HALOHYDANTOINS Toxicology Chapter TABLE OF CONTENTS 1.0 HAZARD CHARACTERIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2.0 REQUIREMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 3.0 DATA GAPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4.0 HAZARD ASSESSMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4.1 Acute Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4.2 Subchronic Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 4.3 Prenatal Developmental Toxicity 10 4.4 Reproductive Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 4.5 Chronic Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 4.6 Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 4.7 Mutagenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 4.8 Neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 4.9 Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 5.0 TOXICITY ENDPOINT SELECTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 5.1 See Section 9.2 for Endpoint Selection Table. . . . . . . . . . . . . . . . . . . . . . . . . . . 27 5.2 Dermal Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 5.3 Classification of Carcinogenic Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 6.0 FQPA CONSIDERATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 6.1 Special Sensitivity to Infants and Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 6.2 Recommendation for a Developmental Neurotoxicity Study . . . . . . . . . . . . . . . 27 7.0 ENDOCRINE DISRUPTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 8.0 OTHER ISSUES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 9.0 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 10.0 APPENDICES 10.1 Toxicity Profile Summary Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 10.1.1 Acute Toxicity Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 10.1.2 Subchronic, Chronic and Other Toxicity Tables . . . . . . . . . . . . . . . . . . . 32 10.2 Summary of Toxicological Dose and Endpoints . . . . . . . . . . . . . . . . . . . . . . . . 38 Page 3 of 41 1.0 HAZARD CHARACTERIZATION The hydantoins are a group of chemicals composed of several halogenated and nonhalogenated compounds. This group of chemicals includes the following: 1 Bromo 3 chloro 5,5 dimethylhydantoin, 1.3 Dibromo 5,5 dimethylhydantoin, 1,3 Dichloro 5,5 dimethylhydantoin, 1,3 Dichloro 5 ethyl 5 methylhydantoin, 1,3 bis( hydroxymethyl) 5,5 dimethylhydantoin, and 1 hydroxymethyl) 5,5 dimethylhydantoin. In addition to these four hydantoins, the Agency determined that the 5,5 Dimethylhydantoin and 5,5 Ethylmethylhydantoin metabolites of the halogenated hydantoins are appropriate test substances for assessing the toxicity of the hydantoins. However, since the hydroxymethylhydantoins as mentioned above have the potential for release of formaldehyde, the risks associated with this release need to be assessed. The Agency has determined that the risks from exposure to formaldehyde via the hydroxymethylhydantoins will be addressed when the active ingredient formaldehyde is assessed for reregistration eligibility. Therefore, this reregistration eligibility decision ( RED) document assesses the eligibility of the halogenated hydantoins and their metabolites for reregistration.

These antimicrobial chemicals are registered for use in indoor food, indoor non food, indoor residential, aquatic non food residential, aquatic food, aquatic non food, and aquatic non food industrial sites for control of bacteria, fungi, and algal slimes.

The acute toxicity of dimethylhydantoin is low by the oral and dermal routes of exposure ( Toxicity categories III and IV, respectively). Acute lethality by the inhalation route is more significant ( Toxicity category II). The dimethylhydantoins are significant eye and skin irritants ( Toxicity category I and II, respectively). Positive dermal sensitization has also been observed.

Non acute toxicity testing of dimethylhydantoins ( including subchronic, developmental, reproductive, and chronic toxicity testing) all show the presence of non specific toxicity only at relatively high doses of the test chemical. Developmental and reproductive toxicity data demonstrate no increase in susceptibility to the toxic effects of dimethylhydantoin with the exception of one study, where fetal and litter effects ( increased incidence of 27th presacral vertebrae) in rabbits were observed at a lower dose level than that which resulted in maternal toxicity ( decreased body weight and food consumption during the dosing period) following treatment. In a prenatal developmental toxicity study conducted in rabbits with ethylmethylhydantoin, there was no increased susceptibility of the fetuses observed.

Chronic toxicity testing of dimethylhydantoins in rats showed a decrement in body weight and body weight gain in females and increased mortality, particularly in females. Males showed increased incidences of hyperplasia of submandibular lymph nodes and testicular fibrinoid vascular degeneration in early decedents. Both sexes showed increased mammary galactoceles in early decedents and enlarged pituitary glands. In two chronic toxicity studies conducted in dogs, dimethylhydantoin produced slight ( enlarged pituitary glands) to no toxicity at or above the limit dose of 1000 mg/ kg/ day. Page 4 of 41 Cancer studies in rats and mice indicated no systemic effects other than decreased body weight and body weight gains in females ( rats) and males ( mice) and increased hyperplasia of submandibular lymph nodes in males ( rats). No evidence of tumorigenicity of the test material was reported. 5,5 dimethylhydantoin is classified as ` not likely' to be a carcinogen based upon the negative evidence for carcinogenicity in both the rat and mouse studies as well as the negative evidence of mutagenicity.

The data on mutagenicity of dimethylhydantoins shows, in large part, negative responses in the studies conducted. Literature reports indicate a positive effect for 2 in vitro mammalian cytogenetic assays in Chinese Hamster Ovary cells.

Available metabolism data indicate that the dimethylhydantoins are excreted unchanged in the rat. However, it is known that methylhydantoins are formaldehyde releasers. Therefore, any risk assessment involving dietary or non dietary exposure to the methylhydantoins will also involve calculating risk from formaldehyde exposure.

On July 25, 2000 , the Health Effects Division ( HED) Hazard Identification Assessment Review Committee ( HIARC) reviewed the recommendations of the toxicology reviewer for 5,5 dimethylhydantoin ( a chemical representative of hydantoin toxicity) with regard to the acute and chronic Reference Doses ( RfDs) and the toxicological endpoint selection for use as appropriate in occupational/ residential exposure risk assessments. The potential for increased susceptibility of infants and children from exposure to this hydantoin was also evaluated as required by the Food Quality Protection Act ( FQPA) of 1996. Endpoints were established for the Halohydantoinss and a report issued. A summary of the endpoints selected is provided at the end of this chapter. Page 5 of 41 2.0 TOXICOLOGY DATA REQUIREMENTS The requirements ( CFR 158.340) for the registered uses for the Halohydantoinss are in Table 1. Use of the new guideline numbers does not imply that the new ( 1998) guideline protocols were used.

Table 1. Toxicity Data Requirements for Halohydantoinss Test Technical Required Satisfied 870.1100Acute Oral Toxicity . . . . . . . . . . . . . . . . . . . 870.1200Acute Dermal Toxicity . . . . . . . . . . . . . . . . . 870.1300Acute Inhalation Toxicity . . . . . . . . . . . . . . . 870.2400 Primary Eye Irritation . . . . . . . . . . . . . . . . . . 870.2500 Primary Dermal Irritation . . . . . . . . . . . . . . . 870.2600 Dermal Sensitization . . . . . . . . . . . . . . . . . . . yes yes yes yes yes yes yes yes yes yes yes yes 870.3100 Oral Subchronic ( rodent) . . . . . . . . . . . . . . . 870.3150 Oral Subchronic ( nonrodent) . . . . . . . . . . . . 870.3250 90 Day Dermal . . . . . . . . . . . . . . . . . . . . . . . 870.3465 90 Day Inhalation . . . . . . . . . . . . . . . . . . . . . yes yes yes yes yes no yes no 870.3700 Developmental Toxicity ( rodent) . . . . . . . . . 870.3700 Developmental Toxicity ( nonrodent) . . . . . . . 870.3800Reproduction . . . . . . . . . . . . . . . . . . . . . . . . yes yes yes yes yes yes 870.4100Chronic Toxicity ( rodent) . . . . . . . . . . . . . . . 870.4100Chronic Toxicity ( nonrodent) . . . . . . . . . . . . 870.4200 Oncogenicity ( rat) . . . . . . . . . . . . . . . . . . . . 870.4200 Oncogenicity ( mouse) . . . . . . . . . . . . . . . . . . yes yes yes yes yes1 yes yes yes 870.5100Mutagenicity C Gene Mutation  bacterial . . . 870.5375Mutagenicity C Structural Chromosomal Aberrations . . . . . . . . . . . . . . . . . . . . . . . . . . 870.5550Mutagenicity C Other Genotoxic Effects . . . . yes yes yes yes yes yes 870.6100 Acute Delayed Neurotox. ( hen) . . . . . . . . . . 870.6300 Developmental Neurotoxicity . . . . . . . . . . . . no no

870.7485 General Metabolism . . . . . . . . . . . . . . . . . . . yes yes 1Satisfied by chronic/ oncogenicity study Page 6 of 41 3.0 DATA GAPS A 90 day inhalation toxicity study in rats is required because of the concerns for inhalation exposure to formaldehyde as a result of the degradation of hydroxymethyl 5,5 dimethylhydantoin and 1,3 bis( hydroxymethyl 5,5 dimethylhydantoin) into free formaldehyde and dimethylhydantoin. Data on the inhalation toxicity of formaldehyde are also relevant for assessing risks posed by inhalation 5,5 DMH. The Agency for Toxic Substances and Disease Registry has recently published a Toxicology Profile for Formaldehyde ( July, 1999). Minimal Risk Levels ( MRLs) have been developed for inhalation exposure to formaldehyde. Acute, Intermediate, and Chronic MRLs of 0.04 ppm, 0.03 ppm, and 0.008 ppm have been selected for formaldehyde based on human data ( acute and chronic MRLs) or animal data ( intermediate). These can be used in assessing risk from inhalation exposure to formaldehyde as a result of its formation from breakdown of dimethylhydantoins.

4.0 HAZARD ASSESSMENT 4.1 Acute Toxicity Adequacy of data base for acute toxicity: The data base for acute toxicity is considered complete. No additional studies are required at this time.

The acute toxicity data on technical Halohydantoins is summarized below in Table 2.

Table 2. Acute Toxicity Profile for the Halohydantoins Guideline No./ Study Type MRID No. ( TRID No.) Results Toxicity Category 5,5 Dimethylhydantoin 870.1100 Acute oral ( gastric intubation) toxicity ( limit test) Mouse 45738401 LD50 ( combined) > 5,000 mg/ kg IV 1 Bromo 3 chloro 5,5 dimethylhydantoin 870.1100 Acute oral toxicity Rat 93074006, 00128244 ( 4226 010 01) LD50( males) = 1,350 mg/ kg LD50( females) = 1,520 mg/ kg LD50( combined) = 1,390 mg/ kg III 870.1100 Acute oral toxicity Rat 93077008, 00147325 ( 4600 950 21) LD50( males) = 1,037 mg/ kg LD50( females) = 860 mg/ kg LD50( combined) = 929 mg/ kg III 870.1300 Acute inhalation toxicity Rat 43654101 LC50( males) = 0.157 mg/ L LC50( females) = 0.213 mg/ L LC50( combined) = 0.168 mg/ L II Page 7 of 41 870.2500 Acute dermal irritation Rabbit 93074011, 93075014, 00128242 ( 4225 014 10) severe skin irritant I 870.2500 Acute dermal irritation Rabbit 93077009, 00147326 ( 4600 950 22) severe skin irritant I 870.2600 Skin sensitization Guinea pig 41670001 positive sensitizer N/ A 1,3 Dibromo 5,5 dimethylhydantoin 870.1100 Acute oral toxicity Rat 93076011, 00137105 ( 4334 012 01) LD50 = 760 mg/ kg III 870.1200 Acute dermal toxicity Rabbit 93076025, 00137110 ( 4334 012 07) LD50 cannot be ascertained ( study is classified as Unacceptable/ non guideline

870.2500 Acute dermal irritation Rabbit 93076017, 00137109 ( 4334 012 05) severe skin irritant I 1,3 Dichloro 5,5 dimethylhydantoin 870.1200 Acute dermal toxicity Rabbit 93076013, 00084176 ( 2402 448 05) LD50 > 20,000 mg/ kg IV 870.2500 Acute dermal irritation Rabbit 93076017, 00137109 ( 2402 448 01) severe skin irritant I 4.2 Subchronic Toxicity Adequacy of data base for subchronic toxicity: The data base for subchronic toxicity of Halohydantoins is complete.

