sodium 4,4-dimethyl-2,5-dioxoimidazolidin-1-ide

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed to GLP and guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

5,5-dimethylhydantoin

EC Number:

201-051-3

EC Name:

5,5-dimethylhydantoin

Cas Number:

77-71-4

IUPAC Name:

5,5-dimethylimidazolidine-2,4-dione

Constituent 2

Reference substance name:

dimethylhydantoin

IUPAC Name:

dimethylhydantoin

Details on test material:

- Name of test material (as cited in study report): 5,5-Diemthylhydantoin (DMH)
- Structural formula attached as image file (if other than submission substance): see Data Matrix attachment in Section 13 for DMH structural formula
- Physical state: white crystalline solid
- Stability under test conditions: stable under laboratory storage conditions

Test animals

Species:

rat

Strain:

other: outbread albino CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Testing Lab
- Age at study initiation: (P) 6 wks
- Weight at study initiation: (P) Males: 199.0-199.5 g; Females: 162.3-163.1 g
- Housing: individually, except during the cohabitation and the lactation period
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: ~ 2 weeks

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh diet was prepared and offered to the animals each week.
- Mixing appropriate amounts with (Type of food): Crystalline DMH was milled prior to diet preparation for one hour. A concentrated premix was then prepared by direct addition of milled DMH to ground rodent feed and mixed for one hour. The test diets were prepared by appropriate dilutions of the concentrated premix or next higher diet concentration with ground rodent feed and were mixed in a Hobart mixer for 15 minutes.
- Storage temperature of food: Diets were stored in polyethylene containers at room temperature until they were fed to the animals.

Details on mating procedure:

- M/F ratio per cage: One male to one female
- Length of cohabitation: Animals were paired for seven days; after the first seven days of the mating period, females of unsuccessfully mated pairs were placed with males of other unmated paris within the same dose group; after an additional seven days, unsuccessfully mated pairs were switched again for a period of seven days or until successful mating had occurred, whichever came first, allowing for a period of 21 days to mate.
- Proof of pregnancy: Dropped copulation plug.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Results on stability analyses conducted on diets containing 2000 or 20000 ppm DMH indicated that DMH was stable for at least 14 days in open glass feed jars and for at least 21 days in closed polyethylene containers when stored at room temperature. Results of the homogeneity analyses indicated that the distribution of DMH in the test diets was uniform. Concentration verification analyses of the prepared test diets showed analytical values ranging from 91.0 % to 109.0% of nominal.

Duration of treatment / exposure:

P and F1 generation were exposed to the diet for a 10 week pre-breed period, a 21 day mating period and through gestation, parturition and lactation.
F2 generation were exposed through lactation and until one week after weaning.

Frequency of treatment:

Not specified

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 70 days of age.
- Age at mating of the mated animals in the study: 10 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
2000 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
6000 ppm
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
20000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

28/sex/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
Selection of dose levels was based on the results of previous toxicity studies in rats including a 90-day oral gavage study in rats, a 14-day palatability study, and interim results from a chronic dietary study conducted at BRRC.

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality and moribund status and once daily for any clinical signs of toxicity. Detailed clinical examinations were conducted weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly.

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

Oestrous cyclicity (parental animals):

Not evaluated

Postmortem examinations (parental animals):

HISTOPATHOLOGY
The following tissues from the control and high dose groups were prepared for microscopic examination : vagina, uterus, ovaries, testis, epididymis, seminal vesicle, prostate and other tissues with gross lesions.
The following tissues from the low and intermediate dose groups were prepared for microscopic examination : testes and epididymides from males which did not sire litters and any tissues which exhibited gross lesions.

ORGAN WEIGHTS
Not evaluated

Postmortem examinations (offspring):

HISTOPATHOLOGY
The following tissues from the control and high dose groups were prepared for microscopic examination : vagina, uterus, ovaries, testis, epididymis, seminal vesicle, prostate and other tissues with gross lesions.
The following tissues from the low and intermediate dose groups were prepared for microscopic examination : testes and epididymides from males which did not sire litters and any tissues which exhibited gross lesions.

