Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
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:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 5,5-Dimethylhydantoin
- Physical state: white crystalline solid
- Analytical purity: 99.8%
- Lot/batch No.: NO432543
- Stability under test conditions: Not stated
- Storage condition of test material: ambient temperature

Test animals

Species:
rat
Strain:
other: CD
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Age at study initiation: 8 weeks at mating
- Weight at study initiation: females: 217.8-218.6 g
- Housing: 2 per stainless steel cage.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 66-77 °F
- Humidity (%): 40-70 %
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Each dosing solution was prepared by dissolving the appropriate amount of DMH with Milli-Q filtered water. Due to the high purity of the test substance, concentrations were not adjusted for percent active ingredient of test substance. Dosing solutions were prepared once and stored at room temperature.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of DMH in Milli-Q filtered water was analysed using HPLC.

HPLC parameters:
Instrument: Waters HPLC
Column: Waters µBondapack C-18 (3.9 mm x 30 cm) 10 micron
Column temperature: ambient
Mobile phase: 5% methanol/95% Milli-Q filtered weater, isocratic
Flowrate: 1.0 ml/min
Detector: Waters lambda Max 481 variable wavelength.
Wavelength: 230 nm
Sensitivity: 0.05 absorbance units full scale.
Injection volume: 20 µl
Limit of detection: ca. 0.01 mg/ml.

Differences between duplicate analyses did not exceed 15% and individual analyses were within ± 15% of nominal.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: 4 days
- Proof of pregnancy: the day when vaginal copulation plug was observed, was referred to as day 0 of pregnancy.
Duration of treatment / exposure:
Rats were dosed daily from gestation day 6 through 15
Frequency of treatment:
Single daily dose
Duration of test:
21 days in total
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
nominal in water
No. of animals per sex per dose:
25 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels of 0 (control), 100, 300, and 1000 mg/kg/day were selected by the Sponsor. The limit dose of 1000 mg/kg/day recommended for developmental; toxicity studies was selected as the high dose level. The mid and low dose levels were chosen on a half-log scale below the highest dose level.

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily (twice daily during dosing)

BODY WEIGHT: Yes
- Time schedule for examinations: gestation day 0, 6, 9, 12, 15, 18 and 21.

FOOD CONSUMPTION: Yes
- Time schedule: measured at three day intervals throughout the study (gestation day 0 to gd 21)

WATER CONSUMPTION: No

COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: liver, see ovaries and uterine contents.

LITER RESPONSE
- litter size, number of dead foetuses, sex ratio
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: cervix, vagina, peritoneal and thoracic cavities, uterus examined for signs of hemorrhage and dissected longitudinally to expose the contents, all live and dead fetuses, uteri from females that appeared nongravid were placed in a 10% ammonium sulfide solution for detection of early resorptions.
Fetal examinations:
- External examinations: Yes: all of the live fetuses per litter
- Soft tissue examinations: Yes: all of the live fetuses per litter
- Skeletal examinations: Yes: all of the live fetuses per litter
- Head examinations: Yes: half of the live fetuses per litter
- Thoracic and abdominal visceral abnormalities: Yes: approximately half of the live fetuses per litter
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 equal 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. Frequency data were compared using the Fischer's Exact Test. With the exception of the data analysis for fetal malformations an dvariations, all statistical analyses were performed using BMDP statistical software (Dixon, 1990). Frequency data for fetal malformations and variations were analysed using an internal computer program. For all statistical tests, the probability value of < 0.05 (two-tailed) was used as the critical level of significance.
Indices:
Pregnancy rate was equivalent for all groups and ranged from 92 to 100 %.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No females died, aborted, delivered early or were removed from the study prior to sacrifice. All females that bore litters had one or more viable fetuses.
No treatment-related clinical signs were observed during or subsequent to treatment at any dose level.
There were no dose related changes in maternal body weights or body weight changes throughout gestation. Statistically significant reductions in average weight gain for days 9-12 at 1000mg/kg/day were not considered to be related to treatment but rather were considered to reflect stabilization of body weight in the 1000mg/kg/day group which exhibited a slightly higher weight gain for days 6-9. There were no differences in body weight gain at 1000mg/kg/day for the entire treatment period.
There were no treatment-related effects on food consumption during or subsequent to dosing.
There were no treatment related findings observed at necropsy. There were no effects on terminal body weight, gravid uterine weight, corrected body weight, corrected weight change, or relative and absolute liver weights.

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No treatment-related effects on fetal body weights for male and female were observed in any group.
There were no differences in individual external, visceral or skeletal malformations by category, or in total malformations among groups. There were no treatment-related increases in the incidences of individual fetal external, visceral or skeletal variations by category, or of total variations among groups. Statistically significant decreases in 1 external variation, excessive bleeding at the umbilicus, and 1 skeletal variation, majority of proximal phalanges unossified at 1000mg/kg/day were not considered to be treatment related or biologically significant due to the lack of a dose-related trend.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1. Results for maternal effects

Parameters

Control group

100 mg/kg bw/d

300 mg/kg bw/d

1000 mg/kg bw/d

0 mg/kg bw/d

Number of dams examined

25

25

25

25

Mortality of dams (%)

0

0

0

0

Abortions

0

0

0

0

Body weight gain (mean (g))

 Days 15-21 (post treatment)

 Days 0 -21 (gestation)

88.29

150.96

 

86.85

153.57

 

88.15

154.01

 

85.50

151.05

Food consumption (mean (g))

Day 0 to 6 (pre-treatment)

Day 6 to 15 (treatment)

Day 15 to 21 (post treatment)

19.93

22.04

24.82 

 

19.42

22.00

25.01

 

19.82

21.50

24.35

 

19.89

21.66

24.55

Pregnancies (%)

92

100

100

100

Table 2. Results for teratogenic effects: litter response (Caesarean section data)

Parameters

Control group

100 mg/kg bw/d

300 mg/kg bw/d

1000 mg/kg bw/d

0 mg/kg bw/d

Corpora lutea (mean)

14.8

14.8

15.6

15.5

Implantations (mean)

14.2

14.2

14.6 

13.9

Early Resorptions (mean)

0.5

0.5

0.6

0.4

Late Resorptions (mean)

0.0

0.0

0.0

0.0

Live fetuses (%)

96.5

95.5

95.8

97.2

Dead fetuses (%)

0.0

0.1

0.0

0.0

pre-implantation loss (state %)

5.5

5.2

7.0

10.4

fetus weight (mean) [g]

5.076

5.088

5.064

4.975

Fetal sex ratio [% male fetuses]

52.0

47.0

47.8

51.5

Table 3: Examination of the fetuses

Parameters

Control group

100 mg/kg bw/d

300 mg/kg bw/d

1000 mg/kg bw/d

 0 mg/kg bw/d

External malformations*[%]

0.3

0.0

0.0

0.0

Soft tissue malformations*[%]

3.7

4.0

3.3

2.9

Skeletal malformations*[%]

0

0.0

0.6

0.0

External variations*[%]

13.3

13.9

15.1

13.0

Soft tissue variations*[%]

50.3

39.5

47.2

39.7

Skeletal variations*[%]

100

100

100

100

   

 

Applicant's summary and conclusion

Conclusions:
The substance is not a developmental toxin as no maternal toxic effects and no teratogenic/embryotoxic effects were observed.