fetuses come from only one or two litters, then it is possible to attribute the effect to exposure when in fact it is due to variation among the mother animals The use of litters as the unit of measure in studies of developmental effects in animals is recommended by the EPA in its Guidelines ( Ex. 5 153), and this unit of measure enjoys wide support in the literature ( see, for example, Ex. 5 018)

OSHA's decision to include in an overall measure of response only those effects which are statistically significant when considered individually is based on its belief that by so restricting inclusion of an effect in an overall measure one obtains a more accurate measure of overall response. Inclusion of effects which are not dose related and are attributable solely to chance dilutes the overall measure of the potency of a developmental toxicant This position is easily illustrated. Hanley et al counted all the rabbit litters in the control and exposed groups which had at least one fetus with any major malformation. The incidence of major malformations was found to be 6/ 23 in controls ( 26%), 4/ 23 in the 3 ppm group ( 17%), 3/ 24 in the 10 ppm group ( 13%) and 20/ 22 in the 50 ppm group ( 91%). If only those major malformations which were statistically significant in exposed litters had been included, the overall incidence of major malformations would have been 0/ 23 in the control group ( 0%), 2/ 23 in the 3 ppm group ( 13%) 1/ 24 in the 10 ppm group ( 4%) and 20/ 22 in the 50 ppm group ( 91%). While the NOEL is 10 ppm regardless of how the overall incidence of major malformations is measured, the effect attributable to exposure at 50 ppm is clearer and starker when those major malformations attributable to chance are excluded Tables VI F, VI G, and VI H present the overall incidence of developmental effects in rats, rabbits, and mice, respectively. For rabbits, resorptions were not included in the overall measure of response because the rate for all three exposure groups ( 42% to 67%) was well within the historical range reported by Hanley et al ( mean 55%, range 38% to 74%) and only the rate of resorption among controls ( 22%) was outside of that range. The tables clearly indicate that 2 ME induces developmental effects in all three species at 50 ppm and that the NOEL for all three species in these studies is 10 ppm Table VI F Overall Incidence of Developmental Effects Observed in Litters of Fisher 344 Rats Exposed to 2 ME Days 6 through 15 of Gestation ( a)

Control 3 ppm 10 ppm 50 ppm Minor Skeletal Alterations( b):

Litters 14/ 29 12/ 28 16/ 28 27/ 30 **

a  Data from Hanley et al, Ex. 4 042a b  The numerator is the number of litters with at least one fetus presenting either delayed ossification of centra or rib spurs; the denominator is the number of litters at risk **  Significantly different than controls at the p < .01 level Table VI G Overall Incidence of Developmental Effects Observed in Litters of New Zealand White Rabbits Exposed to 2 ME Days 6 through 15 of Gestation ( a)

Control 3 ppm 10 ppm 50 ppm Major External Alterations( b)

Litters 0/ 23 1/ 23 0/ 24 16/ 22 **

Minor External Alterations( c)

Litters 0/ 23 0/ 23 0/ 24 15/ 22 **

Major Soft Tissue Alterations( d)

Litters 0/ 23 1/ 23 1/ 24 20/ 22 **

Minor Soft Tissue Alterations( e)

Litters 3/ 23 5/ 23 2/ 22 18/ 22 **

Major Skeletal Alterations( f)

Litters 0/ 23 0/ 23 0/ 24 5/ 22 *

[ page 15562]

Minor Skeletal Alterations( g) Litters 1/ 23 4/ 23 2/ 24 13/ 22 **

Any Major Alterations( h)

Litters 0/ 23 2/ 23 1/ 24 20/ 22 **

Any Minor Alterations( i)

Litters 4/ 23 7/ 23 4/ 24 20/ 22 **

Any Alterations( j)

