Methylhydrazine

Toxicity to reproduction

Administrative data

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.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Ancient study prior to GLP, non-guideline and non-specific analytical follow-up. Reported data are reliable but a large amount of required data are missing or not reported.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

A series of up to 3-month MMH chronic exposures to three animal species was undertaken to evaluate the safety factor and appropriateness of the current TLV for health of workmen.

GLP compliance:

no

Remarks:

prior to GLP

Limit test:

no

Species:

other: see below

Strain:

other: see below

Details on test animals or test system and environmental conditions:

Each dose-group included female beagle dogs, female rhesus monkeys, male albino rats (Sprague- Dawley).
food ad libitum during exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Remarks:

continuous

Vehicle:

other: nitrogen-air mixture

Remarks on MMAD:

MMAD / GSD: not applicable (vapour)

Details on inhalation exposure:

Liquid MMH was expressed from a 20 ml glass syringe into a flow of 1 liter/minute dry nitrogen. Exposure was to a MMH-nitrogen vapour mixture.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

MMH dissolved in the absorber liquid reacted with iodine in a colorimetric reaction.
No real validation was reported, but the standard curve was always a straight line through the origin for concentration versus absorbance.
Each of the three exposure domes was sampled sequentially for 40 minutes around the clock, giving a 2-hour cycle for monitoring all three exposure domes (control, low-dose, high-dose).

Duration of treatment / exposure:

Rats: 45 days or 90 days
dog, monkey: 90 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0.100 and 0.0462 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

Rat, 45 days: 30
Rat, 90 days: 50
Dogs: 8
Monkeys: 4

Control animals:

yes

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes in rat

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 0, 2, 4, 6, 8, 10, 12 and 13 (terminal) weeks in dogs/monkeys; days 45 and 90 in rats
- Parameters checked: hematocrit, hemoglobin, RBC count, WBC count (all species), reticulocytes, Heinz bodies (dog/monkey only), RBC fragility (dog in week 13 only)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 0, 2, 4, 6, 8, 10, 12 and 13 (terminal) weeks in dogs/monkeys; days 45 and 90 in rats
- Parameters checked: total inorganic phosphorus, alkaline phosphatase

All other: not mentioned

Sacrifice and pathology:

20 rats per group were used for gross pathology at 90 days; weights of heart, spleen, lung, liver and kidney recorded.
dogs and monkeys at 90 days: gross and histopathology

Clinical signs:

no effects observed

Description (incidence and severity):

clinical signs not reported

Mortality:

no mortality observed

Description (incidence):

clinical signs not reported

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

rat 0.1 ppm, dog/monkey not reported

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:

effects observed, treatment-related

Description (incidence and severity):

rat and dog 0.0462 and 0.1 ppm

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

rat and dog 0.0462 and 0.1 ppm

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

rat only reported

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

dog 0.1 ppm

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No data on clinical signs. One monkey in the 0.0462 ppm exposure group died on the 10th day of exposure; there was no evidence of any relationship of the MMH exposure to death.
Other species: no data

BODY WEIGHT AND WEIGHT GAIN
Rat, 90 days: slightly reduced growth at 0.1 ppm only
Other species: no data

HAEMATOLOGY
Rat: hematocrit, hemoglobin, RBC count were slightly, up to 9-10% lower (most sensitive: RBC count, all significant) at both test concentrations on day 45 and RBC count was 13% lower (significant) at 0.1 ppm at 90 days.
Dog: Same effects at 90 days at 0. 1 ppm; non significant but notable at 0.0462 ppm (-14% RBC count); increased reticulocytes (x3) at both doses; increased osmotic RBC fragility at 0.1 ppm.
Monkey: no noteworthy effect

CLINICAL CHEMISTRY
Rat: slight dose-related increase in phosphorus (8% and 13% at 0.0462 and 0.1 ppm) on day 90.
Dog: idem, but only significant at 0.1 ppm (because of low number of animals; +18% at 0.0462 ppm). Important increase (up to x4) in alkaline phosphatase levels at 0.1 ppm.
Monkey: no noteworthy effect

GROSS PATHOLOGY
dogs: at 0.1 ppm, livers had a nutmeg appearance consistent with passive congestion
Monkey, rat: no noteworthy effect

Dose descriptor:

LOAEC

Remarks:

Male rat, female beagle dog

Effect level:

0.046 ppm

Based on:

test mat.

Remarks:

achieved concentration; continuous exposure

Sex:

male/female

Basis for effect level:

other: minimal regenerative (investigated in dog only) hemolytic anemia and increase in phosphorus.

Dose descriptor:

NOEC

Remarks:

female monkey

Effect level:

0.1 ppm

Based on:

test mat.

Remarks:

achieved concentration; continuous exposure

Sex:

female

Basis for effect level:

other: No effect but several effects not investigated or not reported

Critical effects observed:

not specified

Executive summary:

Subchronic MMH inhalation leads to decreased body weight gain (rat), regenerative (not investigated in rats) hemolytic anemia (in rats/dogs/monkeys) with fragilisation of RBC (dogs), elevation of phosphate and/or alkaline phosphatase levels sis (rat and dog).

The 3-month continuous exposure (24 h/day, 7 days/week) LOAEC was 0.0462 ppm as achieved concentration in both rats and dogs. Monkeys did not show any noteworthy effect (NOEC = 0.1 ppm), but a large variety of required investigations were not carried out or reported.

The nature and amplitude of the effects at the investigated dose-levels does not warrant repeated-dose toxicity classification.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Report date:

1973

Materials and methods

Test guideline

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

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

Details on test material:

monomethylhvdrazine (MMH)

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

not examined

Details on results (P0)

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.

Effect levels (P0)

Dose descriptor:

LOAEL

Effect level:

12 mg/kg bw/day

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

no offspring (only male "parents")

Effect levels (F1)

Remarks on result:

not measured/tested

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

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).