Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study, also largely meeting current standards, documentation limited, acceptable for assessment.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Toxicological Research of Methanol as a fuel for Power Station. Summary Report on Tests with Monkeys, Rats and Mice.
Author:
New Energy Development Organization
Year:
1987
Bibliographic source:
New Energy Development Organization, Tokyo
Reference Type:
publication
Title:
Long-term effects of methanol vapor at low concentration.
Author:
Takeda, K. and Katoh, N.
Year:
1988
Bibliographic source:
Proc. of the 8th Int. Symp. Alcohol Fuels: 1051-1056, Tokyo, Japan

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
- limited documentation; copulation time was too long (21 days); not all parameters mentioned in the guideline were investigated
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): methanol (reagent special grade from Junsei Chemicals Co.)
- Analytical purity: no data
- Impurities (identity and concentrations): < 1 ppm vinyl chloride, < 3 ppm formaldehyde

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Inc.
- Age at study initiation: 8 weeks
- Diet: Solid Chow for rat (CRF-1, Charles River Japn Inc.)
- Water: sterilised and filtrated water (ad libitum)
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 2
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: multi-stage inhalation chamber (Hazleton 1000 exposure chamber)
- Temperature, humidity in air chamber: 24 ± 2 °C; 55 ± 5 %


TEST ATMOSPHERE
- Nominal exposure levels were prepared by generating methanol gas and then mixing it with fresh air.
- A methanol gas analyser measured the concentration in the chamber.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 21 d
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Other: The pairs without evidence of insemination within 21 d were again cohabited with untreated animals (2nd mating) to determine the fertility of each animal, in this case without exposure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical concentration values of methanol were close to nominal ones (the monthly variation remained less than 5%).
Duration of treatment / exposure:
F0: 103 -108 d
F1: 61 -62 d and 145 -153 d
F2: 54 -56 d
further informations see "any other information on materials and methods"
Frequency of treatment:
continuously
Details on study schedule:
- F1 parental animals not mated until 12 weeks after selected from the F1 litters.
- Female F1 animals were examined for sexual cycle at 12-weeks or thereafter and mated with males of the same group.
Doses / concentrations
Remarks:
Doses / Concentrations:
0.013; 0.13; 1.3 mg/L (corresponding to 10; 100; 1000 ppm)
Basis:
nominal conc.
No. of animals per sex per dose:
30 (F0 generation)
Additionally, 15 animals were reared for a second mating.
Control animals:
yes, sham-exposed

Examinations

Parental animals: Observations and examinations:
Observations of F0 and F1 parental animals.

CAGE SIDE OBSERVATIONS:
- clinical signs, mortality, any sign of abortion and premature delivery
- Time schedule: at least once a day, 5 days a week


BODY WEIGHT:
- Time schedule for examinations: animals were weighed weekly: day 0, 7, 14 and 20 of gestation and day 0, 4, 7, 14 and 21 of delivery


FOOD AND WATER CONSUMPTION::
- consumption measured by cage (on the same days as body weight measurements)


OTHER:
- Generally sexual cycle, mating time, fertility, pregnancy rate were documented. During the lactation period, maternal animals were observed for nursing behaviour including lactation, nest building and presence/absence of pup-eating.
Sperm parameters (parental animals):
Histological examination of morphology of sperms was not included.
Litter observations:
Observations of F1 and F2 litters.

Litters were examined on the day of birth for live pups, dead pups, sex and any external abnormalities. The observations were done daily until weaning and thereafter 5 days a week.
Each litter was weighed on day 0 and 4 (before reduction) of birth by sex and respective mean value calculated. After adjustment of litter size, pups were weighed individually on day 4, 7, 14 and 21. From weaning to week 14 of birth, the measurements were done weekly.

All surviving pups were observed for post-natal morphological differentiation indices: pinna unfolding, eruption of incisors, open eyes, descensus testis (males), vagina opening (females).
As for movement function test, all surviving pups after adjustment of litter size were tested for righting on a surface, ipsilateral flexor reflex, pinna reflex, auricular startle response, visual recognition response, pain response, corneal reflex and suspension abililty on a particular day before weaning. Also emotional tests, learning ability tests, and movement coordination tests were included.

In 9-week old F1 pups, blood methanol was measured, but not formate.
Postmortem examinations (parental animals):
Examinations of F0 and F1 parental animals.

After mating all males were necropsied, and testes, epididymis, seminal vesicle and prostate gland were removed and preserved.

After 2 weeks of rearing, the females at the 2nd mating were necropsied and examined for pregnancy status. After termination of mating, the not inseminated females were necropsied and the ovary, uterus and vagina were preserved.
21 days after delivery, all dams were necropsied and examined for implantation. The vagina, uterus and ovary were preserved.

Any organ with any abnormality was subjected to a histopathological examination, if necessary.

26 days after evidence of insemination, females which had not yet delivered were necropsied and subjected to the same examinations as described above.
Postmortem examinations (offspring):
Examinations of F0 and F1 litters.

After termination of movement function tests, pups were sacrificed and necropsied.
The pups which were selected for examination and not used for the movement function test were necropsied on a day of the same age at 8 weeks old or thereafter and principal organs were weighed.
Statistics:
All data obtained were analysed by t-test, Fischer's exact test, U-test of Mann-Whitney or Armitage's chi²-test.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

No treatment-related alterations in general observations and in all reproductive parameters.
None of the fertility indices including sexual cycle, days needed for insemination, insemination rate and pregnancy rate showed statistically significant differences. There were no differences for body weight, food consumption and water consumption during gestation and lactation period, either.
No abnormalities were observed in findings on delivery and nursing behavior and necropsy data of F0 animals.