Litters 4/ 23 7/ 23 5/ 24 20/ 22 **

a  Data from Hanley et al, Ex. 4 042a b  The numerator is the number of litters with at least one fetus presenting any of the following major external alterations: arthrogryposis, anonychia, brachydactyly, ectrodactyly, or kinky tail; the denominator is the number of litters at risk c  The numerator is the number of litters with at least one fetus presenting any of the following minor external alterations: misalignment of the palatine rugae or narrowed tip of tail; the denominator is the number of litters at risk d  The numerator is the number of litters with at least one fetus presenting any of the following major soft tissue alterations: coarctation of the aortic arch, ventricular septal defect, hypoplastic spleen, or dilated renal pelvis; the denominator is the number of litters at risk e  The numerator is the number of litters with at least one fetus presenting any of the following minor soft tissue alterations: patent ductus arteriosis, hypoplastic gall bladder, pale spleen, dilated ureter, convoluted ureter, or parovarian cyst; the denominator is the number of litters at risk f  The numerator is the number of litters with at least one fetus presenting any of the following major skeletal alterations: missing phalange or missing metatarsal; the denominator is the number of litters at risk g  The numerator is the number of litters with at least one fetus presenting any of the following minor skeletal alterations: delayed ossification of the hyoid, delayed ossification of the tarsal, or extra lumbar ribs; the denominator is the number of litters at risk h  The numerator is the number of litters with at least one fetus presenting any major external alterations, any major soft tissue alteration or any major skeletal alterations listed above; the denominator is the number of litters at risk i  The numerator is the number of litters with at least one fetus presenting any minor external alterations, any minor soft tissue alteration or any minor skeletal alterations listed above; the denominator is the number of litters at risk j  The numerator is the number of litters with at least one fetus presenting any major or minor external alterations, soft tissue alteration or skeletal alterations listed above; the denominator is the number of litters at risk *  Significantly different than controls at the p < .05 level **  Significantly different than controls at the p < .01 level Table VI H Overall Incidence of Developmental Effects Observed in Litters of CF 1 mice Exposed to 2 ME days 6 through 15 of Gestation ( a)

Control 10 ppm 50 ppm Minor Skeletal Alterations( b)

Litters 14/ 26 14/ 23 21/ 24 *

a  Data from Hanley et al, Ex. 4 106 b  The numerator is the number of litters with at least one fetus presenting extra lumbar ribs; the denominator is the number of litters at risk *  Significantly different than controls at the p< .05 level In its comments to OSHA's ANPR, Du Pont states that it is " appropriate to look at all adverse effects, but not to accumulate these effects into one endpoint" ( Ex. 7 28). OSHA seeks additional information on how the approach suggested by Du Pont could be used for quantitative risk assessment. Specifically, it is unclear how different NOELs for different endpoints from the same study would be treated. In addition, OSHA seeks comment on whether adverse developmental effects should be combined, and if so, how this should be done Given that the NOEL for 2 ME has been derived from animal studies, OSHA believes that an uncertainty factor of 100 is appropriate for derivation of the acceptable daily intake ( ADI) i. e. the dose at which humans are unlikely to exhibit effects similar to those observed in animals. An uncertainty factor of 100 provides a factor of 10 for inter species variability and a factor of 10 for intra species variability ( i. e. individual human sensitivity to 2 ME). Therefore, based on the studies of Hanley et al, OSHA estimates the ADI to be 10 ppm/ 100 or 0.1 ppm. That is, at 0.1 ppm of 2 ME, humans are unlikey to exhibit effects similar to those observed in animals b. 2 EE Table VI D and VI E show clearly that exposure to 2 EE had an effect on developing animals although the results of the 2 EE bioassay are not as strong as those from the 2 ME bioassay. While most of the developmental effects observed in the 2 EE bioassay were observed in the high dose groups for both rats and rabbits ( 250 ppm and 175 ppm respectively), there were statistically significant effects in the 50 ppm group of rats and the 10 ppm group of rabbits. Thus, determination of the NOEL for 2 EE is more difficult than for 2 ME Table VI I presents the overall incidence of developmental effects in fetal rats exposed to 2 EE. Only those effects which were statistically significant when measured in litters are included in the overall measure of incidence.

[ page 15563]

Table VI I Overall Incidence of Developmental Effects Observed in Litters of Wistar Rats Exposed to 2 EE Days 6 through 15 of Gestation ( a)

Control 10 ppm 50 ppm 250 ppm Late Intra Uterine Deaths ( b)

Litters 2/ 23 2/ 24 2/ 22 7/ 21 *

Minor Skeletal Defects ( c)

Litters 17/ 23 21/ 24 20/ 22 21/ 21 *

Skeletal Variants ( d)

Litters 20/ 23 23/ 24 22/ 22 21/ 21 Minor External and Visceral Defects ( a)

Litters 12/ 23 14/ 24 12/ 22 18/ 21 *

Unclassified Defects ( f)

Litters 1/ 23 2/ 24 2/ 22 7/ 21 *

Any Death, Defect or Variant ( g)