870.3100 90 Day Oral Toxicity  Rat Executive Summary: In a subchronic toxicity study ( MRID 42009201), Dimethylhydantoin (> 99.5% a. i.) was administered orally by gavage to 15 Crl: CDBR ( Sprague Dawley) rats/ sex at dose levels of 0, 100, 300, or 1000 mg/ kg/ day, 5 days/ week for 13 weeks.

There were neither treatment related deaths not treatment related clinical signs of toxicity in any animal. No biologically significant effects were seen on body weight, body weight gain, or food consumption in either sex. Hematology and clinical chemistry analyses revealed no significant differences from controls; urinalysis was not performed. Page 8 of 41 Statistically significant decrements in absolute and relative to body liver weights were reported for high dose males, but the decrement in liver relative to brain weight was not statistically significant nor were the differences considered toxicologically significant in the absence of histopathological or clinical chemistry correlates indicative of toxicity. A 16% ( p< 0.05) increase in absolute liver weight in the high dose female group and increases relative to body weight in the high dose and mid dose females were not considered toxicologically significant in the absence of significantly increased relative to body or brain weight or histopathological or clinical chemistry correlates. The 8.7% ( p< 0.05) increase in kidney weight of the high dose female group was considered toxicologically insignificant in the absence of increased relative to body or brain weight and the absence of clinical chemistry or histopathological findings. The higher mean kidney weight was likely related to the higher body weight of this group. There were no treatment related gross or microscopic lesions in any examined tissue. Since the highest dose and evaluation of higher doses was not necessary.

No treatment related neoplastic findings were reported.

The Subchronic Toxicity NOAEL is greater than or equal to 1000 mg/ kg/ day ( highest dose tested). The LOAEL is greater than 1000 mg/ kg/ day ( not established).

This subchronic toxicity study is classified as Acceptable guideline as it satisfies the guideline requirement for a subchronic oral study [ OPPTS 870.3100 ( § 82 1)] in rats 870.3100 28 Day Oral Toxicity  Mouse Executive Summary: The test compound, Dimethylhydantoin[ 100% a. i.], was offered on a continuous basis in the diet to four ( 4) groups of 5 animal/ sex of Charles River CD 1 mice for a period of 4 weeks [ MRID 45738402]. Dose levels of 0, 1000 [ males: 177, females: 289 mg/ kg/ day], 5,000 [ males: 945, females: 1231 mg/ kg/ day], 10,000 [ males: 1612, females: 2866 mg/ kg/ day] and 50,000 [ males: 10,057, females: 14,972 mg/ kg/ day] ppm were selected for the study. All mice were observed twice daily for mortality and signs of over toxicity. Detailed physical examinations, individual body weights and food consumption were recorded weekly. Clinical pathology parameters were evaluated at study termination. Complete gross necropsy examinations were performed at the scheduled sacrifice and selected organs were weighed. A microscopic examination was conducted on selected tissues from all animals.

No compound related effect was apparent at any dose level on survival, clinical observations, body weight data, food consumption, hematology parameters organ weight, or macroscopic or microscopic findings. Mean serum alkaline phosphatase in the female 50,000 ppm group was significantly increased ( 60%, p< 0.05) when compared to the control group. No other differences in serum chemistry parameters were apparent between the control and treated groups.

The Subchronic Toxicity NOAEL is greater than or equal to 50,000 ppm [ males: 10,057, females: 14972 mg/ kg/ day] ( highest dose tested). The LOAEL is greater than 50,000 ppm [ males: 10,057, females: 14,972 mg/ kg/ day] ( not established). Page 9 of 41 870.3150 90 Day Oral Toxicity  Dog No study is available. However, a chronic toxicity study is available in the dog.

870.3200 21/ 28 Day Dermal Toxicity B Rat No study is available. However, a 90 day dermal toxicity study is available.

870.3250 90 Day Dermal Toxicity B Rat Executive Summary: In a 90 day subchronic toxicity study ( MRID # 43173901), male and female CD rats were treated with 5,5 dimethylhydantoin ( DMH) by dermal occlusion at doses of 0, 39, 130, or 390 mg/ kg/ day for 6 hours/ day, 5 days/ week for 13 weeks.

No statistically significant differences in group mean body weight or cumulative weight gain was observed at any dose level tested. There were no treatment related effects on mortality, skin irritation, or clinical signs of toxicity in males or females at any dose level tested. DMH caused no significant changes in hematology, clinical chemistry, or gross and microscopic pathology in male or female rats. Absolute weight of the adrenal gland was significantly decreased in male rats at the 39 and 390 mg/ kg/ day dose levels ( 12.5% and 13.6%, respectively). Adrenal to body weight ratio was also decreased by 11.1% in female rats at 130 ppm. The differences in adrenal weight were considered unrelated to treatment as there was no definitive dose response and there was no evidence of microscopic lesions.

In this study , the NOAEL was determined to be 390 mg/ kg/ day ( the highest dose tested), and the LOAEL is greater than 390 mg/ kg/ day ( not established).

This study is classified as Acceptable Guideline and it satisfies the guideline requirements for a subchronic toxicity study [ OPPTS 870.3250 ( § 82 1b)] in rats. Higher doses of DMH were not possible in this study; thus, although a limit dose was not tested, the study is considered acceptable.

870.3465 90 Day Inhalation B Rat This study is required to assess risks from formaldehyde exposure, but will be assessed in the RED assessment for formaldehyde. Page 10 of 41 4.3 Prenatal Developmental Toxicity Adequacy of data base for Prenatal Developmental Toxicity: The data base for prenatal developmental toxicity is considered complete. No additional studies are required at this time.

Developmental toxicity testing in the rat showed no increase in susceptibility of the fetus to the toxic effects of DMH because maternal toxicity occurred at the highest dose tested in the absence of fetal effects. In a prenatal developmental study in rabbits, there is quantitative evidence of increased susceptibility for this species; fetal and litter effects ( increased incidence of 27th presacral vertebrae) occurred at a lower dose level than that for maternal toxicity ( decreased body weight and food consumption during the dosing period) following exposure to dimethylhydantoin. However, in a second prenatal developmental toxicity study that used ethylmethylhydantoin as the test material, rabbits showed no increase in susceptibility of the fetuses.

870.3700 Prenatal Developmental Toxicity Study  Rat Executive Summary: In a developmental toxicity study ( MRID # 42432701), mated female CD rats were administered DMH ( 99.8% a. i.) by gavage ( in filtered water) at dose levels of 0, 100, 300, or 1000 mg/ kg/ day on gestation days 6 15, inclusive.

There were no treatment related effects on mortality, clinical signs, food consumption, gross pathology or Caesarean section observations in dams administered DMH at any dose level tested. Maternal toxicity was observed at 1000 mg/ kg and consisted of a transient decrease in body weight gain ( 16%) on gestation days 12 15 for this dose group. The maternal toxicity NOAEL is 300 mg/ kg/ day and the maternal toxicity LOAEL is 1000 mg/ kg/ day based on the transient decrease in body weight gain during gestation days 9 12.

No statistically significant differences were observed between the treated and control groups for number of corpora lutea/ doe, implantations/ doe, pre or postimplantation loss, resorptions/ doe, or fetal sex ratios.

There was no evidence of developmental toxicity in fetuses at any dose level tested; external, visceral and skeletal malformations occurred at similar incidences across dose groups or occurred as single, sporadic events. Based on the results of this study, the developmental toxicity NOAEL is greater than or equal to 1000 mg/ kg/ day ( highest dose tested), and the developmental toxicity LOAEL is greater than 1000 mg/ kg/ day ( not established).

This study is classified as Acceptable Guideline and satisfies the guideline requirements for a developmental toxicity study [ OPPTS 870.3700 ( § 83 3)] in rats.

Executive Summary: In a developmental toxicity study ( MRID # 42413101), groups of artificially inseminated New Zealand White Rabbits ( 20/ dose) were administered 5,5 dimethylhydantoin ( DMH) by gavage on gestation days 6 through 18 at doses of 0, 100, 500, and 1000 mg/ kg/ day in a dose volume of 5 ml/ kg. Page 11 of 41 Maternal toxicity was observed at the 1000 mg/ kg/ day dose level as decreased body weight gain and decreased food consumption during the dosing period. The Maternal Toxicity NOAEL is 500 mg/ kg/ day. The Maternal Toxicity LOAEL is 1000 mg/ kg/ day based on decreased body weight and food consumption during the dosing period.

No statistically significant differences were observed between the treated and control groups for number of corpora lutea/ doe, implantations/ doe, pre or postimplantation loss, resorptions/ doe, or fetal sex ratios.

Developmental toxicity was observed at the 500 mg/ kg/ day dose level as an increased incidence of skeletal variations of the 27th presacral vertebrae ( fetal incidence of 39/ 134, 42/ 140, 82/ 145, and 91/ 123; litter incidence of 11/ 16, 11/ 18, 17/ 19, and 17/ 17 respectively). The increased incidence at 500 mg/ kg/ day exceeded both the concurrent and historical control incidence. The Developmental Toxicity NOAEL is 100 mg/ kg/ day and the Developmental toxicity LOAEL is 500 mg/ kg/ day based on the increased incidence of 27th presacral vertebrae.

This study is classified as Acceptable Guideline and satisfies the guideline [ 870.3700; ( § 83 3)] for a developmental toxicity study in the rabbit.

870.3700 Prenatal Developmental Toxicity Study  Rabbit EXECUTIVE SUMMARY: In a developmental toxicity study ( MRID 42205401), 20 presumed pregnant New Zealand rabbits per group were administered 5 Ethyl 5 methylhydantoin ( EMH) ( 99.5% a. i.; Batch No.: II) by gavage at doses of 0, 50, 125, or 375 mg/ kg/ day on gestation days ( GD) 6 18, inclusive ( phase I). When no toxicity was observed at the highest dose tested, an additional control group and a 1000 mg/ kg/ day group were evaluated later ( phase II). On GD 29, all surviving does were sacrificed and all fetuses were weighed and examined for external malformations variations. Each fetus was examined viscerally by gross dissection and the sex determined. The gross pathology of the brain of each fetus was examined. All carcasses were processed for skeletal examination.