Effect levels (P0)

Dose descriptor:
NOAEC
Effect level:
1.3 mg/L air (nominal)
Sex:
male/female
Basis for effect level:
other: reproductive parameter

Results: F1 generation

Details on results (F1)

F1-GENERATION:
CLINICAL SIGNS (F1-OFFSPRING)
In male pups of the 1.3 mg/L group, post-natal morphological differentiation appeared to be influenced with respect to the descensus testis occurring 0.5 to 1 d earlier (see same effect in F2 generation)[not mentioned by Takeda and Katoh, 1988]: This time-dependent parameter was evaluated by relating the completion of downward migration of the testes (final length of the gubernaculum reached) to the post-natal body-weight gain (The more reliable body length was not available):
In the F1 pups derived from the 1.3 mg/L group (108 males), this process was completed within 16 through 20 post-natal days with the climax at day 17 and 18 (32 and 39%, respectively), while in the respective control (113 males), descent was complete from 16 through 21 days with the maximum at day 19 (32%), but also relatively high percentages on the days before and after: day 18 (22%), day 17 (19%), day 20 (18%).

SEXUAL MATURATION (F1-OFFSPRING)
None of the fertility indices including sexual cycle, mating time, fertility and pregnancy rate showed a significant difference from untreated F1 controls.

ORGAN WEIGHTS (F1-OFFSPRING)
Absolute and relative brain weights were significantly lowered in the high-dose groups of either sex at an age of 8 and 16 weeks. This was still found in females necropsied after 24 weeks. Also other organs showed slight shifts in weights: thymus, pituitary (lower), heart, lung, liver (higher).

HISTOPATHOLOGY (F1-OFFSPRING)
no histopathological manifestations; no effects on testes or ovaries reported

OTHER FINDINGS (F1-OFFSPRING):
There were no significant differences in functional tests (movement, emotion, learning) as compared with the control or the other groups.



F2-GENERATION:
CLINICAL SIGNS (F2-OFFSPRING)
As in F1 males, an apparently dose-related earlier descensus testis was noted after 1.3 mg/L exposure: F2 (94 males) on day 16 (42%), day 17 (40%), day 18 (15%) vs. control (91 males) on day 16 (10%), day 17 (39%), day 18 (31%), day 19 (14%).
After 0.13 mg/L, descensus testis in male F2-progeny was about 0.5 d earlier than in male control F2 pups. Detailed data not specified and not addressed under "Discussion" of the study.

ORGAN WEIGHTS (F2-OFFSPRING)
Organ weights showed similar tendencies as found in the F1-generation.

HISTOPATHOLOGY (F2-OFFSPRING)
no histological changes; no effects on testes or ovaries reported.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
0.13 mg/L air (nominal)
Sex:
male/female
Dose descriptor:
LOAEC
Generation:
F1
Effect level:
1.3 mg/L air (nominal)
Sex:
male/female
Basis for effect level:
other: reproductive parameter, brain weight

Results: F2 generation

Effect levels (F2)

open allclose all
Dose descriptor:
NOAEC
Generation:
F2
Effect level:
0.13 mg/L air (nominal)
Sex:
male/female
Dose descriptor:
LOAEC
Generation:
F2
Effect level:
1.3 mg/L air (nominal)
Sex:
male/female
Basis for effect level:
other: reproductive parameter, brain weight

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Blood levels of methanol measured in the F1-offsprings (age 9 weeks) (NEDO, 1987):

controls (baseline): approx. 2 - 3 mg/L

0.013 mg/L methanol: approx. 3 - 3.5 mg/L

0.13 mg/L: approx. 1 - 4.2 mg/L

1.3 mg/L: approx. 53 (males)-100 (females) mg/L

There are no data on formate.

Note: Exposure per day was 20 h, which implies that prolonged steady-state blood levels were reached which may have been even higher than in studies using the same exposure concentration, but shorter exposure times.

Applicant's summary and conclusion

Conclusions:
No firm conclusions can be drawn about fertility of either sex, as the copulation time of 21 d was comfortably long for successful insemination and gametogenesis was not considered.
In the F1 and F2 progeny (both sexes), a decrease in brain weights was evident at 1.3 mg/L methanol, but without noticeable histological changes and functional impairments. This phenomenon is believed to represent a change occurring during the perinatal period (Takeda and Katoh, 1988). But no quantitative data and statistical level are documented for organ weights. The information about organ weights is lacking, with much weight placed on the "descensus testis". The meaning of an apparent shift of testis descent in male offspring in relation to body-weight development of the pups in the two following generations is unclear and was not directly addressed by Takeda and Katoh (1988), but detailed by NEDO (1987) and considered a significant difference from untreated controls. Furthermore, it is obvious that this parameter showed considerable variation also between the control groups of both generations.
Although there was no obvious pathological effect in the progeny of 1.3 mg/L exposed groups, the effects observed may be considered as biologically relevant under these test conditions and, therefore, 1.3 mg/L is established as LOAEC and 0.13 mg/L as NOAEC for post-natal development while for parental effects, the NOAEC is 1.3 mg/L.
The NOAEC(develop) = 0.13 mg/L is also compatible with a NOEC.