Reaction mass of methyl acetate and methanol

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The reaction mass of methyl acetate and methanol is assessed on the basis of these individual substances methyl acetate and methanol in a read-across approach.

 

Methyl acetate

There are no data on reproductive toxicity of methyl acetate. However, due to the rapid hydrolysis of this compound it is justified to base hazard assessment with respect to reproduction on the toxicological properties of the immediate metabolites acetic acid and methanol. Acetic acid appears to be of less relevance, since there are no indications of a fetotoxic or teratogenic potential in literature. For methanol some embryo-/fetotoxic and teratogenic effects were demonstrated in rodents at relatively high maternal toxic concentrations. A NOEC/fertility for methanol of 1300 mg methanol/m³ was derived from a 2-generation inhalation study in rats. This value can be converted to NOAEC/fertility of about 3000 mg methyl acetate/m³. In a one generation study in monkeys, the NOAEC for maternal toxicity/reproductive performance and growth and physical development of the offspring were considered both to be 2390 mg/m³ which can be converted to 5515 mg/m³ methyl acetate.

 

Methanol

Effects of methanol on reproduction were tested in a two generation reproductive toxicity study similar to OECD guideline 416 in male and female rats after inhalative administration. The highest dose of 1300 mg/m³ did induce earlier post-natal descensus morphological differentiation in males of the F1 and F2 generation ( descensus tests occurring 0.5 to 1 d earlier). 1300 mg/m³ is therefore established as LOAEC and 130 mg/m³ as NOAEC for post-natal development. For parental effects, the NOAEC is established as 1300 mg/m³. In a one generation reproductive toxicity study similar to OECD guideline 415, male and female monkeys were treated for 12 months by daily inhalative administration. Methanol exposures were associated with a reduction in the length of pregnancy in this animal model, thus shortening the gestation length of the offspring. The reduction in the length of pregnancy was not dose dependent. Based on the study results, the NOAEC for maternal toxicity/reproductive performance and the NOAEC for growth and physical development of the offspring were considered both to be 2390 mg/m³.

Conclusion: No profound effects on fertility were observed.

 

Conclusion

No experimental data are available for the target substance itself. Thus, the assessment is based on a read-across and weight of evidence approach, using the source substances methyl acetate and methanol. As methanol is the more critical substance, the assessment of the target substance is based on results of source substance 1 (methanol), following a worst-case scenario. Due to no profound effects on fertility and very high effect doses of source substance 1, the reaction mass of methyl acetate and methanol is not classified toxicity on reproduction.

Link to relevant study records

Reference

Reference 1

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

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

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
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12/12

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
for 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.

Dose / conc.:

0.013 mg/L air

Remarks:

corresponding to 10 ppm

Dose / conc.:

0.13 mg/L air

Remarks:

corresponding to 100 ppm

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

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.

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: 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.

Key result

Dose descriptor:

NOAEC

Effect level:

1.3 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

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: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Key result

Dose descriptor:

NOAEC

Based on:

test mat.

Sex:

male/female

Remarks on result:

not measured/tested

Key result

Critical effects observed:

not specified

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.

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

0.13 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

other: reproductive parameter, brain weight

Key result

Dose descriptor:

LOAEC

Generation:

F1

Effect level:

1.3 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

other: reproductive parameter, brain weight

Key result

Critical effects observed:

no

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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEC

Generation:

F2

Effect level:

0.13 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

other: reproductive parameter, brain weight

Key result

Dose descriptor:

LOAEC

Generation:

F2

Effect level:

1.3 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

other: reproductive parameter, brain weight

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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.

Reference 2

Endpoint:

one-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

Remarks:

limitation due to low number of animals.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Principles of method if other than guideline:

One generation reproduction toxicity study: Adult female monkeys were exposed to methanol vapour daily during prebreeding, breeding and pregnancy.

GLP compliance:

not specified

Limit test:

no

Species:

monkey

Strain:

Macaca fascicularis

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Cohort 1
- Source: All feral born
- Age at assignment to project: 5.5-11 years old (estimated on the basis of dental records)
- Weight at assignment to project: 2.3-3.7 kg
Cohort 2
- Source: Feral born (n=15), colony born (n=9, Texas Primate Center, Charles River Primates, CV Primates or Johns Hopkins University)
- Age at assignment to project: 5-13 years old
- Weight at assignment to project: 2.2-5.7 kg
Both cohorts
- Housing: Individual (social contact through wire mesh)
- Diet: Purina Laboratory Fiber-Plus® Monkey Diet, once per day in the afternoon
- Water: Ad libitum
- Acclimation period: The females were transferred to and from the laboratory (inhalation chamber) in a transfer cage on a daily basis.