The only treatment related clinical sign of toxicity among surviving animals was the observation of small amount or no stool in 16/ 20 does in the 1000 mg/ kg/ day group vs 6/ 20 in the concurrent control group. A high dose doe was killed on GD 22 and another was found dead on GD 21 following apparent abortions by both of these animals. In addition, three high dose does were sacrificed moribund, one each on GD 15, 16, and 18, after the observations of emaciation, hypoactivity, and/ or prostration. All other does survived to terminal sacrifice. No treatmentrelated findings were observed in any animal at necropsy.

No effects on maternal body weights, body weight gains, or food consumption were observed at doses of 375 mg/ kg/ day and lower. In the high dose group, lower body weight gains occurred in the high dose group ( p

0.05 or 0.01) during the intervals of GD 9 12, 12 15, and 18 21. The high dose group also had an overall decrease in the rate of weight gain during the entire dosing interval ( 183.3 gm vs. 39.9 gm in the control; p

0.01) and during the entire study ( 47.7 gm vs. 274.3 gm in the control; p

0.05). Page 12 of 41 Food consumption by the high dose group was significantly ( 37 63% vs. controls; p

0.01) during the intervals of GD 6 9, 9 12, 12 15, 15 18, and 18 21. This resulted in significantly ( p

0.01) lower food consumption by the high dose group during the entire dosing interval and overall for the study ( 51% and 24% vs. controls, respectively). Therefore, the Maternal Toxicity LOAEL is 1000 mg/ kg/ day based on mortality, reduced body weights gains and reduced food consumption. The Maternal Toxicity NOAEL is 375 mg/ kg/ day.

No statistically significant differences were observed between the treated and control groups for number of corpora lutea/ doe, implantations/ doe, pre or postimplantation loss, resorptions/ doe, or fetal sex ratios.

Body weights of the high dose female fetuses were significantly ( 12%; p

0.05) less than the concurrent controls. Body weights of the high dose male fetuses were not statistically different from the concurrent controls; however, the mean weight of the treated group was also 12% vs. the control level. No dose or treatment related external, visceral, or skeletal malformations/ variations were observed in any fetus. Therefore, the Developmental Toxicity LOAEL is 1000 mg/ kg/ day based on decreased fetal body weights and the Developmental Toxicity NOAEL is 375 mg/ kg/ day.

This study is classified as Acceptable/ guideline and satisfies the guideline requirements for a developmental toxicity study ( § 83 3) in rabbits.

Reproductive Toxicity Adequacy of data base for Reproductive Toxicity: The data base for reproductive toxicity is considered complete. No additional studies are required at this time.

No increased susceptibility was observed in the reproduction studies.

870.3800 Reproduction and Fertility Effects  Rat Executive Summary: In a two generation reproduction study ( MRID 42462502), groups of 28 male and 28 female F0 and F1 rats were administered 5,5 dimetylhydantoin ( 0, 2000, 6000 or 20000 ppm) in their diets for 10 weeks before mating and during mating, gestation, and lactation. Calculated doses were 136 and 127, 408 and 379, and 1396 and 1322 mg/ kg/ day, respectively, for F0 and F1 males ( premating/ postmating periods) and 176 and 158, 516 and 475, and 1775 and 1602 mg/ kg/ day, respectively for F0 and F1 females ( premating periods only).

There was no evidence that dietary administration of 5,5 dimethylhydantoin resulted in treatmentrelated effects on mortality, clinical signs of toxicity, gross lesions, or microscopic lesions in Fo or F1 adult rats of either sex. Body weights and body weight gain were significantly increased in Fo males fed 20,000 ppm of the test material during the mating and postmating periods. The increased body weight is not considered to be a toxic response, because food consumption was Page 13 of 41 also significantly increased during the same period. There were no treatment related effects on body weight or body weight gain in F0 and F1 females during the premating period. Body weights and body weight gain in F0 and F1 females during gestation and lactation were similar to their corresponding controls except for a significantly elevated body weight gain ( 213%) during lactation of F0 females receiving 20,000 ppm of DMH. There was a nonsignificant increase ( 122%) in body weight gain during lactation of the F1 females receiving 20,000 ppm of DMH. The increased body weight gain is not considered to be a toxic effect. The parental/ systemic toxicity NOAEL is equal to or greater than the highest dose tested, 20,000 ppm ( 1322 and 1602 mg/ kg/ day, respectively) for male and female rats. The parental/ systemic toxicity LOAEL cannot be established ( greater than 20,000 ppm) because of the absence of toxicity at any dose tested. In addition, the lack of toxicity at doses greater than the limit dose of 1000 mg/ kg/ day, suggests that the test material has low systemic toxicity.

The indices ( mating, fertility, and fecundity) of reproductive performance in F0 and F1 animals were similar to those of their corresponding controls; no treatment related effects were observed on the indices of reproductive performance as well as litter size, pup viability, pup survival or sex ratio. The NOAEL for reproductive toxicity is equal to or greater than 20,000 ppm ( 1322 and 1602 mg/ kg/ day for males and females, respectively) and the corresponding reproductive toxicity LOAEL is greater than 20,000 ppm ( not established).

Growth of F1 and F2 pups was reduced as indicated by lower body weights ( 7 to 8%; males and females combined) and body weight gain ( 7 to13%) between day 7 of lactation and weaning in high dose ( 20,000 ppm) pups compared with the controls. Body weights were still significantly lower on day 28 in F1 and F2 females weanlings compared with controls. However, these effects may be explained as a transient rejection of the treated diets by the pups since the decreased body weight and body weight gain were seen only during a two week period that coincides with the period in time that the pups begin eating solid food. In the absence of any other reproductive effect, the fact that the body weight effects were not observed at birth or during the first week after birth rules out any relationship to reproduction. The opinion of the study authors is supported by US EPA Guideline for Reproductive Toxicity Risk Assessment ( 61 Fed. Reg. 56273 56322, October 31, 1996) that do not list isolated changes in growth during the latter stages of lactation as adverse reproductive findings. Therefore, the NOAEL for offspring toxicity is equal to or greater than 20,000 ppm ( 1322 and 1602 mg/ kg/ day for males and females, respectively) and the corresponding offspring toxicity LOAEL is greater than 20,000 ppm ( not established).

This study is classified as Acceptable Guideline and it satisfies the guideline requirements for for a two generation reproductive toxicity study [ 870.3800 ( § 83 4] in rats. Page 14 of 41 4.5 Chronic Toxicity Adequacy of data base for chronic toxicity: The data base for chronic toxicity is considered complete.

The combined chronic/ carcinogenicity studies in rat showed a decrement in body weight and body weight gain in females and increased mortality, particularly in females. Males showed increased incidences of hyperplasia of submandibular lymph nodes, and testicular fibroid vascular degeneration in early decedents. Both sexes showed increased mammary galactoceles in early decedents and enlarged pituitary glands. In two chronic toxicity studies conducted in dogs, dimethylhydantoin produced slight ( hypertrophy in the adrenal cortex of male dogs upon microscopic examination) to no toxicity at or above the limit dose of 1000 mg/ kg/ day.

870.4100a ( 870.4300) Chronic Toxicity B Rat Executive Summary: In a chronic toxicity/ carcinogenicity study ( MRID # 43397702), 5,5 dimethylhydantoin ( DMH) was administered in the diet to groups of 60 CD rats/ sex/ dose for 104 weeks at dietary levels of 0 ( first control), 100, 300, 1000, or 0 ( second control) mg/ kg/ day. These dosage levels correspond to dietary concentrations of DMH over the course of the study that ranged from 1080 26794 ppm ( males) and from 945 20890 ( females). [ Note: 5,5 dimethylhydantoin is the organic moiety of 1 bromo 3 chloro 5,5 dimethylhydantoin. The Agency agreed that toxicology studies on the technical should be performed on the organic moiety.]

In both sexes at the highest dose tested, there was no evidence of compound related effects on clinical signs, mortality, food consumption and organ weights, or on clinical pathology. At the highest dose level, statistically significant ( p< 0.01 for the first control and p< 0.05 for the second control groups) treatment related effects on body weight and body weight gain were observed in females only at weeks 90, 92, and 96. During weeks 90 to 96 of the study, body weights were 14 15% lower than control 1 and 9% lower than control 2 and the body weight gains were 23 24% lower than the control 1 and 16% lower than control 2. There were statistically significant increased incidences of hyperplasia of submandibular lymph nodes [ 5/ 19 ( 26%) vs. 0/ 31 to 1/ 33 ( 0 3%) of both controls] at week 104 ( p, 0.05 and p< 0.01 for the controls 1 and 2, respectively) in the high dose males only. This increased incidence was considered to be related to the administration of the DMH.

The Chronic Toxicity NOAEL is 300 mg/ kg/ day. The LOAEL is 1000 mg/ kg/ day, based on decreases in body weight and body weight gain in females and hyperplasia of submandibular lymph nodes in males.

There were no increases in tumor incidences for the DMH treated groups when compared to the control group. This study is classified as Acceptable guideline and satisfies the guideline requirement for a chronic toxicity/ carcinogenicity study ( 870.4300 § 83 5) in rats. Page 15 of 41 Executive Summary: In a chronic/ oncogenicity toxicity study ( MRID 44095901), dimethylhydantoin ( DMH) ( 97.3%, 97.1% and 93.5% a. i. for lot # 6, # 2412 67 D1, and # 2412 67 D2, respectively) was administered to 80 Crl: CDBR rats/ sex/ dose in the diet at dose levels of 0, 100, 300, or 1000 mg/ kg/ day for 104 105 weeks. The same doses of DMH were given to 20 rats/ sex/ dose in a 52 week interim study. The concentration of DMH in the diets was adjusted weekly according to body weight and food consumption measurements to achieve the target doses.

No treatment related statistically significant increases in the incidences of any lesion were seen at the termination of this 105 week study. However, the animals that died during the study tended to die earlier at the high dose. Mortality during treatment weeks 52 79 was 11% in control males and 15% in high dose males; 7% in control females, and 27% ( p

0.001) in high dose females. Significantly increased incidences of enlarged pituitary glands were found in high dose early decedent males ( control, 20%; high dose, 38%, p

0.05) and females ( control, 33%; high dose, 61%, p

0.05). Pituitary pars distalis adenomas were the most common cause of early deaths in the study and contributed to the increased mortality seen during weeks 52 79 at the high dose. Increased incidences of mammary galactoceles occurred in high dose early decedent females ( control, 31%; high dose, 59%, p

0.01). The incidence of testicular fibrinoid vascular degenerations was increased in high dose males ( control, 13%; high dose, 8%, p

0.01). The incidence of iliac lymph node lymphangiectasia was increased in high dose males ( control, 13%; high dose, 26%) and females ( control, 1%; high dose, 9%). The number of times that dried yellow matting in the urogenital area was observed over the 24 month treatment period increased about 3 fold in high dose males and females compared to controls. Urine volume roughly doubled in high dose males and females, and specific gravity decreased during week 77. Kidney tubule dilation was increased in high dose males at 52 weeks ( control, 65%; high dose, 100%, p

0.01). Lung mineralization at 52 weeks was increased in males at 1000 mg/ kg/ day ( 68%, p

0.05) compared to the controls ( 30%). The mean absolute brain weight was slightly decreased in high dose males ( 4%, p

0.05) compared to the controls at 52 weeks.

The Chronic Toxicity NOAEL is 320 mg/ kg/ day. The LOAEL is 1000 mg/ kg/ day, based on enlarged pituitary glands in decedents of both sexes, increased mortality occurring early in ( especially) females, increased mammary galactoceles in early decedents of both sexes and testicular fibroid vascular degeneration in early decedent males.

