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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1981
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
The doses exceeded the upper limit stated in the OECD TG 414. Materials and method section is incomplete and in parts not comprehensive. Maternal effects were not specified. Main focus of this publication is the comparison of the teratogenic potential within a group of hydantoin derivatives, not a valid study of the substance hydantoin.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1982

Materials and methods

Test guideline
Qualifier:
no guideline followed
Version / remarks:
No guideline is stated in the publication.
Principles of method if other than guideline:
CD-1 dams were treated ip with the drugs on Days 8, 9, and 10 or Days 11, 12, and 13 of gestation (plug-positive day = Day 1). The drugs were administered as a suspension in methylcellulose (0.5%, w/v, 1 ml / 100 g body wt), with control animals receiving vehicle alone. Dams were killed by cervical dislocation on Day 18 and uterine contents were examined (Brown et al., 1979). Each live fetus was weighed and examined for gross and visceral abnormalities (Staples, 1974). Fetal heads were fixed and examined by the method of Wilson (1965) and skeletons were stained and examined by the method of Staples and Schnell (1964).
The doses utilized in the teratogenicity study were selected following evaluation of the adult lethality. The highest dose was within the 95% confidence limits of the calculated LD01, and a geometric progression of lower doses was utilized until no significant teratogenic or embryolethal effects were observed.
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Hydantoin
EC Number:
207-313-3
EC Name:
Hydantoin
Cas Number:
461-72-3
Molecular formula:
C3H4N2O2
IUPAC Name:
hydantoin
Test material form:
not specified
Specific details on test material used for the study:
Material was greater than 98% pure, as determined by GLC, TLC, and NMR techniques.

Test animals

Species:
mouse
Strain:
CD-1

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
other: methylcellulose (suspension, 0.5%, w/v, 1 mL/100 g body wt)
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The doses utilized in the teratogenicity study were selected following evaluation of the adult lethality (from the acute toxicity study). The highest dose was within the 95% confidence limits of the calculated LD01, and a geometric progression of lower doses was utilized until no significant teratogenic or embryolethal effects were observed.
Details on mating procedure:
Not specified
Duration of treatment / exposure:
6 days: Days 8, 9, and 10 or Days 11, 12, and 13 of gestation (plug-positive day = Day 1)
Frequency of treatment:
Daily on 3 consecutive days
Duration of test:
18 days
Doses / concentrationsopen allclose all
Dose / conc.:
20 other: mmol/kg
Remarks:
equivalent to 2001.54 mg/kg bw/day
Dose / conc.:
30 other: mmol/kg
Remarks:
equivalent to 3002.31 mg/kg bw/day
Dose / conc.:
40 other: mmol/kg
Remarks:
equivalent to 4003.08 mg/kg bw/day
No. of animals per sex per dose:
Treatment on Days 8, 9 and 10 of pregnancy:
Treated: Pregnant:
Control: 208 Control: 192
20 mmol/kg: 21 20 mmol/kg: 18
30 mmol/kg: 28 30 mmol/kg: 27
40 mmol/kg: 10 40 mmol/kg: 10

Treatment on Days 11, 12 and 13 of pregnancy:
Treated: Pregnant:
Control: 135 Control: 134
30 mmol/kg: 15 30 mmol/kg: 14
40 mmol/kg: 19 40 mmol/kg: 17
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
Not specified
Ovaries and uterine content:
Number of conceptuses: number/litter, resorptions and dead, live, malformed;
percentage embryo mortality/litter, percentage malformations/litter
Fetal examinations:
Fetal weight/litter, number of affected fetuses: external abnormalities (cleft palate, exen-cephaly, open eyes, others), visceral abnormalities (cardiovascular, others), skeletal abnormalities (ribs or vertebrae, others)
Statistics:
Fetal weights, embryo mortality, and malformation percentages are reported as mean values/litter +/- SE, with statistical comparisons to control parameters by the Mann-Whitney U test (Siegel, 1956). Dose-response relationships for adult lethality, teratogenicity, embryolethality, and combined teratogenicity and embryonic death were examined by probit analysis, using an SAS program (SAS, 1979).
x2 tests were utilized to determine the goodness of fit of the observed data to the probit model. Probability values (p) are computed from standard distribution of x2 with k - 2 degrees of freedom (k = number of doses used). When little heterogeneity was observed (p > 0.10) confidence limits were computed using a t value of 1.98 (probability 0.05, co df). When probability from the x2 test was less than 0.10, the heterogeneity factor, h, was introduced (h = x2/(k - 2), Finney, 1971a).
Variances and covariances were multiplied by this factor, and we computed confidence limits using the t value at probability 0.05, k - 2 df. This procedure has the effect of widening considerably the computed confidence limits when heterogeneity is observed (Finney, 1971a).
The relative teratogenic index was calculated at LD01/tD05 as discussed elsewhere (Fabro et al., 1982).
Tests for parallelism between dose-response curves were performed by a computer program based upon the method of Finney (1971a).
The linear free energy related (LFER), or physiochemical, approach to quantitative structure-activity relationships (QSAR) was taken in the analysis of structure-toxicity variations.
Indices:
Not specified

