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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
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:
EPA OPP 83-4 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 5,5-Diemthylhydantoin (DMH)
- Physical state: white crystalline solid
- Analytical purity: 99.8%
- Lot/batch No.: NO432543
- 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 and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- 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 / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
6000 ppm
Basis:
nominal in diet
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 assessed in the study report. The study in question was performed according to EPA guideline 83-4 in 1994. The EPA guideline at this time would have been equivalent to OECD 416 (1983). OECD 416 was however updated in 2001 7 years after the study had been performed. This situation is the same for the EPA guideline aswell.
Sperm parameters (parental animals):
Not assessed in the study report. The study in question was performed according to EPA guideline 83-4 in 1994. The EPA guideline at this time would have been equivalent to OECD 416 (1983). OECD 416 was however updated in 2001 7 years after the study had been performed. This situation is the same for the EPA guideline aswell.
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 animals)

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
Description (incidence and severity):
Not assessed in the study report. The study in question was performed according to EPA guideline 83-4 in 1994. The EPA guideline at this time would have been equivalent to OECD 416 (1983). OECD 416 was however updated in 2001 7 years after the study had b
Gross pathological findings:
no effects observed
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)

open allclose all
Dose descriptor:
NOEL
Effect level:
1 395 mg/kg bw/day
Sex:
male
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
Description (incidence and severity):
Not assessed in the study report. The study in question was performed according to EPA guideline 83-4 in 1994. The EPA guideline at this time would have been equivalent to OECD 416 (1983). OECD 416 was however updated in 2001 7 years after the study had b
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)

open allclose all
Dose descriptor:
NOEL
Generation:
F1
Effect level:
379 mg/kg bw/day
Sex:
male
Dose descriptor:
NOEL
Generation:
F1
Effect level:
475 mg/kg bw/day
Sex:
female

Results: F2 generation

Effect levels (F2)

open allclose all
Dose descriptor:
NOEL
Generation:
F2
Effect level:
6 000 ppm (nominal)
Sex:
male
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.

The reason that the robust summary dose not contain any information on organ weights among parental animals and offspring, estrous cyclicity among parental animals, and sperm parameters among parental animals is because they were not assessed in the study report. The study in question was performed according to EPA guideline 83-4 in 1994. The EPA guideline at this time would have been equivalent to OECD 416 (1983). OECD 416 was however updated in 2001 7 years after the study had been performed. The study should therefore be assessed according to the older guideline (1983) rather than the revised guideline as the 1983 guideline did not require the assessment of organ weights, estrous cyclicity and sperm parameters which are described as missing. This situation is the same for the EPA guideline aswell. The study should be considered as acceptable as it fulfilled the requirements of the testing guideline in existence at the time the study was performed.

This study has also been submitted for two Annex I BPD dossiers: Bromochlorodimethylhydantoin (BCDMH) which has been evaluated by The Netherlands CA and Dimethylol Dimethyl Hydantoin (DMDMH) which has been evaluated by the Polish CA.

The Netherlands evaluation of this study stated ‘The study was essentially performed according to OECD 416 (1983) the applicable guideline at the time. The fertility parameters oestrus cycle and sperm parameters and organ weights were not determined. As no indication that reproductive performance was affected and no histopathological effects were seen, this is acceptable’ and the study has been assigned a Klimish reliability score of 1 (Document IIIA6.8.2_1 dCAR published on the confidential part of CIRCA 4th February 2011)