At the doses tested, there was not a treatment related increase in tumor incidence after 105 weeks of treatment with DMH. The increase at 52 weeks in the incidences of pituitary adenomas in the pars distalis in high dose females ( control, 10%; high dose, 50%, p

0.01) and males ( control, 10%; high dose, 32%, N. S.) was not dose related. There were no differences in pituitary adenoma incidences between control and high dose groups by the end of the 105 week study. The high dose of 1000 mg/ kg/ day meets the limit dose requirement for non toxic agents.

This study is classified as Acceptable Guideline, and does satisfy the guideline requirements for a chronic/ carcinogenicity study [ OPPTS 870.4300 ( § 83 5)] in rats. Page 16 of 41 870.4100b Chronic Toxicity  Dog EXECUTIVE SUMMARY:

In a chronic toxicity study in dogs ( MRID No. 43813301), 5,5 dimethylhydantoin ( 97.4% a. i.) was administered to 32 beagle dogs ( 4/ sex/ dose) by capsule at dose levels of 0, 250, 500, or 1000 mg/ kg/ day for 52 weeks ( capsules given 7 days/ week).

There were no treatment related effects on mortality, clinical signs, body weight, body weight changes, food consumption, hematology, clinical chemistry, urinalysis, organ weights, organ/ body weight or organ/ brain weight ratios, ophthalmological parameters, or gross or microscopic results from histologic examination of selected tissues. The LOAEL is greater than 1000 mg/ kg/ day ( HDT), based on the lack of any observable toxic effects. The NOAEL is greater than or equal to 1000 mg/ kg/ day.

This chronic toxicity study in the dog is classified as Acceptable guideline and does satisfy the guideline requirement ( 870.4100) for a chronic oral study in the dog.

EXECUTIVE SUMMARY:

In this chronic ( one year) toxicity study ( MRID No. 43553101), purebred beagle dogs, 4/ sex/ group, were administered 5,5 dimethylhydantoin ( 98.9% a. i.) in the diet att levels of 0, 4000, 12000, or 40000 ppm ( corresponding to measured dose levels of 0, 120, 342, or 1506 mg/ kg body wt./ day for males and 0, 121, 414, and 1352 mg/ kg body wt/ day for females, respectively).

All animals survived to scheduled sacrifice. At all doses tested, 5,5 dimethylhydantoin had no effect on body weight compared with control values at any time period. Statistically significant increases in the absolute weight of the adrenals and adrenal / body weight and adrenal brain weigh ratios were observed in the Group IV ( 40000 ppm) males; no other statistically significant effects on organ weight or organ to body or brain weight ratios were observed. Increases in food consumption during the latter half of the study ( weeks 26 52) by Group IV ( 40000 ppm) males most likely resulted from inaccurate measurements due to digging in the food hoppers by two of the four dogs in this group; decreases in food consumption by females in all dose groups was neither dose nor time related and are not considered to be related to treatment with 5,5 diethyl hydantoin. Neither the apparent increases or decreases in food consumption were reflected by corresponding changes in body weight. 5,5 dimethylhydantoin, at all dose levels tested, had no statistically significant effect on any of the hematology, clinical chemistry, or urinalysis parameters examined for either males or females. Treatment related clinical observations included discolored feces and oily coat in both males and females, with the latter observation most prevalent in males. Primarily in the mid ( Group III; 12000 ppm) and high ( Group IV; 40000 ppm) dose groups, gross necropsy revealed treatment related effects on the ear ( nodule, scab, red color), lymph nodes ( enlarged), skin ( alopecia, red color), and spleen ( red foci). Microscopic histopathology revealed hypertrophy of the adrenal cortex in all four males in the high dose group ( Group IV; 40000 ppm). This microscopic finding correlates with the increased weight of adrenals and Page 17 of 41 adrenal weight to body and brain weight ratios found in this same treatment group. This finding was not observed in males or females in the control group or mid ( Group III; 12000 ppm) or low ( Group II; 4000 ppm) dose treatment groups. The NOAEL is 12000 ppm ( 342 mg/ kg/ day) and the LOAEL is 40000 ppm ( 1506 mg/ kg/ day), based on enlarged adrenal glands in male dogs, shown by microscopic examination to exhibit hypertrophy in the adrenal cortex.

4.6 Carcinogenicity Adequacy of data base for Carcinogenicity: The data base for carcinogenicity is considered complete. No additional studies are required at this time.

In carcinogenicity testing of rats and mice there is some evidence of target organ toxicity, but no evidence of positive tumorigenicity for the test material.

870.4200a Carcinogenicity Study  rat See chronic toxicity/ carcinogenicity studies above ( MRID # 43397702 and 44095901).

870.4200b Carcinogenicity ( feeding)  Mouse EXECUTIVE SUMMARY: ( MRID # 43397701) DMH was administered in the diets of CD 1 mice at dietary concentrations of 0, 400, 1850, or 8500 ppm ( target doses of 0, 100, 300, or 1000 mg/ kg/ day) for 78 weeks. At the highest dose tested, there was no evidence of carcinogenicity, nor were there compound related clinical signs, effects on mortality or on clinical pathology. In males at the highest dose level, there was a statistically significant ( 4 9%) decrease in the mean absolute body weight and in mean body weight gain ( 19 and 28%, respectively, when compared to controls 1 and 2) which persisted from week 16 of the study. Based on the decreases in body weight and body weight gain in males, the NOAEL is 300 mg/ kg and the LOAEL is 1000 mg/ kg. No signs of toxicity were reported in females.

The study satisfies the guideline requirements for a mouse oncogenicity study as set forth in Subdivision F, Guideline 83 2 ( 870.4200).

EXECUTIVE SUMMARY: In a mouse oncogenicity study ( MRID 4463901), DMH ( 97.1 97.3% a. i.) was administered to CD 1 mice ( 80/ sex/ group) for 18 months at 0, 100, 320, or 1000 mg/ kg/ day. No significant differences were observed in survival rates in male or female mice in any of the treated groups throughout the study when compared to the respective control groups. Overall body weight gains, leukocyte differential counts, absolute and relative organ weights, gross necropsy, as well as microscopic findings for both sexes at all doses were unaffected by treatment with DMH. Page 18 of 41 Negligible systemic toxicity was characterized by statistically significant decreases in mean body weights in males receiving the 1,000 or 320 mg/ kg/ day diets (  3 5%; p< 0.05 or 0.01) and increases in mean feed consumption by the high dose animals (  3.8 9.8%; p< 0.05 or 0.01). The biological significance of these small, but statistically significant differences in mean body weights and feed consumption is unknown.

The Systemic Toxicity NOAEL is greater than or equal to 1000 mg/ kg/ day ( highest dose tested) and the Systemic Toxicity LOAEL is greater than 1000 mg/ kg/ day ( not established).

Under the conditions of this study, there was no evidence of carcinogenic potential in mice following treatment with DMH and the dosing was considered adequate because the highest dose of 1000 mg/ kg/ day represents a " limit dose."

This study is classified as Acceptable Guideline and does satisfy the guideline requirements for a carcinogenicity study [ OPPTS 870.4200 ( § 83 2b)] in mice.

4.7 Mutagenicity Adequacy of data base for Mutagenicity: The data base for Mutagenicity is considered ( in) adequate based on pre 1991 mutagenicity guidelines. A battery of mutagenicity assays were performed; including reverse mutation, mammalian cell gene mutation, chromosome aberration and unscheduled DNA damage. In most assays no mutagenicity was observed; however, two in vitro mammalian cytogenetics assays in Chinese Hamster Ovary cells indicated positive results.

Substance: Dimethylhydantoin Salmonella Reverse Gene Mutation Assay In an Ames Salmonella assay ( MRID No. 164036, Accession number 265457), 5,5 dimethylhydantoin ( DHM) was tested in S. tyhimurium strains TA1535, TA 100, TA1538, TA98 and TA1537 in the absence and presence of S9 metabolic activation at concentrations of 100, 500, 2500, 5000, and 10,000  g/ plate. There was neither a cytotoxic nor a mutagenic response in this assay at any dose level tested in the absence or presence of metabolic activation.

Substance: Ethylmethylhydantoin Salmonella Reverse Gene Mutation Assay EXECUTIVE SUMMARY: In a reverse gene mutation assay in bacteria ( MRID No. 137095, TRID No. 433401118), S. typhimurium strains: TA98, TA100, TA1535, TA1537 and TA1538 were exposed to Ethylmethylhydantoin ( Test Article 447: 34 4) ( purity not provided) in water at concentrations of 67, 333, 1667, 3333 and 6666 µ g/ plate in the presence and absence of mammalian metabolic activation ( S9 mix). All platings were in triplicate. The S9 fraction was obtained from Aroclor 1254 induced male Sprague Dawley rat liver. Page 19 of 41 Ethylmethylhydantoin was tested up to a limit concentration of 6666 µ g/ plate. In a preliminary cytotoxicity assay with TA100 in the absence of S9 mix, no cytotoxicity ( as determined by a reduction in the number of revertants per plate or by a thinning or absence of the background lawn of bacteria) was seen at concentrations ranging from 1.0 to 3333 µ g/ plate. In the mutagenicity assay, Ethylmethylhydantoin did not affect the background lawn of bacteria or increase the number of revertants over solvent control values in any tester strain at any concentration, with or without S9 mix. The solvent and positive control values were appropriate for each of the five tester strains. In this study, there was no evidence of induced mutant colonies over background.

This study is classified as Acceptable/ nonguideline. It satisfies the requirement for FIFRA Test Guideline § 84 2 for in vitro mutagenicity ( bacterial reverse gene mutation) data with the exception that no confirmatory assay was conducted. In the absence of any evidence of an increase in the number of revertants per plate over solvent control values in any strain at any concentration up to 10,000 µ g/ plate, and with acceptable control values, this omission does not make the study unacceptable.

Substance: Dimethylhydantoin Salmonella typhimurium Reverse Mutation Assay EXECUTIVE SUMMARY: In a reverse gene mutation assay in bacteria ( MRID No. 137100, TRID 433401124), S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were exposed to Test Article 447: 34 2, Dimethylhydantoin ( purity not provided) in water at concentrations of 100, 500, 2500, 5000 and 10,000 µ g/ plate in the presence and absence of mammalian metabolic activation ( S9 mix). All platings were in triplicate. The S9 fraction was obtained from Aroclor 1254 induced male Sprague Dawley rat liver.

DMH was tested up to a limit concentration of 10,000  g/ plate. In a preliminary cytotoxicity assay with TA100 in the absence of S9 mix, no cytotoxicity ( as determined by a reduction in the number of revertants per plate or by a thinning or absence of the background lawn of bacteria) was seen at concentrations ranging from 2.9 to 10,000 µ g/ plate. In the mutagenicity assay, DMH did not affect the background lawn of bacteria or increase the number of revertants over solvent control values in any tester strain at any concentration, with or without S9 mix. The solvent and positive control values were appropriate for each of the five tester strains. In this study, there was no evidence of induced mutant colonies over background.