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
In the group treated with 40 mmol hydantoin/kg on days 11, 12 and 13, two dams died before day 18. No other mortalities occurred after hydantoin exposure.
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
Treatment on Days 8, 9 and 10 of pregnancy:
Resorptions and dead (number of litters):
Control: 243 (125)
20 mmol/kg: 16 (11)
30 mmol/kg: 30 (18)
40 mmol/kg: 8 (7)

Treatment on Days 11, 12 and 13 of pregnancy:
Resorptions and dead (number of litters):
Control: 192 (92)
30 mmol/kg: 21 (11)
40 mmol/kg: 52 (13)
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
Treatment on Days 8, 9 and 10 of pregnancy:
Live (number of litters):
Control: 2143 (192)
20 mmol/kg: 215 (18)
30 mmol/kg: 318 (27)
40 mmol/kg: 109 (9)

Treatment on Days 11, 12 and 13 of pregnancy:
Live (number of litters):
Control: 1473 (134)
30 mmol/kg: 163 (14)
40 mmol/kg: 142 (16)
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Treatment on Days 8, 9 and 10 of pregnancy:
Treated: Pregnant:
Control: 208 Control: 192
20 mmol/kg: 21 20 mmol/kg: 18
30 mmol/kg: 28 30 mmol/kg: 27
40 mmol/kg: 10 40 mmol/kg: 10

Treatment on Days 11, 12 and 13 of pregnancy:
Treated: Pregnant:
Control: 135 Control: 134
30 mmol/kg: 15 30 mmol/kg: 14
40 mmol/kg: 19 40 mmol/kg: 17
Other effects:
not specified

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment on Days 8, 9 and 10 of pregnancy:
Fetal weight / litter:
Control: 0.97 +/- 0.01
20 mmol/kg: 0.89 +/- 0.01**
30 mmol/kg: 0.92 +/- 0.02*
40 mmol/kg: 0.83 +/- 0.02**

Treatment on Days 11, 12 and 13 of pregnancy:
Fetal weight / litter:
Control: 0.98 +/- 0.01
30 mmol/kg: 0.95 +/- 0.03*
40 mmol/kg: 0.87 +/- 0.02**

*,** Significantly different from control values, p < 0.05 and p > 0.01, respectively; Mann-Whitney U test
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
Treatment on Days 8, 9 and 10 of pregnancy:
Percentage embryo mortality/litter:
Control: 10.4 +/- 0.7
20 mmol/kg: 6.9 +/- 1.7
30 mmol/kg: 8.3 +/- 1.5
40 mmol/kg: 15.4 +/- 9.5

Treatment on Days 11, 12 and 13 of pregnancy:
Percentage embryo mortality/litter:
Control: 11.5 +/- 1.1
30 mmol/kg: 13.3 +/- 3.6
40 mmol/kg: 25.6 +/- 6.5**

** Significantly different from control values, p > 0.01; Mann-Whitney U test
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Treatment on Days 8, 9 and 10 of pregnancy:
Number of conceptuses per litter:
Control: 12.4 +/- 0.2
20 mmol/kg: 12.8 +/- 0.5
30 mmol/kg: 12.9 +/- 0.5
40 mmol/kg: 11.7 +/- 1.3

Treatment on Days 11, 12 and 13 of pregnancy:
Number of conceptuses per litter:
Control: 12.4 +/- 0.2
30 mmol/kg: 13.1 +/- 0.9
40 mmol/kg: 11.4 +/- 0.9
Changes in postnatal survival:
not specified
External malformations:
effects observed, treatment-related
Description (incidence and severity):
Treatment on Days 8, 9 and 10 of pregnancy (numbers (number of litters)):
Malfomed: Percentage malformations/litter: Cleft palate: Exencephaly: Open eyes: Others:
Control: 24 (23) Control: 1.2 +/- 0.2 7 (7) 5 (5) 0 2 (2)
20 mmol/kg: 1 (1) 20 mmol/kg: 0.4 +/- 0.4' 0 1 (1) 0 0
30 mmol/kg: 22 (8) 30 mmol/kg: 6.4 +/- 1.7** 1 (1) 4 (3) 2 (2) 1 (1)
40 mmol/kg: 8 (4) 40 mmol/kg: 8.1 +/- 3.8** 1 (1) 4 (2) 4 (2) 0

