Registration Dossier

No relevant data were available: a minimal effect on mouse sperm cells was noted, but:

- the dosing route (i.p.) was not relevant for a chemical

- effect was at most 4% abnormal cells at doses as high as 40% of the LD50 (12 mg/kg, i.p.)

- there was no impact on total sperm cell number, testes weight, testes histopathology

- as animals were not paired, relevance towards fertility is unknown

Reference

Endpoint:

toxicity to reproduction

Remarks:

other: effects on sperm cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non guideline study, prior to GLP, unknown relevance for fertility assessment in absence of pairing. General pathology and hematology data were ignored as it was unclear in which study design and with which test item these investigations were done.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Examination of sperm parameters after repeated exposure by inhalation

GLP compliance:

no

Remarks:

prior to GLP

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

age from 11 to 18 weeks,
caged in groups of 3 to 5 animals each in plastic cages
air conditioned rooms
automated light-dark cycles (10:14 hours)
food and water ad libitum

Route of administration:

intraperitoneal

Vehicle:

water

Remarks:

distilled

Details on mating procedure:

not performed

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

dose prepared <0.5 h before injection

Frequency of treatment:

5 injections

Details on study schedule:

Study 1): time-dependency: Five animals of each group were sacrificed by cervica! dislocation at weekly intervals.
Study 2): dose-dependency: 0.8 and 3 weeks after exposure

Remarks:

Doses / Concentrations:
3 mg/kg
Basis:
other: nominal injected

No. of animals per sex per dose:

Study 1): 50 males at 3 mg/kg
Study 2): 2 or 3 males at 7.5 or 12 mg/kg

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Study 1): body weights recorded

Sperm parameters (parental animals):

Study 1): number and percent of abnormally shaped sperm-per cauda epididymis were scored
Study 2): abnormally shaped sperm in the cauda epididymis 3.5 weeks after injection

The average of 1000 to 2000 sperm were analyzed on two separate air-dried smears prepared from a pooled suspension of the sperm from the cauda epididymides of two or three mice. All countings of the sperm were conducted as blind experiments.

Postmortem examinations (parental animals):

Study 1): ratios of testis to body weight, histopathology of the testes (PAS-hematoxylin)

Clinical signs:

not examined

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:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

testes

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

not examined

Body weight:
Study 1): decreased until 5 weeks after exposure; weight gain unaffected after exposure

Sperm:
Study 1): maximum effect of more than doubling the background incidence of abnormal sperm shape (i.e.: at most 4% in treated animals), between 1 and 3 weeks after the end of exposure. Reversible in 7 weeks. No clear effect on sperm count.
Study 2): same effects, dose-dependent: > doubling (<4% abnormal) at 12 mg/kg, < doubling (<3% abnormal) at 7.5 mg/kg.

Dose descriptor:

LOAEL

Effect level:

12 mg/kg bw/day

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

no offspring (only male "parents")

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

Executive summary:

MMH has the ability to increase sperm malformation, however this effect is minimal:

- at most 4% abnormal cells at doses as high as 40% of the LD50 (12 mg/kg, i.p.)

- no impact on total sperm cell number, testes weight, testes histopathology

The fertility was not assessed in this study (no pairing of animals).

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

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).

Reference

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

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

Specific details on test material used for the study:

Provider: Eastman, Rochester, N.Y.

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

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

Dose / conc.:

2.5 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

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

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.

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

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

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

Abnormalities:

not examined

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

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

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

Developmental effects observed:

no

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.

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.

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

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.