Litters 23/ 23 23/ 24 22/ 22 21/ 21 a  Data from Tinston, Doe et al, Ex. 4 038a b  The numerator is the number of litters with at least one late intra uterine death; the denominator is the number of litters at risk c  The numerator is the number of litters with at least one fetus presenting any of the following minor skeletal defects: frontal partially ossified, parietal partially ossified, interparietal partially ossified, odontoid not ossified, third, fourth, fifth, sixth or seventh cervical centrum not ossified, eighth, ninth, tenth, twelfth, or thirteenth thoracic centrum partially ossified, first lumbar centrum partially ossified, fourth right lumbar traverse process partially ossified, or first or second sternebra partially ossified; the denominator is the number of litters at risk d  The numerator is the number of litters with at least one fetus presenting any of the following skeletal variants: first sternebra bipartite, first or second cervical centrum not ossified, or extra ( 14th) rib bilaterally short; the denominator is the number of litters at risk e  The numerator is the number of litters with at least one fetus presenting renal pelvic dilation; the denominator is the number of litters at risk f  The numerator is the number of litters with at least one fetus presenting a posteriorly moved pelvic girdle ( 27 pre sacral vertebrae); the denominator is the number of litters at risk. The defect " pelvic girdle moved posteriorly" was not classified as a major or minor external and visceral defect, a major or minor skeletal defect, or a skeletal defect. Thus it is " unclassified." g  The numerator is the number of litters with at least one fetus dying late in utero or presenting any minor skeletal defect, skeletal variant, minor external or visceral defect, or unclassified defect listed above; the denominator is the number of litters at risk *  Significantly different than controls at the p< .05 level The surprising result in table VI I is that when all the effects are combined, it would appear that 2 EE has no effect on developing rats despite the fact that there is a significant excess of late intra uterine deaths in the 250 ppm group, a significant excess of minor skeletal defects in the 250 ppm group, and a significant excess of external and visceral defects in the 250 ppm group. Furthermore, Table VI D shows that there were twenty four effects that were significantly elevated in the 250 ppm group in both litters and fetuses The reason the overall incidence of effects in fetal rats exposed to 2 EE is not significant is that the study authors looked at the developmental effects in such minute detail. While this results in there being a large number of effects which are statistically significant in the 250 ppm group when each effect is considered individually, when combined the incidence of these effects in the other exposure groups dilutes the association between dose and response Examination of the data reveals that the incidence of non ossification of the cervical centra is one effect which is diluting the overall dose response relationship. When ossification of each of the seven cervical centra is considered individually, it is clear that exposure to 250 ppm of 2 EE in fetal rats results in non ossification of each of the centrum. Incidence of non ossification of each of the centrum in the 250 ppm group is statistically significant at the p=. 014 level or lower. If all the centra were combined into a category " delayed ossification of the cervical centra", however, this effect would not be statistically significant when measured in litters. The litter incidence of delayed ossification of any cervical centrum is 19/ 23 in control ( 83%), 23/ 24 in the 10 ppm group ( 96%), 21/ 22 in the 50 ppm group ( 95%), and 21/ 21 in the 250 ppm group ( 100%). Thus, when each centrum is considered individually, there is clearly a dose related effect at the 250 ppm level, but when the centra are considered together, this effect disappears. Because of the high background incidence of the effect and the small number of litters in each group, the overall incidence of developmental effects in the 250 ppm group appears to be unrelated to dose OSHA does not believe that there is no developmental effect on fetal rats exposed to 2 EE. When the incidence of late intra uterine deaths is considered, when the overall incidence of minor skeletal defects is considered and when the overall incidence of minor external and visceral defects are considered, it is clear that exposure to 250 ppm of 2 EE results in detrimental effects on the developing fetus. No such effect is seen at 50 ppm or lower, and therefore OSHA concludes that the NOEL for 2 EE is 50 ppm in rats The results presented in Table VI J demonstrate that 50 ppm is also the NOEL for fetal rabbits exposed to 2 EE. The overall incidence of developmental effects is statistically significant for the 175 ppm group at the p= 0.004 level. This indicates that exposure below the 250 ppm level, the level observed to have an adverse effect on developing rats, can have an adverse effect on developing rabbits Although fewer effects were observed in the rabbit at 175 ppm than in the rat at 250 ppm, dose related adverse effects were nonetheless induced at the 175 ppm level.

Table VI J Overall Incidence of Developmental Effects Observed in Litters of Dutch Rabbits Exposed to 2 EE Days 6 through 18 of Gestation ( a)

Control 10 ppm 50 ppm 175 ppm Minor Skeletal Defects ( b)

Litters 3/ 21 4/ 20 2/ 16 10/ 22 *

[ page 15564] Skeletal Variants ( c)

Litters 7/ 21 10/ 20 9/ 16 18/ 22 **

Any Defect or Variant ( d)

