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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

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

Link to relevant study records
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:
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 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).

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

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).

Link to relevant study records
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:
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 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:
clinical signs
body weight and weight gain
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 ± 3b

2 ± 4b

- 3 ± 3b

Days 11-16

21 ± 5

19 ± 10

15 ± 5b

12 ± 8b

Days 17-20

30 ± 6

29 ± 10

25 ± 10

26 ± 12

Litter data

 

 

 

 

Implants/littera

7.8 ± 3.6

8.8 ± 3.4

7.3 ± 2.5

7.6 ± 3.4

Viable fetuses/littera

6.8 ± 4.0

7.5 ± 3.4

6.2 ± 2.7

6.1 ± 3.9

Fetal weighta

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 examc

2(2)

1(1)

3(4)

2(2)

Soft-tissue exam

1(1)d

2(2)e

6(9)e

3(4)f

Skeletal examg

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

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.

Justification for classification or non-classification