Methylhydrazine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1984

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

intraperitoneal route of administration, treatment during days 6-15 of gestation, less than 20 females/group

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methylhydrazine

EC Number:

200-471-4

EC Name:

Methylhydrazine

Cas Number:

60-34-4

Molecular formula:

CH6N2

IUPAC Name:

methylhydrazine

Test material form:

liquid

Specific details on test material used for the study:

Provider: Eastman, Rochester, N.Y.

Test animals

Species:

rat

Strain:

Fischer 344

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass
- Housing: 3 per plastic cages containing woodchip bedding
- Diet (e.g. ad libitum): pelleted feed (Ralston Purina Co., St. Louis, Mo.), ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature : 72 ± 2°F
- Photoperiod (hrs dark / hrs light): 12-h light/dark cycle

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

physiological saline

Details on exposure:

All dose volumes were 2 mL/kg. Solutions were prepared daily in physiologic saline.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Two females were placed overnight with each male and checked for presence of sperm by vaginal wash the next morning. The day on which sperm was detected was considered d 0 of pregnancy.

Duration of treatment / exposure:

Days 6-15 of gestation

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2.5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

10 mg/kg bw/day (nominal)

No. of animals per sex per dose:

13 to 16

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Appropriate dose ranges for the three compounds were established during preliminary studies using groups of three or four pregnant animals. Parameters of toxicity observed in the higher dose preliminary treatment groups were maternal mortality and weight loss or markedly reduced weight gains and or convulsions (ranging from moderate to severe/resulting in mortality)
- The pregnant rats were sacrificed on gestation day 20 by overdose of halothane

Examinations

Maternal examinations:

The pregnant rats were weighed daily.

Ovaries and uterine content:

The uterus was opened and the numbers and positions of implants, dead fetuses, live fetuses, and resorptions were recorded

Fetal examinations:

Live fetuses were blotted dry, weighed, examined for external abnormalities, and divided into groups for visceral and skeletal examination (two-thirds and one-third of each litter, respectively). Fetuses for visceral examination were fixed in Bouin solution for at least 2 wk and were then examined by the freehand razor sectioning technique of Wilson (1965). The remaining fetuses were processed by an Alizarin red staining technique for skeletal examination (Dawson, 1926).

Statistics:

Maternal and fetal body weights and numbers of implants and resorptions were analyzed by the Student's t-test and are listed as mean ± standard deviation. Incidence of abnormalities was analyzed with Fisher's exact test. The litter was used as the experimental unit for those data that could be analyzed using either litter or fetus as the experimental unit, and a probability of p<0.05 was accepted as significant in all analyses.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Four of the pregnant females in the high-level treatment group convulsed following methylhydrazine injection on one or more occasions during the treatment period.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Maternal weight gains were significantly less in all treatment groups during the early (days 6-10) part of the treatment period and significantly less for the two higher treatment groups during the latter (days 11-16) part of the treatment period (See Table 1). Significant decreases in weight gains for treatment groups were not seen after cessation (days 17-20) of treatment; however, overall weight gains were lower than those of the control group (-14%, -29% and -41% of controls in the 2.5, 5 and 10 mg/kg bw/day groups, respectively).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

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:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

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:

effects observed, treatment-related

Description (incidence and severity):

High incidence of resorptions in the two higher dose groups

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

2.5 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

early or late resorptions

Maternal abnormalities

Abnormalities:

not examined

Results (fetuses)

Fetal body weight changes:

not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

not examined

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Intermediate dose group had a moderate increase in abnormalities involving the eyes (anophthalmia and/or microophthalmia) but the higher dose group had only a slight increase in abnormalities involving the eye

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not examined

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

2.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

Fetal abnormalities

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: eye

Description (incidence and severity):

Intermediate dose group had a moderate increase in abnormalities involving the eyes (anophthalmia and/or microophthalmia) but the higher dose group had only a slight increase in abnormalities involving the eye

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: Effects of methylhydrazine given intraperitoneally on days 6 -15 of gestation in the rat

	Methylhydrazine dose (mg/kg bw/day)
Effect	0 (13 litters)	2.5 (15 litters)	5 (15 litters)	10 (16 litters)
Gestation weight gain of females (g)^a
Days 6-10	8 ± 5	3 ± 3^b	2 ± 4^b	- 3 ± 3^b
Days 11-16	21 ± 5	19 ± 10	15 ± 5^b	12 ± 8^b
Days 17-20	30 ± 6	29 ± 10	25 ± 10	26 ± 12
Litter data
Implants/litter^a	7.8 ± 3.6	8.8 ± 3.4	7.3 ± 2.5	7.6 ± 3.4
Viable fetuses/litter^a	6.8 ± 4.0	7.5 ± 3.4	6.2 ± 2.7	6.1 ± 3.9
Fetal weight^a	3.1 ± 0.3	3.3 ± 0.3	3.1 ± 0.3	3.2 ± 0.3
Litters with ≥ 33% fetuses resorbed	0	2	3	3
Incidence of abnormalities:
Litters (fetuses) affected
Gross exam^c	2(2)	1(1)	3(4)	2(2)
Soft-tissue exam	1(1)^d	2(2)^e	6(9)^e	3(4)^f
Skeletal exam^g	0(0)	1(1)	1(1)	2(2)

^aMean ± standard deviation.

^bSignificant at ≤ 0.05 level.

^cAII nanoids (≤ control mean fetal weight minus 3 standard deviations).

^dOne fetus with anophthalmia and hydrocephalus.

^eAII anophthalmia or severe microophthalmia.

^fHydronephrosis and dilated ureter in 1 fetus, hydrocephalus in another, and 2 fetuses with anophthalmia.

^gAll unfused ossification centers of vertebrae.

Applicant's summary and conclusion

Conclusions:

All data that might suggest an embryotoxic or teratogenic effect were obtained with doses producing significant/marked maternal toxicity (marked decrease in bodyweight gain at the 2 higher doses and convulsions at the highest dose)

Executive summary:

Pregnant Fischer female rats were treated daily with methylhydrazine intraperitoneal injections during gestation Days 6 -15 at 2.5, 5 and 10 mg/kg bw/day. The pregnant rats were weighed daily. The uterus was opened and the numbers and positions of implants, dead fetuses, live fetuses, and resorptions were recorded. Live fetuses were blotted dry, weighed, examined for external abnormalities, and divided into groups for visceral and skeletal examination (two-thirds and one-third of each litter, respectively).

Four of the pregnant females in the high-level treatment group convulsed following methylhydrazine injection on one or more occasions during the treatment period. Maternal weight gains were significantly less in all treatment groups during the early (days 6-10) part of the treatment period and significantly less for the two higher treatment groups during the latter (days 11-16) part of the treatment period (See Table 1). Significant decreases in weight gains for treatment groups were not seen after cessation (days 17-20) of treatment; however, overall weight gains were lower than those of the control group (-14%, -29% and -41% of controls in the 2.5, 5 and 10 mg/kg bw/day groups, respectively). High incidence of resorptions in the two higher dose groups was observed. Intermediate dose group had a moderate increase in abnormalities involving the eyes (anophthalmia and/or microophthalmia) but the higher dose group had only a slight increase in abnormalities involving the eye.

All data that might suggest an embryotoxic or teratogenic effect were obtained with doses producing significant/marked maternal toxicity (marked decrease in bodyweight gain at the 2 higher doses and convulsions at the highest dose). Therefore NOAEL for maternal toxicity and developmental toxicity were both identified at 2.5 mg/kg bw/day.