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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Influence of dietary folic acid on the developmental toxicity of methanol and the frequency of chromosomal breakage in the CD-1 mouse.
Author:
Fu, S.S., Sakanashi, T.M., Rogers, J.M., Keen, C.L.
Year:
1995
Bibliographic source:
Teratology 51, 162-163
Reference Type:
publication
Title:
Influence of dietary folic acid on the developmental toxicity of methanol and the frequency of chromosomal breakage in the CD-1 mouse.
Author:
Fu, S.S., Sakanashi, T.M., Rogers, J.M., Hong, K.H., Keen, C.L.
Year:
1996
Bibliographic source:
Reprod. Toxicol. 10, 455-463
Reference Type:
publication
Title:
Folate deficiency alone does not produce neural tube defects in mice.
Author:
Heid, M.K. et al.
Year:
1992
Bibliographic source:
J. Nutr. 122, 1198-1200
Reference Type:
publication
Title:
Plasma formate levels in methanol treated dams are not markedly influenced by dietary folate.
Author:
Hong, K.H., Sakanashi, S.S., Fu, S.S., Rogers, J.M., Keen, C.L.
Year:
1997
Bibliographic source:
Teratology Society Abstract 55, 57

Materials and methods

Principles of method if other than guideline:
Treatment of pregnant female mice on GD 6-10 with test substance by oral administration twice daily, investigation of the influence of folate in the diet on incidence of developmental defects in the offspring.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
CD-1
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles Rivers, Inc., Gilroy, CA
- Age at study initiation: 8 weeks
- Weight at study initiation: group weights on GD0 (mean ± SEM): 25.72 ± 0.48 to 27.19 ± 0.73 g
- Fasting period before study: no data
- Housing: stainless steel wire-bottomed cages
- Diet: amino-acid based, folic-acid free diet supplemented with either 400 or 1200 nmol folic acid/kg diet and 1 % succinylsulfathiazole for 5 weeks prior to mating and throughout breeding and gestation
- Water (e.g. ad libitum): no data
- Acclimation period: 5 weeks (already fed with special diet during this period)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 50
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: not applicable
Vehicle:
water
Details on exposure:
VEHICLE
- Concentration in vehicle: 15.65 % in deionized water
- Amount of vehicle (if gavage): no data
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2 or 1/3
- Length of cohabitation: 12 h
- Further matings after two unsuccessful attempts: no (second breeding period was continued for up to 10 days)
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
GD 6-10
Frequency of treatment:
twice daily 2500 mg/kg bw
Duration of test:
until GD 18
Doses / concentrations
Dose / conc.:
5 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
21 to 24 dams per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: knowledge from previous experiments

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 5, 10, 12, 15, 18
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 18
- Organs examined: liver, kidney, gravid uterus, blood (plasma and hematocrit, folate concentrations, MN formation in maternal reticulocytes)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: fetal weights, crown-rump lengths, fetal liver folate concentrations, fetal hematocrit, MN formation in fetal reticulocytes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
The pregnant dam and the litter were considered the units for comparisons. Continuous variables were analyzed using the two-way analysis of variance procedure and the Fisher PLSD for multiple comparisons of means. These analyses were carried out on STATVIEW SE+ (Abacus, Berkeley, CA). Incidences of fetal malformations and frequency of micronuclei, based on affected litters, were analyzed using binomial statistics.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
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 examined
Changes in number of pregnant:
not examined
Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
5 000 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced mean fetal weight and reduced mean crown-rump length
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
not examined
Changes in sex ratio:
not examined
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
not specified
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
skeletal abnormalities (cleft palate, exencephaly)
Visceral malformations:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
LOAEL
Effect level:
5 000 mg/kg bw/day (nominal)
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
external: cranium

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
50 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
not specified

Any other information on results incl. tables

A. Folate levels (measured on gd 18, 8 d after the final methanol treatment):

Low-folate diet produced a decline in folate in the maternal liver (total folate approx. -30%), in maternal plasma (approx. -30%), in maternal erythrocytes (approx. -30%) vs. normal-folate diet and in the fetal liver (approx. -60 to -70%) (Fu et al., 1996).

Methanol had no marked influence on maternal and folate levels irrespective of folate supplementation, except in maternal plasma where there was some evidence of a reduction of about 20 % . Methanol treatment was slighty fetotoxic (reduced mean fetal weight and reduced mean crown-rump length), but had no impact other reproductive parameters. It showed some evidence of a teratogenic effect (increased incidences of cleft palate and exencephaly) under folate-adequate supply, but this was hardly statistically significant: cleft palate (2/222 vs. 0/282) and execephaly (5/222 vs. 1/282 in the respective high-folate control).

Likewise, folate deficiency failed to produce significant malformations (in accordance with previous reports: e.g. Heid et al., 1992): cleft palate (5/215 vs. 0/282) and execephaly (2/215 vs. 1/282 in the respective high-folate control). However, cleft palate, but not exencephaly was significantly increased in the presence of methanol: 39/235 vs. 5/215 and 8/235 vs. 2/215 of folate-poor control, respectively. Methanol did not induce micronuclei in maternal or fetal blood.

B. In pregnant CD-1 mice given methanol (5 g/kg/d) from gestation day 6 - 15, one group receiving folate deficient, the other folate supplemented diet, there were no differences in the formate-blood levels between both groups: 5.13 +-0.68 mmol/L (folate-def.) and 3.90 +-0.94 mmol/L (folate-suppl.) vs. 0.36 +-0.13 mmol/L (untreated control). But developmental toxicity was significantly higher in folate-deficient dams (Hong et al. 1997). The results indicate that increased plasma formate levels in dams do not underlie the increased developmental toxicity of methanol in mice fed low dietary folate (Hong et al. 1997).

CONCLUSION:

Folate deficiency enhanced the teratogenic effects of methanol in mice.

Applicant's summary and conclusion