The four adult males were feral-born with age estimates between 10 and 12 years. The males weighed between 5 and 7.6 kg, and each had sired an offspring during the project.

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

TYPE OF INHALATION EXPOSURE: Whole body

GENERATION OF TEST ATMOSPHERE / CHAMPER DESCRIPTION
- Exposure apparatus: Inhalation chamber housing 1 animal in a cage (47, 61, 80 cm (w, h, d))
- Source and rate of air: Dayton Model 5K901C blower (Dayton Corporation, Moraine, OH), 420 L/min

TEST ATMOSPHERE
Methanol vapour was generated by passing compressed air through gas dispersion bottles filled with methanol. The methanol was heated by placing the bottles in a water bath set at a temperature of approximately 36 °C. The methanol vapour was delivered to the chamber via insulated polypropylene tubing that ran from the bottles to the vapour inlet port of each chamber.

ANALYSIS OF METHANOL AND CARBON DIOXIDE CONCENTRATIONS
Methanol, carbon dioxide, and dew point were measured by withdrawal of an air sample through a polypropylene tube located in the chamber at a level 5 cm above the monkey cage. The air sample was drawn at a rate of approximately 1.5 L/minute. Methanol and carbon dioxide were measured by a General Analysis Corporation (Norwalk, CT) infrared analyzer. Dew point was measured by a General Eastern Instruments (Woburn, MA) hygrometer, and chamber temperature was measured via resistance temperature detectors placed in each chamber. Dew point and temperature were used to calculate the relative humidity(RH) of each chamber.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 4 hours each day on the 11th, 12th and 13th day of the menstrual cycle after exhibiting a minimum of 7 menstrual cycles (3 cycles prior to methanol exposure and 4 cycles after initial exposure)
- Proof of pregnancy: Blood progesterone analysis (pregnancy was confirmed by 18 to 20 days of gestation)
- Further matings after two unsuccessful attempts: Yes (for 3 additional days, if necessary, a 3rd and 4th breeding took place for 5 consecutive days (days 10 through 14 of cycle)) - always using the same male

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Average chamber concentrations obtained for 11 samples taken from 14 minutes after onset of methanol flow until 6 minutes prior to offset of methanol flow were all within 5 % of the target concentrations.

Duration of treatment / exposure:

Duration prebreeding: Approximately 120 days; during breeding: approximately 70 days; during pregnancy: approximately 165 days.

Frequency of treatment:

Daily for 2.5 hours (7 days/week) before breeding, during breeding and during pregnancy.
At the end of each 2-hour exposure, the animals remained in the chamber for another 30 minutes while the methanol dissipated.

Details on study schedule:

18-November 1992 to 17-December- 1994 (Cohort 1)
23-November-1994 to 07-November-1996 (Cohort 2)

Dose / conc.:

0.27 mg/L air

Remarks:

corresponding to 200 ppm

Dose / conc.:

0.8 mg/L air

Remarks:

corresponding to 600 ppm

Dose / conc.:

2.39 mg/L air

Remarks:

corresponding to 1800 ppm

No. of animals per sex per dose:

11-12 adult females

Control animals:

yes

Details on study design:

- Dose selection rationale: The target air concentrations were chosen to provide a range of blood methanol concentrations from just above background to just below that reported to cause nonlinear clearance kinetics in primates (Horton et al., 1992).

The two-cohort study design utilized 48 adult females (24 females/cohort), 4 adult males (2 males/cohort), and their offspring. This design minimized the number of subjects tested simultaneously, yet achieved a sufficient sample size to detect subtle changes. For each cohort, adult females were initially separated into 6 groups, with 4 animals per group based on known or estimated age, size, and colony parity. Females from each of the 6 groups were then randomly assigned to one of four methanol-exposure groups.


Reference:

Horton VL, Higuchi MA, Rickert DE (1992). Physiologically based pharmacokinetic model for methanol in rats, monkeys and humans. Toxicol Appl Pharmacol 117: 26–36.