This study is classified as Acceptable/ nonguideline. It satisfies the requirement for FIFRA Test Guideline § 84 2 for in vitro mutagenicity ( bacterial reverse gene mutation) data with the exception that no confirmatory assay was conducted. In the absence of any evidence of an increase in the number of revertants per plate over solvent control values in any strain at any concentration up to 10,000 µ g/ plate, and with acceptable control values, this omission does not make the study unacceptable. Page 20 of 41 Substance: 5 Ethyl, 5 methylhydantoin Mammalian cells in Culture Gene Mutation Assay in L5178Y +/ Mouse Lymphoma Cells EXECUTIVE SUMMARY: In a mammalian cell gene mutation assay, L5178Y TK+/ mouse lymphoma cells ( MRID No. 137089, TRID No. 433401121) cultured in vitro were exposed to Ethylmethylhydantoin ( purity not provided) in F 0 P medium ( Fisher's media for leukemic cells of mice with 0.1% Pluronic) at concentrations of 751, 1001, 1335, 1780, 2373, 3164, 4219, 5000, 5625, 7500 µ g/ mL in the absence of mammalian metabolic activation ( S9 mix) and at concentrations of 563, 751, 1001, 1780, 2373, 3164, 4219, 5625, 7500, 10,000 µ g/ mL in the presence of S9 mix. The S9 fraction was obtained from Aroclor induced rat liver.

Ethylmethylhydantoin was tested up to limit concentrations of 7500 µ g/ mL in the absence of S9 mix and of 10,000 µ g/ mL in the presence of S9 mix. In the preliminary cytotoxicity assay, concentration of ethylmethylhydantoin ranging from 0.01 to 5000 µ g/ mL were tested. In the absence of S9 mix, no cytotoxicity was seen at any concentration with suspension growth ranging from 92% to 104% of the solvent control with no dose relationship. In the presence of S9 mix, the suspension growth ranged from 62% to 98% of the solvent control, but again with no dose relationship ( the 62% value was seen at the lowest concentration tested while the value at the highest concentration tested was 97%). In the mutagenicity assay, suspension growth, measured following the expression period, ranged from 93% to 109% of the solvent control in the absence of S9 mix ( discounting a value of 38% at 1001 µ g/ mL, the next to lowest dose tested) and from 94% to 118% of the solvent control in the presence of S9 mix. The mutant frequencies in cultures treated with the test article in the absence of S9 mix ranged from 0.3 to 0.4 mutants per 104 surviving cells in a non dose dependent manner compared to the solvent control value of 0.3 mutants per 104 surviving cells. The mutant frequencies in cultures treated with ethylmethylhydantoin in the presence of S9 mix ranged from 0.9 to 1.6 mutants per 104 surviving cells in a non dose dependent manner compared to the solvent control value of 1.3 mutants per 104 surviving cells. The solvent and positive controls induced the appropriate response. In this study, there was no evidence of induced mutant colonies over background.

This study is classified as Acceptable/ nonguideline. It satisfies the requirement for FIFRA Test Guideline § 84 2 for in vitro mutagenicity ( mammalian forward gene mutation) data with the exception that no confirmatory assay was conducted. This omission did not render the study unacceptable because no hint of an increase in mutant frequency was seen at any test material concentration up to 10,000 µ g/ mL, and the solvent and positive control data were acceptable.

Substance: Dimethylhydantoin Mammalian Cells in Cullture Gene Mutation Assay in L5178Y +/ Mouse Lyphoma Cells EXECUTIVE SUMMARY: In a mammalian cell gene mutation assay, L5178Y TK+/ mouse lymphoma cells ( MRID No. 132165, TRID No. 433401127) cultured in vitro were exposed to Dimethylhydantoin ( purity not provided) in F 0 P medium ( Fisher's media for leukemic cells of mice with 0.1% Pluronic) at concentrations of 563, 1001, 1335, 1780, 2373, 3164, 4219, 5625, 7500, Page 21 of 41 10,000 diethel hydantoin  / mL in the absence of mammalian metabolic activation ( S9 mix) and at concen trations of 751, 1001, 1335, 1780, 2373, 3164, 4219, 5625, 7500, 10,000  g/ mL in the presence of S9 mix. The S9 fraction was obtained from Aroclor induced rat liver.

Dimethylhydantoin was tested up to a limit concentration of 10,000  g/ mL in the absence of any significant cytotoxicity. No cytotoxicity was seen at concentrations up to 5000  g/ mL, with or without S9 mix, in the preliminary cytotoxicity test. In the mutagenicity assay, suspension growth ( measured following the expression period) without S9 mix was at least 90% of the solvent control value at all concentrations except 10,000  g/ mL where the suspension growth was 75% of the solvent control. In the presence of S9 mix, the suspension growth was at least 93% of the solvent control value at all tested concentrations. The concurrent solvent controls for the nonactivated part of the study were lost due to contamination; therefore, the historical solvent control value obtained from 170 previous tests in the absence of S9 mix was used for comparative calculations ( 0.46 mutants per 104 surviving cells with a standard deviation of 0.19). The mutant frequencies in cultures treated with Dimethylhydantoin in the absence of S9 mix ranged from 0.4 to 0.7 mutants per 104 surviving cells in a non dose dependent manner. The mutant frequency of the concurrent solvent control in the presence of S9 mix was 0.8 mutants per 104 surviving cells compared to a range of mutant frequencies in dimethylhydantoin treated cultures ranging from 0.4 to 0.9 mutants per 104 surviving cells in a non dose dependent manner. The positive controls induced the appropriate response. In this study, there was no evidence of induced mutant colonies over background.

This study is classified as Acceptable/ nonguideline. It satisfies the requirement for FIFRA Test Guideline § 84 2 for in vitro mutagenicity ( mammalian forward gene mutation) data with the exception that no confirmatory assay was conducted. This omission did not render the study unacceptable because no hint of an increase in mutant frequency was seen at any test material concentration up to 10,000  g/ mL with or without S9 mix, and the solvent and positive control data were acceptable.

Substance: 5,5 Dimethylhydantoin In Vitro Mammalian Cytogenics Assay in CHO Cells EXECUTIVE SUMMARY: In a mammalian cell cytogenetics assay ( MRID No. 164037, TRID No. 265457), CHO cell cultures were exposed to 5,5 dimethylhydantoin at concentrations of 200 to 2000 µ g/ ml.

In this study, there was no evidence of structural chromosomal aberration induction over background.

This study, therefore, is classified as Acceptable/ guideline. It satisfies the requirement for FIFRA Test Guideline § 84 2 for in vitro cytogenetic mutagenicity data. Page 22 of 41 Substance: 1,3 Dichloro 5,5 ethylmethylhydantoin In Vitro Mammalian Cytogenetics Assay in CHO Cells ( MRID 40348201)

In a mammalian cell cytogenetics assay ( MRID 40348201), CHO cell cultures were exposed to 1,3 Dichloro 5,5 ethylmethylhydantoin [ purity not provided] dissolved in McCoy's 5A medium at concentrations of 60, 30, 15, and 8 µ g/ ml for 22 hours without metabolic activation ( S9 mix) and at concentrations of 120, 60, 30, and 15 µ g/ ml for 16 and 22 hours with S9 mix. The S9 fraction was obtained from Arochlor 1254 induced male Sprague Dawley rat liver.

1,3 Dichloro 5,5 ethylmethylhydantoin was tested up to cytotoxic concentrations. A preliminary cytotoxicity test was performed over the dose range 0.02 to 200 µ g/ ml; treatment was for 6 hours without activation and for 2 hours with activation. 1,3 Dichloro 5,5 ethylmethylhydantoin reduced cell growth in a dose dependent manner without metabolic activation; with activation,

only the highest dose reduced cell growth. Metaphase cells were rare or absent at 200 µ g/ ml with and without metabolic activation.

In the cytogenetic assay, 200 cells/ dose/ treatment time/ activation condition were scored for structural chromosomal aberrations. Significant increase in the percentage of cells with structural chromosomal aberrations ( mainly chromatid and chromosome breaks and dicentrics) was seen at the highest dose tested without S9 mix; the percentage of cells with structural chromosomal aberrations, excluding gaps, reached 9.5% at 60 µ g/ ml. This result meets the testing laboratory's criterion for a " suspect" positive. The frequency of cells with structural chromosome aberrations was significantly increased in cultures treated with 120 and 60 µ g/ ml for 16 hours with S9 mix, and at all dose levels in cultures treated for 22 hours with S9 mix. Positive and solvent control values were appropriate throughout the study. There was evidence of structural chromosomal aberration induction greater than background.

This study is classified as Acceptable/ guideline. It satisfies the requirement for FIFRA Test Guideline § 84 2 for in vitro cytogenetic mutagenicity data.

Substance: Ethylmethylhydantoin In Vitro Mammalian Cytogenics Assay in CHO Cells EXECUTIVE SUMMARY: In a mammalian cell cytogenetics assay ( MRID No. 137096, TRID No. 433401119), CHO cell cultures were exposed to Ethylmethylhydantoin [ purity not provided] at concentrations of 10,204.08, 7653.06, and 5739.80 µ g/ ml for 4 hours with and without metabolic activation ( S9 mix). The S9 fraction was obtained from livers of Aroclor 1242/ 1254 induced Sprague Dawley rats of unspecified sex.

Ethylmethylhydantoin was tested up to the maximum soluble dose. A preliminary cytotoxicity test was performed using seven doses ranging from 10.204.08 down to 10.36 µ g/ ml. Ethylmethyhydantoin did not cause dose dependent reduction in cell growth with or without Page 23 of 41 metabolic activation. At the highest dose, relative cloning efficiency was 88.78% without activation and 113.71% with activation.

In the cytogenetic assay, 50 cells/ dose/ treatment time/ activation condition were scored for visible structural and numerical frequency of chromosomal aberrations. A two fold or greater increase in the percentage of cells with structural chromosomal aberrations, excluding gaps, was seen at two nonconsecutive doses in cultures treated with S9 mix. No dose response was evident. Results from the treatment without S9 mix were negative at all doses. Chromosome numbers were recorded but no increase in polyploid cells was observed in treated or in positive control cultures. Positive and solvent control values were appropriate throughout the study. There was evidence of structural chromosomal aberration induction over background in the presence of metabolic activation.

This study is classified as Acceptable/ guideline. It satisfies the requirement for FIFRA Test Guideline § 84 2 for in vitro cytogenetic mutagenicity data.

Substance: 5,5 Dimethylhydantoin In Vitro Mammalian Cytogenics Assay in CHO Cells EXECUTIVE SUMMARY: In a mammalian cell cytogenetics assay ( MRID No. 137101, TRID No. 433401125), CHO cell cultures were exposed to dimethylhydantoin [ purity not provided] in the absence of metabolic activation at concentrations of 20,000, 15,000, and 11,250 µ g/ ml and in the presence of metabolic activation at concentrations of 15,000, 11,250 and 8457.5 µ g/ ml. All exposures were for 4 hours. The S9 fraction used for metabolic activation was obtained from livers of Aroclor 1242/ 1254 induced Sprague Dawley rats of unspecified sex.

Dimethylhydantoin was tested up to the maximum soluble dose. A preliminary cytotoxicity test was performed using nine doses ranging from 10,000 down to 0.99 µ g/ ml. Dimethylhydantoin did not cause dose dependent reduction in cell growth with or without metabolic activation. At the highest dose, relative cloning efficiency was 81.82% without activation and 88.89% with activation.