ORGAN WEIGHTS
Not evaluated

Statistics:

The data for quantitative continuous variables were intercompared for the 3 treatment groups and the control group by use of Levene's test for equality of variances, analysis of variance (ANOVA), and t-tests. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated similar variances, and the ANOVA was significant, a pooled t-test was used for pairwise comparisons. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by a separate variance t-test for pairwise comparisons.
Non-parametric data were statistically evaluated using the Kruskal-Wallis test followed by the Mann-Whitney U test when appropriate. Incidence data were compared using the Fischer's exact test. Statistical analyses were performed using BMDP statistical software (Dixon, 1990). For all statistical tests, the probability value of < 0.05 (two-tailed) was used as the critical level of significance.

Reproductive indices:

Mating index, fertility index, no pregnancy, gestational length, fecundity index, gestation index.

Offspring viability indices:

Number and sex of pups
Weight gain
Physical abnormalities

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS (PARENTAL ANIMALS)
No mortality or treatment-related clinical signs of toxicity were observed for males and females during the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Gestational Body Weights and Food Consumption:
Parent males: In the 20000 ppm treatment group consistently increased food consumption values and increases in body weight were noted throughout the treatment period.
Parent females: maternal food consumption was consistently increased in the 20000 ppm treatment group during gestation. No differences in maternal weights or weight gains were observed in any dose groups throughout the gestational period.

Lactational Body Weights and Food Consumption:
Maternal body weights were equivalent across groups during lactation. However, the routine body weights were equivalent across groups during lactation. However, the routine body weight loss experienced during the last week of lactation for all groups of females was less for females in the 20000 ppm group than for females in the control group.

There were no treatment-related differences in food consumption during lactation.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no effects of treatment on mating, fertility, gestational indices or gestational length.

GROSS PATHOLOGY & HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related findings from necropsy and histopathologic evaluations of treated F0 males and females. All F0 parents survived to scheduled sacrifice.

Effect levels (P0)

1

Dose descriptor:

NOEL

Effect level:

1 395 mg/kg bw/day

Sex:

male

2

Dose descriptor:

NOEL

Effect level:

1 774 mg/kg bw/day

Sex:

female

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFF-SPRING)
There were no treatment-related effects on F1 pup viability or survival.

CLINICAL SIGNS (OFFSPRING)
F1 males: No treatment-related clinical signs of toxicity were observed in F1 males fed the DMH test diets. One male from the 20000 ppm dose group was found dead on day 104 (following the mating period). The cause of death for this animal could not be determined from clinical signs of toxicity or necropsy findings.
F1 females: No treatment-related mortality or clinical signs of toxicity were observed in females from any dose group. One female from the 6000 ppm group was sacrificed after weaning of her litter due to clinical signs (ulceration in the inguinal region).

BODY WEIGHT (OFFSPRING)
F1 (males and females): Reduced body weights in the 20000 ppm treatment group in the last two weeks of lactation and one week after weaning (postnatal day 28). Weight gains substantially reduced from postnatal days 7 to 14 and slightly reduced from postnatal days 14 to 28. Occasional statistically significant increases in body weight gain were not considered to be treatment related due to the lack of a dose-response relationship or a consistent pattern of effects.
F2 (males): Reduced body weights in the 20000ppm treatment group for days 14-28 but not sufficient to reach statistical significance.
F2 (females): Reduced body weights in the 20000ppm treatment group for lactation days 14, 21 and one week after weaning.
Overall the body weight effects observed for F2 offspring were less than or no more severe than those observed in the F1 offsrping.

REPRODUCTIVE PERFORMANCE (OFFSPRING)
There were no treatment-related effects on reproduction parameters.
See table on reproductive performance in "Remarks on results including tables and figures"

SEXUAL MATURATION (OFFSPRING)
The number of pregnancy and fecundity index in F1 females were found similar to F0 parental number of pregnancy and fecundity index.

GROSS PATHOLOGY AND HISTOPATHOLOGY (OFFSPRING)
There were no lesions observed at scheduled necropsy of F1 weanlings which were attributed to treatment. There were no treatment-related lesions observed at necropsy of F1 pups which died during the lactational period.