Litters 8/ 21 11/ 20 9/ 16 18/ 22 **

a  Data from Tinston, Doe et al, Ex. 4 039a b  The numerator is the number of litters with at least one fetus presenting 27 pre sacral vertebrae; the denominator is the number of litters at risk c  The numerator is the number of litters with at least one fetus presenting an extra ( 13th) rib unilaterally short or bilaterally normal; the denominator is the number of litters at risk d  The numerator is the number of litters with at least one fetus presenting any minor skeletal defect or skeletal variant listed above; the denominator is the number of litters at risk *  Significantly different than controls at the p< .05 level **  Significantly different than controls at the p< .01 level Given, as with 2 ME, that the NOEL for 2 EE has been derived from animal studies, OSHA believes that an uncertainty factor of 100 is again appropriate for derivation of the ADI. As with 2 ME, an uncertainty factor of 100 provides a factor of 10 for inter species variability and a factor of 10 for intra species variability. Therefore, based on the studies by Tinston et al, OSHA estimates the ADI to be 50 ppm/ 100 or 0.5 ppm. That is, at 0.5 ppm of 2 EE, humans are unlikely to exhibit adverse effects similar to those observed in animals 5. Alternative Uncertainty Factors As discussed in a previous section, an ADI ( i. e the levels at ewhich humans are unlikely to exhibit effects similar to those observed in humans) is derived by dividing a NOEL by an uncertainty factor. To derive ADIs for glycol ethers, OSHA has used an uncertainty factor of 100; a factor of ten to account for inter species variability and a factor of 10 to account for intra species variability. Several of the commentors who responded to OSHA's ANPR for glycol ethers advocated use of a lower uncertainty factor ( see, for example, Kodak, Ex. 7 16, and Du Pont, Ex. 7 28). In its comments, CMA argues that an uncertainty factor well below 100 should be used to arrive at new PELs ( Ex. 7 17). While CMA presents a number of reasons for its position, OSHA does not find any of its reasons sufficiently compelling to depart from the traditional uncertainty factor for derivation of ADIs across species. What follows here is a brief description of each of the arguments presented by CMA and OSHA's response to these arguments To begin, CMA states that lower safety factors are appropriate with high quality animal studies. OSHA agrees that the Hanley et al studies and the Tinston et al studies are high quality studies, but the Agency does not see how the quality of these studies reduces either inter species variability or intra species variability. Both types of variability are issues in determining a " safe" occupational exposure level from animal data even when such studies are of high quality CMA argues that lower safety factors are appropriate when NOELs have been established in more than one species. CMA notes that not only have NOELs been established for a number of species but the NOELs and the observed effects have also been quite similar across species While OSHA agrees that one has greater confidence in a NOEL determined in more than one species, the Agency is concerned the CMA has confused the finding of the same NOEL in more than one species with the finding of the same threshold in more than one species. The exposure levels employed by Hanley et al and by Tinston et al were almost identical in each of the species tested. In the case of 2 ME, for example, rats and rabbits were exposed at identical exposure levels and mice were exposed to two of the three same levels. Given this similarity of exposure, it is not surprising that the same NOEL was identified in all three species. One cannot conclude from this, however, that the exposure threshold for developmental effects is the same in all three species. Based on the data from the Hanley et al bioassays, for example, one cannot reject a hypothesis that the " true" NOELs might be 40 ppm for mice, 25 ppm for rats, and 10 ppm for rabbits. The exposure levels employed do not allow one to reject this hypothesis, thus one cannot conclude there is no inter species variability for 2 ME. The same is true for 2 EE. In addition, the small number of animals used in each of these bioassays, ( no study used more than 30 animals in each exposure group and most used less), limited the power of these bioassays to detect lower NOELs. For these reasons, OSHA cannot support the use of a lower safety factor on these grounds as advocated by CMA In addition, OSHA does not agree that the effects observed across species exposed to the same glycol ethers were quite as similar as CMA maintains. In the case of 2 ME, the only effects observed to occur at a statistically significantly greater rate in the high dose groups of rats and mice were minor skeletal defects. In high dose rabbits, however, the effects were far and away more severe including major external alterations, major soft tissue alterations, and major skeletal alterations. In the case of 2 EE, it is difficult to compare effects across species because of differences in exposure levels used for the high dose group CMA  s third reason for advocating an uncertainty factor well below 100 is that data on inter species metabolism and pharmacokinetics can be employed to justify this. As discussed in the Health Effects section of this preamble, the evidence indicates that the four glycol ethers of interest here are most likely metabolized via the alcohol dehydrogenase ( ADH) pathway in both animals and humans. Although all species seem to share the same metabolic pathway, however, there is also evidence that the species do not metabolize glycol ethers at the same rate. Groeseneken et al found that the biological half life of 2 EE in humans was 21 to 24 hours compared to the biological half life of 8 to 12 hours reported in animals ( Exs. 5 112, 5 113, and 5 114). This finding led the study authors to conclude that the metabolites of 2 EE will not be cleared from the urine by the next morning following exposure and accumulation of the metabolites may be expected through repetitive exposures. In a study of 2 EEA in humans, Groesenken et al found [ page 15565]