In the cytogenetic assay, 50 cells/ dose/ treatment time/ activation condition were scored for structural and numerical chromosomal aberrations. A two fold or greater increase in the percentage of cells with structural chromosomal aberrations, excluding gaps, was not seen at any dose in cultures treated with or without S9 mix. Chromosome numbers were recorded but no increase in polyploid cells was observed in treated or in positive control cultures. Positive and solvent control values were appropriate throughout the study. There was no evidence of structural chromosomal aberration induction over background.

This study is classified as Acceptable/ guideline. It satisfies the guideline requirement for an in vitro cytogenetic mutagenicity assay ( § 84 2). Page 24 of 41 Substance: 5,5 Dimethylhydantoin In Vitro Mammalian Cytogenics Assay in CHO Cells EXECUTIVE SUMMARY: In a mammalian cell cytogenetics assay ( MRID No. N/ A, TRID No. 470264004), CHO cell cultures were exposed to 5,5 dimethylhydantoin in sterile distilled water at concentrations of 800, 400 and 80 µ g/ ml for 20 hours without metabolic activation or for 2 hours with metabolic activation. The S9 fraction was obtained from Arochlor 1254 induced male CD rat livers.

5,5 dimethylhydantoin was tested up to the maximum soluble dose. A preliminary cytotoxicity test was performed with seven doses from 12.5 to 800 µ g/ ml. No dose related effect on mitotic index was observed in the presence or the absence of metabolic activation.

In the cytogenetic assay, 100 cells/ dose/ activation condition were scored for structural chromosomal aberrations. Statistically significant increase in the percentage of cells with structural chromosomal aberrations was not seen, including or excluding gaps, at any dose in the presence or absence of metabolic activation. Positive and solvent control values showed the appropriate results.

throughout the study. In this study, there was no evidence of structural chromosomal aberration induction over background.

This study, therefore, is classified as Acceptable/ guideline. It satisfies the requirement for FIFRA Test Guideline § 84 2 for in vitro cytogenetic mutagenicity data.

Substance: 5,5 Dimethylhydantoin Unscheduled DNA Synthesis in Mamalian Cells in Culture EXECUTIVE SUMMARY: In an unscheduled DNA synthesis ( UDS) assay in HeLa S3 cells ( MRID No. N/ A, TRID No. 470264005), cell cultures were exposed in two duplicate experiments to twelve concentrations of 5,5 dimethylhydantoin ranging [ purity not available] from 10 to 20,480 µ g/ ml. The test material was dissolved in distilled water and cells were exposed for 180 minutes. Autoradiography was used to detect UDS.

No cytotoxicity assessment was made but 5,5 dimethylhydantoin was tested to the maximum soluble dose. Positive and solvent control produced the appropriate results. In one experiment, one relatively low dose of 5,5 dimethylhydantoin produced a statistically significant increase in net nuclear grain count. No increase was seen, however, at higher doses in that experiment and the increase was not reproduced in the second experiment. Therefore, the reproducibility criterion for a positive response was not met. No positive UDS response to 5,5 dimethylhydantoin was seen in the assay. Page 25 of 41 This study is classified as Acceptable/ guideline. It satisfies the requirements for FIFRA Test Guideline § 84 2 for other genotoxic mutagenicity data.

Substance: 5,5 Dimethylhydantoin Unscheduled DNA Synthesis in Mamalian Cells in Culture EXECUTIVE SUMMARY: In an unscheduled DNA synthesis ( UDS) assay in primary rat hepatocytes ( MRID No. 164038, Accession No. 265457), cell cultures were exposed to 5,5 dimethylhydantoin ranging [ purity not available] from 100 to 10,000 µ g/ ml.

No positive UDS response to 5,5 dimethylhydantoin was seen in the assay.

This study is classified as Acceptable/ guideline. It satisfies the requirements for FIFRA Test Guideline § 84 2 for other genotoxic mutagenicity data.

Substance: Ethylmethylhydantoin Unscheduled DNA Synthesis in Mammalian Cells in Culture EXECUTIVE SUMMARY: In an unscheduled DNA synthesis ( UDS) assay in rat primary hepatocytes ( MRID No. 137097, TRID No. 433401120), cultures of primary hepatocytes from adult male Sprague Dawley rats were exposed to Ethylmethylhydantoin at concentrations of 15, 10, 5, 1, 0.5 and 0.1 mg/ mL, selected on the basis of a preliminary cytotoxicity test. The test material was delivered in culture medium and cells were exposed for 18 hours. The autoradiographic technique was used to detect UDS.

Viability as assessed by trypan blue exclusion was used as the measure of cytotoxicity. Cytotoxicity relative to the solvent control was almost 95% at the highest dose, decreasing to 15.5% at the lowest dose. Positive and solvent control values were appropriate. No induction of UDS was seen at any concentration of ethylmethylhydantoin evaluated.

This study is classified as an Acceptable/ guideline. It satisfies the requirements for FIFRA Test Guideline § 84 2 for a UDS study in mammalian cells in culture.

Substance: Dimethylhydantoin Unscheduled DNA Synthesis in Mamalian Cells in Culture EXECUTIVE SUMMARY: In an unscheduled DNA synthesis ( UDS) assay in rat primary hepatocytes ( MRID No. 132166, TRID No. 433401126), cultures of primary hepatocytes from adult male Sprague Dawley rats were exposed to Dimethylhydantoin ( purity not provided) at concentrations of 20, 10, 1, 0.1, 0.01, and 0.001 mg/ ml, selected on the basis of a preliminary cytotoxicity test. The test material was delivered in culture medium and cells were exposed for 18 hours. The autoradiographic technique was used to detect UDS. Page 26 of 41 Viability as assessed by trypan blue exclusion was used as the measure of cytotoxicity. Cytotoxicity relative to the solvent control was 70.4% at the highest dose tested, although the same dose produced relative toxicity of only 26.21% in the preliminary assay. Positive and solvent control values were appropriate. No induction of UDS was seen at any concentration of dimethylhydantoin evaluated.

This study is classified as Acceptable/ guideline. It satisfies the requirements for FIFRA Test Guideline § 84 2 for a UDS study in mammalian cells in culture.

4.8 Neurotoxicity Adequacy of data base for Neurotoxicity: These studies are not required at this time. No neurotoxicity studies were necessary.

870.6100 Delayed Neurotoxicity Study  Hen Study not required, DMH is not an organophosphate.

870.6300 Developmental Neurotoxicity Study Study not required 4.9 Metabolism Adequacy of data base for metabolism: The data base for metabolism is considered to be complete. No additional studies are required at this time.

870.7485 Metabolism  Rat ( MRID # 4217390) Under the conditions of this study, DMH was rapidly absorbed and was excreted primarily in the urine. Most of the radioactivity was recovered in the first 12 hours in both sexes and there was very little accumulation in the tissue.

This study is acceptable. This study alone does not satisfy the guideline requirements for a metabolism study; however, an additional study was conducted to identify the metabolites of DMH in the urine.

( MRID # 42123802) Unmetabolized 14C MEH recovered in the urine represented more than 89% of the administered dose of MEH.

This study is supplementary. The sponsor needs to supply analytical data to demonstrate that the HPLC methodology was validated to confirm the identity of the metabolite in the urine samples. This study may be upgraded upon receipt and evaluation of the requested data. Page 27 of 41 5.0 TOXICITY ENDPOINT SELECTION 5.1 See Section 9.2 for Endpoint Selection Table.

5.2 Dermal Absorption There are no dermal absorption data available for 5,5 dimethylhydantoin.

5.3 Classification of Carcinogenic Potential 5.3.1 5,5 dimethylhydantoin is classified as ` not likely' to be a carcinogen based upon the negative evidence for a carcinogenicity in both the rat and mouse studies as well as the negative evidence of genotoxicity.

Formaldehyde ( a breakdown product of the hydantoins) has previously been classified as a B1 ( probable) carcinogen based upon the limited evidence in humans of site specific respiratory neoplasms following inhalation exposure to formaldehyde, and sufficient evidence in animals, based on squamous cell carcinoma of the nasal cavity following chronic inhalation exposure.

6.0 FQPA CONSIDERATIONS 6.1 Special Sensitivity to Infants and Children The database for 5,5 dimethylhydantoin is considered adequate for a determination as to potential susceptibility of infants and children.

6.2 Recommendation for a Developmental Neurotoxicity Study A developmental neurotoxicity study is not required for 5,5 dimethylhydatoin. The weight of the evidence from the available data on 5,5 dimethylhydantoin indicate no neurotoxic effects of this chemical. There is no evidence to suggest any potential neurotoxic effects from exposure to 5,5 dimethylhydantoin except at unrealistically high exposures.

7.0 ENDOCRINE DISRUPTION The Agency is required under the Federal Food, Drug and Cosmetic Act ( FFDCA), as amended by FQPA, to develop a screening program to determine whether certain substances ( including all pesticide active and other ingredients) " may have an effect in humans that is similar to an effect produced by a naturally occurring estrogen, or other such endocrine effects as the Administrator may designate." Following the recommendations of its Endocrine Disruptor Screening and Testing Advisory Committee ( EDSTAC), EPA determined that there was scientific bases for including, as part of the program, the androgen and thyroid hormone systems, in addition to the estrogen hormone system. EPA also adopted EDSTAC's recommendation that the Program include evaluations of potential effects in wildlife. For pesticide chemicals, EPA will use FIFRA Page 28 of 41 and, to the extent that effects in wildlife may help determine whether a substance may have an effect in humans, FFDCA authority to require the wildlife evaluations. As the science develops and resources allow, screening of additional hormone systems may be added to the Endocrine Disruptor Screening Program ( EDSP).

8.0 OTHER ISSUES Based upon the structural similarity of dimethylhydantoin to diphenylhydantoin ( a known teratogenic and carcinogenic agent), concern was expressed by the Agency as to the valididty of the use of dimethylhydantoin containing products, especially for use in pools and spas. The Agency has considered toxicity data from both diphenylhydantoin and dimethylhydantoin in determining that there is no significant hazard from the use of dimethylhydantoin ( memorandum from Robert P. Zendzian to Arturo Castillo, dated October 21, 1983). First, it is evident that the biotransformation and excretion of dimethylhydantoin and diphenylhydantoin differ. Diphenylhydantoin undergoes extensive metabolism of the phenyl group with the production of an arene oxide intermediate ( believed responsible for the toxicity of the chemical), while dimethylhydantoin is essentially excreted unchanged. Second, the hazard profile for dimethylhydantoin differs significantly from that of diphenylhydantoin. Dimethylhydantoin lacks the toxicity present with diphenylhydantoin and shows no evidence of carcinogenicity or teratogenicity. Page 29 of 41 9.0 REFERENCES in MRID order MRID 00252096 reference information not available MRID 265457( 1) Lawlor, T. E., B. Head, V. O. Wagner, B. E. Carter, S. M. Olewine, and R. J. Plunkett ( 1986). Salmonella/ mammalian microsome plate incorporation mutagenicity assay on 5,5 dimethylhydantoin. Microbiological Associates Inc. Bethesda, MD. Study No. T4638.501. April 1, 1986. Unpublished.