Parental animals: Observations and examinations:

MATERNAL HEALTH ASSESSMENTS
- BODY WEIGHT
- Time schedule: weekly
Females were weighed while being transferred from the inhalation chamber to their homecages.
- CAGESIDE GENERAL OBSERVATIONS
- Time schedule: daily
Each female was observed for signs of lethargy, uncoordinated motor movements (staggering or clumsiness) and laboured or irregular respiration approximately 5 minutes after return to the homecage.
- CLINICAL OBSERVATIONS
- Time schedule: daily
Visual function was assessed by observing whether or not the female could visually orient to and/or follow a syringe filled with apple juice. Fine motor coordination was assessed by observing whether or not the female could reach for and pick up a small piece of fruit using only her thumb and index finger.
- HEALTH CHECK
- Time schedule: daily
Animals were observed for signs of diarrhoea, and medications were administered/recorded.
MATERNAL REPRODUCTIVE ASSESSMENTS
Specific aim of the study was addressed by examining 5 factors in time-mated females: menstrual cycles; frequency of conception; frequency of complications during pregnancy, labour and delivery; duration of pregnancy; and frequency of live births.
- MENSTRUAL CYCLES
- Time schedule: daily
See "Estrous cyclicity" below.
- TIMED MATINGS
See "Details on mating procedure" above.
- PREGNANCY OBSERVATIONS AND DELIVERY EXAMINATIONS
- Time schedule: during the last month of pregnancy (every half hour from 8 p.m. to 6 a.m.)
Females were observed for signs of labour via infrared cameras. Immediately after delivery of an infant, the female was sedated, and the infant was separated from the mother and placed in an isolette. Maternal weights were recorded, and the mother was returned to her home cage for observation while she remained sedated.

Oestrous cyclicity (parental animals):

The onset and duration of menstruation was assessed using a noninvasive observational method to detect menstrual bleeding: the females were trained to present their perinea to an observer for visual evaluation.

Sperm parameters (parental animals):

No adult male monkeys were observed.

Litter observations:

For postnatal developmental evaluation, 8 to 9 infants were available per group, in total 34 infants, among them 26 in-utero treated offsprings.
The birth weight, crown–rump length, and head size of all infants were obtained immediately following delivery. Other infant assessment procedures were also performed at delivery. The results of these assessments on offspring are described in Part II (please refer to section 7.8.2).

Statistics:

Statistical analyses were performed using Systat, SAS, or Splus.Basic hypotheses were developed for the maternal health/reproductive effects part of the study:
3. There will be no significant differences across the methanol exposure groups in overt signs of maternal toxicity.
4. There will be no significant differences across the methanol exposure groups in signs of toxic effects on maternal reproduction.

In addition to these hypotheses, statistical analyses of maternal characteristics (age, weight, crown–rump length, gravidity, parity) at the outset of the study were performed to examine the results of random assignment of adult females to the 4 exposure groups. The general approach to testing the hypotheses was to first assess whether an exposure effect existed, both globally and specifically. A global F test (or equivalent) was used for assessing whether there were detectable differences among the 4 exposure groups. Because this test has less power than specific alternatives, a no exposure–effect hypothesis was also examined that compared the control group with the combination of all methanol-exposure groups. The control group was also compared with each methanol exposure group using pairwise comparisons. Finally, the impact of controlling for cohort was assessed in mean models.

To test Hypothesis 3, four separate procedures were used to detect overt signs of maternal toxicity. Due to the low number of positive responses, descriptive analyses were performed.
To test Hypothesis 4, the following measures of toxic effects on maternal reproduction were used:
(a) menstrual-cycle length
(b) rate of conception
(c) weight gain during pregnancy
(d) frequency of pregnancy and delivery complications
(e) pregnancy duration
(f) frequency of live-birth deliveries
(g) offspring birth size (weight, crown–rump length, head circumference, length, and width)
Repeated measures ANOVA models, one-way ANOVA models and Fisher's exact test were used to assess statistical differences.

Reproductive indices:

Conception rate, live birth delivery rate

Offspring viability indices:

The birth weight, crown-rump length, and head size of all infants were obtained immediately following delivery.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The results of 50 (Cohort 2) to 100 (Cohort 1) clinical observations did not indicate the presence of overt toxicity in the adult females. During the entire study, only 6 females failed to respond to the visual task (3 control females and 3 methanol exposed females). Of the 46 females observed, 23 failed the motor coordination task during the study. Of these 23 females, 15 failed it 3 times or fewer. Of the remaining 8 females, who failed the task 5 to 13 times, 4 were from the control group, 1 was from the 200 ppm exposure group, and 3 were from the 600 ppm exposure group. All of these females failed the task during the baseline period as well as during methanol exposure, and none exhibited a pattern of responses indicative of fine-motor incoordination due to methanol exposure.
The number of females who became ill and required medication was quite low, and unrelated to dose.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The weights of all females were quite stable during the study. The mean weight for each of the 4 methanol exposure groups during the baseline period and through breeding was approximately 3.5 kg. The mean weight at conception for the females in the 4 exposure groups was between 3.2 and 3.7 kg. Mean weight gain during pregnancy varied from 1.3 to 1.8 kg across all exposure groups. Weight gain during pregnancy was calculated for each female and used in ANOVA models to test for differences across the methanol exposure groups. The results did not indicate a significant methanol exposure effect on maternal weight gain during pregnancy (p > 0.12, all tests).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not specified

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

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There was no overt toxicity in adult females from any of the 4 methanol exposure groups. Lethargy, uncoordinated motor movements, and laboured respiration were not observed during the study.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females exhibited 3 menstrual cycles before methanol exposure, 1 cycle during the period in which exposure was started, and 3 cycles after exposure was started. One female exhibited an abnormal cycle length of 88 days prior to methanol exposure. This cycle was not included in the analysis. All females exhibited at least 4 normal cycles (>20 days and 50 days) prior to breeding.
The results of the ANOVA models did not indicate significant differences in the lengths of menstrual cycle of females across the 4 methanol exposure groups during the baseline period (p > 0.12, all tests). There was no statistically significant methanol exposure effect on cycle lengths in the females (p = 0.45). The duration of menstrual cycle remained stable at approximately 30 days.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

The frequency of conception was approximately the same across the 4 exposure groups (82 % in the control group, 75 % at 200 ppm, 82 % at 600 ppm, and 83 % at 1800 ppm). Conception frequencies were not affected by methanol exposure (p = 1.0).
A total of 37 infants were delivered from the 46 females. Two females delivered stillborn infants, 1 infant in the control group and 1 infant at 600 ppm. One female at 1800 ppm required a Caesarean (C) section to deliver a dead fetus.
The rate of complications during pregnancy or labour and delivery were 22 % for the control females and the 200 ppm females, 33 % for the 600 ppm females, and 30 % for the 1800 ppm females. No significant differences across methanol exposure groups were found (p = 1.0). The live-birth delivery rates were between 90 and 100 % for all 4 exposure groups. There were no sire related effects on pregnancy length. The results of the ANOVA model indicated a significant effect on pregnancy length due to methanol exposure (p = 0.03). Post hoc testing indicated that each of the 3 methanol-exposed groups had significantly shorter durations of pregnancy than did the control group (p < 0.04, all tests).

Key result

Dose descriptor:

NOAEC

Effect level:

2.39 mg/L air (nominal)

Sex:

female

Basis for effect level:

other: reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The offspring characteristics analyzed were birth weight, crown–rump length, head circumference, head length, and head width. The results of the ANOVA models did not indicate a significant effect of methanol exposure on offspring size at birth (p > 0.24, all tests). ANOVA models controlling for cohort differences, however, indicated a significant methanol-exposure-group-by-cohort interaction for birth weight (p < 0.002) and head circumference (p < 0.03).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

2.39 mg/L air (nominal)

Sex:

not specified

Basis for effect level:

other: growth and physical development of the offsprings

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Exposure to methanol at concentrations of up to 1800 ppm for over 1 year did not produce overt signs of toxicity (motor incoordination, blindness, and/or respiratory effects) in adult female nonhuman primates. Chronic methanol exposure did not interfere with the menstrual cycle or the ability of females to conceive. The timed-mating procedures used (3 matings/day between days 11 and 13 of the menstrual cycle) typically produce close to 100 % conception rates in normal groups of M. fascicularis females (Mahoney, 1975). The overall conception rate for this study was lower than expected, at 80 %. This was due to a sire effect: 1 of the males in Cohort 2 successfully impregnated only 4 females.

There was no pregnancy-induced folate deficiency. Fetal and newborn mortality frequencies were low for all of the exposure groups. One female at 1800 ppm had to be C-sectioned to deliver a dead fetus, and 2 females (1 control infant, 1 infant at 600 ppm) vaginally delivered full-term stillborn infants. The autopsy on the fetus delivered by C-section indicated the presence of hydrocephalus with significant autolysis in all of the major organs. Autopsies on the 2 stillborn infants indicated that the lungs were not inflated and that they had died close to or during delivery. No malformations were observed, and the cause of death for both infants was asphyxiation.