OTHER FINDINGS (OFFSPRING)
Food consumption:
Females: There were no treatment-related effects on food consumption.
Males: Small increases in food consumption were noted for the F1 males.

Litter size and sex ratio:
There were no effects on litter size or sex ratio for any DMH-treated groups.

Effect levels (F1)

1

Dose descriptor:

NOEL

Generation:

F1

Effect level:

379 mg/kg bw/day

Sex:

male

2

Dose descriptor:

NOEL

Generation:

F1

Effect level:

475 mg/kg bw/day

Sex:

female

Results: F2 generation

Effect levels (F2)

1

Dose descriptor:

NOEL

Generation:

F2

Effect level:

6 000 ppm (nominal)

Sex:

male

2

Dose descriptor:

NOEL

Generation:

F2

Effect level:

6 000 ppm (nominal)

Sex:

female

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1. Effects of DMH oral doses at 2000, 6000 and 20000 ppm, to parental and offspring F1 and F2 reproductive performance and pup survival indice

Parameter			Control 0		low dose 2000 ppm		medium dose 6000 ppm		High dose 20000 ppm
		Generation	m	f	m	f	m	f	m	f
Reproductive Performance		F0 at F1 breed
Mating index (%)			100.0	100.0	100.0	100.0	96.4	100.0	96.4	100.0
Fertility index (%)			92.9	92.9	96.4	96.4	82.1	85.7	89.3	89.3
No. pregnant	Mean		26		27		24		25
Gestational length (days)	Mean		22.0		22.0		21.9		22.3
Fecundity index (%)			92.9	92.9	96.4	96.4	85.2	85.7	92.6	89.3
Gestation index (%)			100.0		100.0		100.0		100.0
Litter size	Mean		15.0		14.7		15.4		15.8
Sex ratio	Male/female		51.1		49.9		53.9		46.4
Pup survival indices
Live birth index		F1 pups	100.0		99.3		99.2		99.5
Lactation index			97.6		96.8		91.1		98.5
28 day survival index			99.5		100.0		100.0		100.0
Reproductive Performance		F1 at F2 breed
Mating index (%)			96.4	96.4	96.4	96.4	100.0	100.0	96.4	96.4
Fertility index (%)			75.0	82.1	82.1	82.1	89.3	89.3	82.1	82.1
No. pregnant	Mean		23		23		25		23
Gestational length (days)	Mean		22.1		22.1		22.1		22.1
Fecundity index (%)			77.8	85.2	85.2	85.2	89.3	89.3	85.2	85.2
Gestation index (%)			95.7		100.0		100.0		100.0
Litter size	Mean		14.0		13.9		14.6		14.3
Sex ratio	Male/female		43.7		51.3		48.9		51.6
Pup survival indices
Live birth index (%)		F2 pups	98.5		98.3		100.0		99.3
Lactation index (%)			95.6		93.5		94.5		96.7
28 day survival index (%)			100.0		99.5		100.0		99.4

Applicant's summary and conclusion

Conclusions:

In conclusion, continuous exposure to DMH in the diet for two generations did not result in parental toxicity or adverse effects on reproduction or reproductive tissues at dietary concentrations as high as 20000 ppm. Small increases in parental food consumption and body weight and slight transient decreases in offspring body weight were observed at the 20000 ppm dose level. The no-observed-effect level (NOEL) for parental animals and offspring in this study was 6000 ppm. The NOEL for reproductive effects was at least 20000 ppm.

The substance DMH is not a reproductive toxin in this study but there may be an effect on lactation.

Executive summary:

This study has been performed on DMH (Dimethyl Hydantoin) and has been used for read-across purposes.

In conclusion, continuous exposure to DMH in the diet for two generations did not result in parental toxicity or adverse effects on reproduction or reproductive tissues at dietary concentrations as high as 20000 ppm. Small increases in parental food consumption and body weight and slight transient decreases in offspring body weight were observed at the 20000 ppm dose level. The no-observed-effect level (NOEL) for parental animals and offspring in this study was 6000 ppm. The NOEL for reproductive effects was at least 20000 ppm. The substance DMH is not a reproductive toxin in this study but there may be an effect on lactation.