Trimethyl orthoacetate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference 1

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 2

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

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

Test material information

Constituent 1

Reference substance name:

Methanol

EC Number:

200-659-6

EC Name:

Methanol

Cas Number:

67-56-1

Molecular formula:

CH4O

IUPAC Name:

methanol

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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 °C
- 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 (p.186).

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

Doses / concentrations 1

Dose / conc.:

0.013 mg/L air

Remarks:

corresponding to 10 ppm

Doses / concentrations 2

Dose / conc.:

0.13 mg/L air

Remarks:

corresponding to 100 ppm

Doses / concentrations 3

Dose / conc.:

1.3 mg/L air

Remarks:

corresponding to 1000 ppm

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 behavior 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 (p. 191).

Postmortem examinations (parental animals):

Examinations of F0 and F1 parental animals.

After mating all males were necropsied, and testes, epididymis, seminal vesicle and postate 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 bagina 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 the 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)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related alterations in general observations.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no differences for body weight.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no differences for food consumption.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

There were no differences for water consumption.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No abnormalities were observed in findings on nursing behavior.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

None of the fertility indices including sexual cycle, days needed for insemination, insemination rate and pregnancy rate showed statistically significant differences.
No abnormalities were observed in findings on delivery and nursing behavior and necropsy data of F0 animals.

Effect levels (P0)

Key result

Dose descriptor:

NOAEC

Effect level:

1.3 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

other: reproductive parameter

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Effect levels (P1)

Key result

Dose descriptor:

NOAEC

Remarks on result:

not measured/tested

Target system / organ toxicity (P1)

Key result

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In male pups of the 1.3 mg/L group, post-natal morphological differentiation appeared to be influenced with respect to the descensus tests 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%).

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

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:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

None of the fertility indices including sexual cycle, mating time, fertility and pregnancy rate showed a significant difference from untreated F1 controls.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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).

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Description (incidence and severity):

no histopathological manifestations; no effects on testes or ovaries reported

Other effects:

no effects observed

Description (incidence and severity):

There were no significant differences in functional tests (movement, emotion, learning) as compared with the control or the other groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

1.3 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

other: reproductive & developmental parameters

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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%) (p. 200).After 0.13 mg/L, "descensus testis" in male F2-progeny was about 0.5 d earlier than in male control F2 pups (p. 195). Detailed data not specified and not addressed under "Discussion".

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

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:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weights showed similar tendencies as found in the F1-generation.

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Description (incidence and severity):

no histological changes; no effects on testes or ovaries reported.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEC

Generation:

F2

Effect level:

1.3 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

other: reproductive & developmental parameter

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Blood levels of methanol measured in the F1-offsprings (age 9 weeks) (NEDO, 1987, p. 191):

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 even may have been higher than in studies using the same exposure concentration, but shorter exposure times.