Treatment on Days 11, 12 and 13 of pregnancy:
Malfomed: Percentage malformations/litter: Cleft palate: Exencephaly: Open eyes: Others:
Control: 12 (10) Control: 1.1 +/- 0.3 8 (8) 2 (2) 2 (2) 2 (2)
30 mmol/kg: 0 (0) 30 mmol/kg: 0 0 0 0 0
40 mmol/kg: 9 (6) 40 mmol/kg: 5.2 +/- 2.0** 5 (5) 0 0 3 (3)

' Significant dose response, p < 0.05; Jonckheere´s test
** Significantly different from control values, p > 0.01; Mann-Whitney U test
Skeletal malformations:
no effects observed
Description (incidence and severity):
Treatment on Days 8, 9 and 10 of pregnancy (numbers (number of litters)):
Cardiovascular: Others:
Control: 1 (1) 0
20 mmol/kg 0 0
30 mmol/kg 0 0
40 mmol/kg 0 0

Treatment on Days 11, 12 and 13 of pregnancy:
Cardiovascular: Others:
Control: 1 (1) 0
20 mmol/kg 0 0
30 mmol/kg 3 (2) 0
40 mmol/kg 0 0
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment on Days 8, 9 and 10 of pregnancy (numbers (number of litters)):
Ribs or vertebrae: Others:
Control: 9 (8) 0
20 mmol/kg 0 0
30 mmol/kg 12 (8) 2 (2)
40 mmol/kg 3 (2) 1 (1)

Treatment on Days 11, 12 and 13 of pregnancy:
Ribs or vertebrae: Others:
Control: 0 0
20 mmol/kg 0 0
30 mmol/kg 0 0
40 mmol/kg 0 0
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
The incidence of malformed fetuses from vehicle-treated dams was 1.2% per litter for Days 8, 9, and 10 and 1.1% per litter for Days 11, 12, and 13.
Hydantoin induced significantly elevated incidences of malformed fetuses per litter with both treatment schedules. The incidence of malformations was higher following treatment on Days 8,9, and 10 than on Days 11, 12 and 13. There was a significant dose response (p < 0.05, Johnckheere’s test) on Days 8, 9, and 10. The incidence of malformations (Days 8, 9, and 10, LD01 dose) was as follows: 8.1% per litter. Following treatment on Days 8, 9, and 10, the most common defects were of the skeletal system. Fused or branched ribs were the most frequent skeletal abnormality for each compound, with fused or branched vertebral arches the next most common. A variety of other defects of the ribs, arches, and centra were also observed. Hydantoin induced cleft palate and exencephaly, which was accompanied by open eyes in most cases. The abnormalities induced by hydantoin treatment on Days 11, 12, and 13 was different from those profiles observed following earlier treatment. Cleft palate was the most common defect, plus low levels of exencephaly (with open eyes) and cardiovascular malformations. Hydantoin also caused intrauterine growth retardation, as shown by the dose-related reduction in mean fetal weight/litter. The effect on fetal weight was more marked with treatment on Days 8, 9, and 10, compared to Days 11, 12, and 13.

Any other information on results incl. tables

The relative teratogenic index gives an estimate of the relationship between general toxicity and specific developmental toxicity by calculating the ratio between adult toxicity (LD01) and teratogenicity (tD05). Thus, a compound with an RTI of 1 induces a significant incidence (5%) of malformation only at the minimally lethal dose while an RTI of 2 indicates effects at half the minimal lethal dose, etc. In this study, the compounds can be ranked as follows, in increasing order of relative teratogenic hazard: mephenytoin,

~0.9; hydantoin, 1.27; ethotoin, 1.39; phenytoin, 1.66; and phenacemide, 3.32.

Applicant's summary and conclusion

Conclusions:
The teratogenic potential of Hydantoin was investigated in pregnant CD-1 mice after animals were treated intraperitoneally on Days 8, 9, and 10 or Days 11, 12, and 13 of gestation. Hydantoin showed a teratogenic potential, however, the doses were well above the recommendation of the OECD TG 414 and maternal effects were not specified. Based on the available information, a classification of Hydantoin as teratogen is not justified.