Registration Dossier

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:
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:
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 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

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
monomethylhvdrazine (MMH)

Test animals

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

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).