MRID 265457( 2) Putman, D. L., M. J. Zito, L. J. Belinsky, D. O. Azorsa, and F. K. Garvert ( 1986). Chromosome aberration assay in Chinese Hamster ovary ( CHO) cells. Microbiological Associates Inc. Bethesda, MD. Study No. T4638.337. May 1, 1986. Unpublished.

MRID 265457( 3) Curren, R. D., L. Dunn, M. Ernst, N. Durvasula, and V. Portner ( 1986). Unscheduled DNA Synthesis in rat primary hepatocytes. Microbiological Associates Inc. Bethesda, MD. Study No. T3638.380. May 5, 1986. Unpublished MRID 42009201 Federici, T. M. ( 1991). A 90 Day Subchronic Oral Toxicity Study in Rats with DMH. Exxon Biomedical Sciences, Inc. East Millstone, N. J. Lab study No. 169070. July 25, 1991.

MRID 42173901 Selim, S. ( 1991). Absorption, Distribution, Metabolism and Excretion ( ADME) Studies of 5,5 Dimethylhydantoin in the Rat. Lonza Inc. Fair Lawn, N. J. Lab study No. P01982. November 17, 1991.

MRID 42123802 Selim, S. ( 1991). Absorption, Distribution, Metabolism and Excretion ( ADME) Studies of 5 Ethyl, 5 Methylhydantoin in the Rat. Lonza, Inc. Fair Lawn, N. J. Study No. PO2000. November 15, 1991.

MRID 42205401 Beyer, B. K. ( 1992). Developmental Toxicity Study in Rabbits with 5 Ethyl 5 Methylhydantoin ( MEH). Exxon Biomedical Sciences, Inc., Toxicology Laboratory, East Millstone, NJ 08875 2350. February 3, 1992. Laboratory Project ID. 166834RB. MRID 42205401. Unpublished.

MRID 42413101 Nemec, M. D. ( 1992). A Developmental Toxicity study of Dimethylhydantoin in Rabbits. WIL Research Laboratories, Inc. Ashland, OH. Lab study No. WIL 12174. July 23, 1992.

MRID 42432701 Driscoll, C. D. and T. L. Neeper Bradley ( 1992). Developmental toxicity Evaluation of 5,5 Dimethylhydantoin ( DMH) Administered by Gavage to CD Rats. Bushy Run Research Center. Export, PA. Study No. 91N0048. July 30, 1992. Page 30 of 41 MRID 42462502 Nemec, M. D. ( 1992). Two generation Reproduction Study of Dimethylhydantoin Administered Orally in Rats. WIL Research Laboratories, Inc. Ashland, OH. Study No. WIL 12153 August 25, 1992.

MRID 43173901 Chun, J. S. and K. A. Loughran ( 1994). Ninety Day Dermal Toxicity Study with 5,5 Dimethylhydantoin ( DMH) in CD Rats. Bushy Run Research Center, Union Carbide Corp. 6702 Mellon Road, Export, PA. Study No. 92N1016. March 10, 1994.

MRID 43290601 Neeper Bradley, T. and M. Kubena ( 1994). Two Generation Reproduction Study in CD Rats with ( inert ingredient) Administered in the Diet. Bushy Run Research Center. Lab project No. 91N0094. Unpublished.

MRID 43397701 Hermansky, S. J. and Loughran ( 1994). Chronic Dietary Oncogenicity Study with 5,5 Dimethylhydantoin ( DMH). Bushy Run Research Center, Union Carbide Corp. 6702 Mellon Road, Export, PA. Lab study No. 91N0112. August 31, 1994.

MRID 43397702 Hermansky, S. J. and C. L. Benson ( 1994). Chronic Dietary Toxicity/ Oncogenicity Study with 5,5 dimethylhydantoin ( DMH) in Rats. Bushy Run Research Center, Union Carbide Corp. 6702 Mellon Road, Export, PA. Lab project No. 91N00113. August 31, 1994.

MRID 43654101 Naas, D. ( 1995). An Acute Inhalation Toxicity Study of BCDMH in Albino Rats. WIL Research Labs, Inc. Lab project No. WIL 12358. Unpublished.

MRID 4553101 Goldenthal, Edwin I. ( 1995). Evaluation of Dimethylhydantoin ( DMH) in a One Year Chronic Dietary Toxicity Study in Dogs. Lonza Inc. 17 17 Route 208, Fair Lawn, NJ. Study No. 647 004.

MRID 44063901 Naas, D. J. ( 1996). 18 Month Dietary Oncogenicity Study in Mice with DMH. WIL Research Laboratories, Inc. Ashland, OH. Lab Study No. WIL 12257. May 23, 1996. Unpublished.

MRID 44095901 Naas, D. ( 1996). Combined 24 month toxicity/ oncogenicity study in rats with DMH. WIL Research Laboratories, Inc. Ashland, Ohio. Lab study No. WIL 12258. July 30, 1996. Unpublished.

MRID 44243001 supplement to multi generation reproduction Page 31 of 41 10.0 APPENDICES Tables for Use in Risk Assessment Page 32 of 41 10.1 Toxicity Profile Summary Tables 10.1.1 Acute Toxicity Table  See Section 4.1 10.1.2 Subchronic, Chronic and Other Toxicity Tables Guideline No./ Study Type/ Substance Purity MRID No. ( year)/ Classification/ Doses Results Subchronic Toxicity 870.3100 90 Day oral toxicity rodents  Rat 1DMH purity > 99.5% 42009201 ( 1991) Acceptable/ guideline0, 100, 300, 1000 mg/ kg/ day NOAEL = greater than or equal to 1000 mg/ kg/ day ( highest dose tested) LOAEL = greater than 1000 mg/ kg/ day ( not established).

28 Day oral toxicity  Mouse DMH purity 100% 45738402 NOAEL = greater than or equal to 50,000 ppm [ males: 10,057, females: 14,972 mg/ kg/ day] ( highest dose tested) LOAEL = greater than 50,000 ppm [ males: 10,057, females: 14,972 mg/ kg/ day] ( not established).

870.3150 90 Day oral toxicity in nonrodents No study available.

870.3200 21/ 28 Day dermal toxicity No study available.

870.3250 90 Day dermal toxicity DMH purity 99.8% 43173901 ( 1994) core/ guideline 0, 39, 130, 390 mg/ kg/ day This study is classified as Acceptable Guideline and it satisfies the guideline requirements for a subchronic toxicity study [ OPPTS 870.3250 ( § 82 1b)] in rats. Higher doses of DMH were not possible in this study; thus, although a limit dose was not tested, the study is considered acceptable. NOAEL = greater than or equal to 390 mg/ kg/ day ( highest dose tested) LOAEL = greater than 390 mg/ kg/ day ( not established) Page 33 of 41 Guideline No./ Study Type/ Substance Purity MRID No. ( year)/ Classification/ Doses Results 870.3465 90 Day inhalation toxicity No study available.

Developmental/ Reproductive Toxicity 870.3700 Prenatal developmental in rats DMH purity 99.8% 42432701 ( 1992) Core Minimum/ Guideline 0, 100, 300, 1000 mg/ kg/ day Maternal Toxicity NOAEL = 300 mg/ kg/ day Maternal Toxicity LOAEL = 1000 mg/ kg/ day based on the transient decrease in body weight gain during gestation days 9 12.

Developmental Toxicity NOAEL = greater than or equal to1000 mg/ kg/ day ( highest dose tested) Developmental Toxicity LOAEL = greater than 1000 mg/ kg/ day ( not established)

870.3700 Prenatal developmental in nonrodents  Rabbits DMH purity 96 98.7% 42413101 ( 1992) core/ guideline 0, 100, 500, 1000 mg/ kg/ day Maternal Toxicity NOAEL = 500 mg/ kg/ day Maternal Toxicity LOAEL = 1000 mg/ kg/ day based on decreased body weight gain and food consumption during the dosing period.

Developmental Toxicity NOAEL = 100 mg/ kg/ day Developmental Toxicity LOAEL = 500 mg/ kg/ day based on the increased incidence of the 27th presacral vertebrae.

870.3700 Prenatal developmental in nonrodents  Rabbits 2EMH purity > 99.5% 42205401 ( 1992) acceptable Phase I: 0, 50, 125, or 375 mg/ kg/ day Phase II: 0 or 1000 mg/ kg/ day Maternal Toxicity NOAEL = 375 mg/ kg/ day Maternal Toxicity LOAEL = 1000 mg/ kg/ day, based on mortality, reduced body weights gains and reduced food consumption.

Developmental Toxicity NOAEL = 375 mg/ kg/ day Developmental Toxicity LOAEL = 1000 mg/ kg/ day, based on decreased fetal body weights.

870.3800 Reproduction and fertility effects DMH purity 96 98.7% 42462502 ( 1992) Core/ Guideline 0, 250, 500, 1000 mg/ kg/ day ( 20,000 ppm)

20,000 ppm ( 1322 and 1602 mg/ kg/ day, respectively) for male and female rats Parental/ Systemic NOAEL = greater than or equal to1000 mg/ kg/ day ( highest dose tested) Parental/ Systemic LOAEL = greater than 1000 mg/ kg/ day ( not established).

Offspring Toxicity NOAEL = greater than or equal to1000 mg/ kg/ day ( highest dose tested) Offspring Toxicity LOAEL = greater than 1000 mg/ kg/ day ( not established).

Chronic Toxicity/ Carcinogenicity Page 34 of 41 Guideline No./ Study Type/ Substance Purity MRID No. ( year)/ Classification/ Doses Results 870.4100 Chronic toxicity rodents DMH purity 99.8% 43397702 ( 1994) core/ guideline [ 870.4300 ( § 83 5)] 0, 100, 300, 1000 mg/ kg/ day Systemic Toxicity NOAEL = 300 mg/ kg/ day Systemic Toxicity LOAEL = 1000 mg/ kg/ day, based on decreases in body weight and body weight gain in females and hyperplasia of submandibular lymph nodes in males.

No evidence of carcinogenicity 870.4100 Chronic toxicity rodents DMH purity 97.3%, 97.1%, and 93.5% 44095901 ( 1996) acceptable/ guideline 0, 100, 320, 1000 mg/ kg/ day Systemic Toxicity NOAEL = 320 mg/ kg/ day Systemic Toxicity LOAEL = 1000 mg/ kg/ day, based on enlarged pituitary glands in decedents of both sexes, increased mortality occurring early in ( especially) females, increased mammary galactoceles in early decedents of both sexes and testicular fibroid vascular degeneration in early decedent males.

At the doses tested, there was not a treatment related increase in tumor incidence after 105 weeks of treatment with DMH. No evidence of carcinogenicity 870.4100 Chronic toxicity dogs DMH purity 97.4% 43813301 ( 1996) NOAEL = greater than 1000 mg/ kg/ day ( highest dose tested LOAEL = greater than 1000 mg/ kg/ day ( not established)

870.4100 Chronic toxicity dogs DMH purity 98.9% 43553101 ( 1995) acceptable Males: 0, 120, 342, or 1506 mg/ kg/ day Females: 0, 121, 414, or 1352 mg/ kg/ day NOAEL = 12000 ppm ( 342 mg/ kg/ day) LOAEL = 40000 ppm ( 1506 mg/ kg/ day), based on enlarged adrenal glands in male dogs, shown by microscopic examination to exhibit hypertrophy in the adrenal cortex.

870.4200 Carcinogenicity rats DMH purity 99.8% 43397702 ( 1994) core/ guideline 0, 100, 300, 1000 mg/ kg/ day See Chronic Toxicity in Rodents  Rats No evidence of carcinogenicity There were no increases in tumor incidences for the DMH treated groups when compared to the control group. This study is classified as 870.4200 Carcinogenicity rats DMH purity 97.3%, 97.1%, and 93.5% 44095901 ( 1996) acceptable/ guideline 0, 100, 320, 1000 mg/ kg/ day See Chronic Toxicity in Rodents  Rats No evidence of carcinogenicity Page 35 of 41 Guideline No./ Study Type/ Substance Purity MRID No. ( year)/ Classification/ Doses Results 870.4300 Carcinogenicity mice DMH purity 99.8% 43397701 ( 1994) Acceptable guideline [ 870.4200 ( § 83 2)] 0, 400, 1850, 8500 ppm ( 0, 100, 300, 1000 mg/ kg/ day) Systemic Toxicity NOAEL = 300 mg/ kg/ day Systemic Toxicity LOAEL = 1000 mg/ kg/ day based on decreases in body weight and body weight gain in males.

No evidence of carcinogenicity 870.4300 Carcinogenicity mice DMH purity 94.9 97.1% 44063901 ( 1996) Acceptable/ guideline [ 870.4200 ( § 83 2b)] 0, 100, 320, 1000 mg/ kg/ day Systemic Toxicity NOAEL = greater than or equal to 1000 mg/ kg/ day ( highest dose tested). Systemic Toxicity LOAEL = greater than 1000 mg/ kg/ day ( not established).

Under the conditions of this study, there was no evidence of carcinogenic potential in mice following treatment with DMH and the dosing was considered adequate because the highest dose of 1000 mg/ kg/ day represents a " limit dose."

No evidence of carcinogenicity Mutagenicity Gene Mutation 870.5100 Ames Salomella Assay DMH purity 97.0% 164036 ( 265457) ( 1986) acceptable 100, 500, 2500, 5000, 10000

g/ plate No evidence of a mutagenic response Gene Mutation 870.5100 Ames Salomella Assay EMH purity not reported 137095 ( 1982) acceptable 67, 333, 1667, 3333 or 6666 µ g/ plate No evidence of a mutagenic response Gene Mutation 870.5100 Ames Salomella Assay DMH purity not reported 137100 ( 1982) acceptable No evidence of a mutagenic response Page 36 of 41 Guideline No./ Study Type/ Substance Purity MRID No. ( year)/ Classification/ Doses Results Gene Mutation 870.5300 In vitro Mammalian Cell Assay EMH purity not reported 137089 ( 1982) acceptable S9: 751, 1001, 1335, 1780, 2373, 3164, 4219, 5000, 5625, 7500 µ g/ mL S9+: 563, 751, 1001, 1780, 2373, 3164, 4219, 5625, 7500, 10,000 µ g/ mL No evidence of genotoxic effects Gene Mutation 870.5300 In vitro Mammalian Cell Assay DMH purity not reported 132165 ( 1983) acceptable S9: 563, 1001, 1335, 1780, 2373, 3164, 4219, 5625, 7500, or 10,000

g/ mL. S9+: 751, 1001, 1335, 1780, 2373, 3164, 4219, 5625, 7500, or 10,000

g/ mL. No evidence of a genotoxic response Cytogenetics 870.5375 In vitro Chromosome Aberration Assay DMH purity 97.0% 164037 ( 265457) ( 1986) acceptable 200, 500, 1000, 2000

g/ mL No cytotoxic or clastogenic effects observed Cytogenetics 870.5375 In vitro Chromosome Aberration Assay 3DEMH purity not reported 40348201 ( 1987) acceptable S9: 8, 15, 30, or 60 µ g/ ml S9+: 15, 30, 60, or 120 µ g/ ml No evidence of structural chromosomal aberration Cytogenetics 870.5375 In vitro Chromosome Aberration Assay EMH purity not reported 137096 ( 1982) acceptable 5739.80, 7653.06, or 10204.08 µ g/ ml A 2 fold or greater increase in the percentage of cells with structural chromosomal aberrations, excluding gaps, was seen at two non consecutive doses after treatment in the presence of S9 mix. No dose response was evident. Results from the treatments in the absence of S9 mix were negative at all doses. No increase in polyploid cells was observed in treated or in positive control cultures. Page 37 of 41 Guideline No./ Study Type/ Substance Purity MRID No. ( year)/ Classification/ Doses Results Cytogenetics 870.5375 In vitro Chromosome Aberration Assay DMH purity not reported 137101 ( 1982) acceptable S9: 11250, 15000, or 20,000 µ g/ ml S9+: 8457.5, 11250, or 15000 µ g/ ml. No evidence of structural chromosomal aberration Cytogenetics 870.5375 In vitro Chromosome Aberration Assay DMH purity not reported MRID: N/ A TRID: 470264004 ( 1986) acceptable 80, 400, or 800 µ g/ ml No evidence of structural chromosomal aberration Cytogenetics 870.5375 In vitro Chromosome Aberration Assay DMH purity 100% MRID: N/ A TRID: 470264005 ( 1986) acceptable 10, 20, 40, 80, 160, 320, 640, 1280, 2560, 5120, 10240, or 20480 µ g/ ml No evidence of structural chromosomal aberration Other Effects 870.5550 Unscheduled DNA Synthesis Assay in Rat Hepatocytes DMH purity 97.0% 164038 ( 265457) ( 1986) acceptable 100, 300, 600, 1000, 3000, 6000, 10000

g/ mL No evidence of a genotoxic response Other Effects 870.5550 Unscheduled DNA Synthesis Assay in Rat Hepatocytes EMH purity not reported 137097 ( 1982) acceptable 0.1, 0.5, 1, 5, 10, or 15 mg/ mL No evidence of a genotoxic response Other Effects 870.5550 Unscheduled DNA Synthesis Assay in Rat Hepatocytes DMH purity not reported 132166 ( 1982) acceptable 0.001, 0.01, 0.1, 1, 10, or 20 mg/ ml No evidence of a genotoxic response Neurotoxicity Page 38 of 41 Guideline No./ Study Type/ Substance Purity MRID No. ( year)/ Classification/ Doses Results 870.6300 Developmental neurotoxicity Not required Metabolism/ Pharmacokinetics 870.7485 Metabolism and pharmacokinetics DMH purity radiolabeled 98.8% nonradiolabeled 99.5% 42173901/ 42123802 ( 1991) acceptable 100, 1000 mg/ kg DMH was rapidly absorbed and excreted, primarily in the urine (> 89%). There was little accumulation in the tissues.

1 DMH= dimethylhydantoin 2 EMH= ethylmethylhydantoin 3 DEMH= dichloroethylmethylhydantoin Page 39 of 41 10.2 Summary of Toxicological Dose and Endpoints for 5,5 dimethylhydantoin for Use in Human Risk Assessment1 Exposure Scenario Dose Used in Risk Assessment, UF FQPA SF and Endpoint for Risk Assessment to be completed later for risk assessment Study and Toxicological Effects Acute Dietary females 13 50 years of age NOAEL = 100 mg/ kg/ day UF = 100 Acute RfD = 1 mg/ kg FQPA SF = 1 aPAD = acute RfD FQPA SF = 1 mg/ kg/ day developmental toxicity  rabbit developmental LOAEL = 500 mg/ kg/ day based on skeletal variations. ( MRID 42413101)

Chronic Dietarya all populations NOAEL= 300 mg/ kg/ day UF = 100 Chronic RfD ( gen Pop.) = 3 mg/ kg/ day FQPA SF = 1 cPAD = chr RfD FQPA SF = 3 mg/ kg/ day chronic toxicity/ carcinogenicity  rats LOAEL = 1000 mg/ kg/ day based on decreased body weight/ weight gain and lymph node hyperplasia. ( MRID 43397702)

Chronic Dietarya females 13 50 NOAEL= 100 mg/ kg/ day UF = 100 Chronic RfD ( females 13 50) = 1 mg/ kg/ day FQPA SF = 1 cPAD = chr RfD FQPA SF = 1 mg/ kg/ day developmental toxicity  rabbit developmental LOAEL = 500 mg/ kg/ day based on skeletal variations. ( MRID 42413101)

Short Term Oral ( 1 7 days)

( Incidental) oral study NOAEL= 500 mg/ kg/ day MOE = 100 ( Residential, includes the FQPA SF) developmental toxicity  rabbit maternal LOAEL = 1000 mg/ kg/ day based on decreased body weight gain in maternal rabbits. ( MRID 42413101)

Intermediate Term Oral ( 1 week  several months)

( Incidental) oral study NOAEL= 300 mg/ kg/ day MOE = 100 ( Residential, includes the FQPA SF) subchronic oral toxicity  rat LOAEL = 1000 mg/ kg/ day based on decreased body weight and liver weight. ( MRID 42009201) Page 40 of 41 Exposure Scenario Dose Used in Risk Assessment, UF FQPA SF and Endpoint for Risk Assessment to be completed later for risk assessment Study and Toxicological Effects Short Term Dermal ( 1 7 days)

( Occupational/ Residential) dermal study NOAEL= 390 mg/ kg/ day ( HDT) MOE = 100 ( Occupational)

MOE = 100 ( Residential, includes the FQPA SF) subchronic dermal toxicity  rats No systemic toxicity at the highest dose tested ( MRID 43173901)

Intermediate Term Dermal ( 1 week  several months)

( Occupational/ Residential) dermal study NOAEL= 390 mg/ kg/ day ( HDT) MOE = 100 ( Occupational)

MOE = 100 ( Residential, includes the FQPA SF) subchronic dermal toxicity  rats No systemic toxicity at the highest dose tested Long Term Dermal ( several months  lifetime)

( Occupational/ Residential) dermal stud1y NOAEL= 390 mg/ kg/ day ( HDT) MOE = 100 ( Occupational)

MOE = 100 ( Residential, includes the FQPA SF) subchronic dermal toxicity  rats No systemic toxicity at the highest dose tested Short Term Inhalation ( 1 7 days)

( Occupational/ Residential) Oral NOAEL= 100b mg/ kg/ day ( inhalation absorption rate = 100%) MOE = 100 ( Occupational)

MOE = 100 ( Residential, includes the FQPA SF) developmental toxicity  rabbit developmental LOAEL = 500 mg/ kg/ day based on skeletal effects in offspring. ( MRID 42413101)

1 UF = uncertainty factor, FQPA SF = FQPA safety factor, NOAEL = no observed adverse effect level, LOAEL = lowest observed adverse effect level, PAD = population adjusted dose ( a = acute, c = chronic) RfD = reference dose, MOE = margin of exposure AThe HIARC selected separate chronic RfDs for females, ages 13 50, and the general population because this was an unusual case where the developmental NOAEL was lower than the lowest Page 41 of 41 NOAEL available for chronic toxicity. The rabbit developmental toxicity was therefore selected to provide adequate protection for the female ( age 13 50) subpopulation.

BA 100% inhalation absorption value is used for route to route extrapolation.