Two methanol-exposed females each at 200 ppm and 600 ppm were C-sectioned following observations of uterine bleeding without productive labour, presumably due to placental detachment. All 4 infants were delivered alive and without complications. Given the small number of animals exhibiting this condition and the lack of a response at the highest exposure concentration, conclusions concerning methanol exposure as a causative factor in uterine bleeding are not warranted.

It was not clear whether the decrease of about 6 to 8 days in duration of pregnancy noted as compared to controls was related to methanol exposure, since there was no dose-response and no differences among offspring groups in body weight or other physical parameters.

Although the average gestation period of the methanol exposed offspring was significantly shorter than that of the controls, methanol exposure did not affect the size of the offspring at birth. The average birth weight, crown–rump length, and head size of infants in the methanol exposure groups were comparable to those of the control infants. These results do not indicate that reduced offspring size at birth is associated with methanol exposure concentrations insufficient to cause overt maternal toxicity.

Prenatal exposure to methanol had no effect on infant growth and physical development for the first 9 months.

 

Reference:

Mahoney C. (1975.) Practical aspects of determining early pregnancy, stage of foetal development, and imminent parturition in the monkey (Macaca fascicularis). Lab Anim 6: 261–274.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 300 mg/m³

Study duration:

chronic

Species:

rat

Quality of whole database:

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

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Methyl acetate

There are no data on reproductive toxicity of methyl acetate. However, due to the rapid hydrolysis of this compound it is justified to base hazard assessment with respect to reproduction on the toxicological properties of the immediate metabolites acetic acid and methanol. Acetic acid appears to be of less relevance, since there are no indications of a fetotoxic or teratogenic potential in literature. For methanol result, see below.

 

Methanol

Two-generation reproductive toxicity in rats, RL2

Effects of methanol on reproduction were tested in a two generation reproductive toxicity study similar to OECD guideline 416 in male and female rats after inhalative administration. The F0 generation was treated for 103 -108 days, the F1 generation for 61 -62 and 145 -153 days and the F2 generation for 54 -56 days (20 hours/day). Treatment doses were 10, 100, 1000 ppm. 30 animals per sex dose were used (F0 generation). Additionally, 15 animals were reared for a second mating.

P0

For the P0 generation, no treatment-related alterations were observed generally. No mortality occurred and there were no differences for body weights or in food and water consumption. There were no haematological, clinical biochemistry, behavior, immunological, gross pathological or neuropathological findings observed and no changes in organ weights. 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. 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.

 

P1

In the P1 generation, results were not specified.

 

F1

In male pups of the 1000 ppm 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 1000 ppm 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%). No mortality and no changes in body weights were observed. There were no hematological or clinical biochemistry findings. None of the fertility indices including sexual cycle, mating time, fertility and pregnancy rate showed a significant difference from untreated F1 controls. 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). Neither gross pathological and histopathological manifestations were observed nor effects on testes or ovaries reported. There were no significant differences in functional tests (movement, emotion, learning) as compared with the control or the other groups and no neurotoxicity effects. Blood levels of methanol measured in the F1-offsprings (age 9 weeks) were approx. 2 - 3 mg/L for control animals (baseline), approx. 3 - 3.5 mg/L for the 10 ppm methanol treatment group, approx. 1 - 4.2 mg/L for the 100 ppm methanol treatment group and approx. 53 (males)-100 (females) mg/L for the 1000 ppm methanol treatment group.

 

F2

As in F1 males, an apparently dose-related earlier descensus testis was noted after 1000 ppm 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. No mortality and no changes in body weights were observed. No hematological findings, no clinical biochemistry findings, no histological findings and no effects on sexual maturation were observed. Organ weights showed similar tendencies as found in the F1-generation.

 

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 1000 ppm 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 the 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 1000 ppm exposed groups, the effects observed may be considered as biologically relevant under these test conditions and, therefore, 1000 ppm (1300 mg/m³) is established as LOAEC and 100 ppm as NOAEC (130 mg/m³) for post-natal development while for parental effects, the NOAEC is 1000 ppm (1300 mg/m³).

 

 

One-generation reproductive toxicity in monkeys, RL2

Effects of methanol on reproduction were tested in a one generation reproductive toxicity study similar to OECD guideline 415 in male and female mokeys after daily inhalative administration. The duration of the study was approx. 12 months (duration prebreeding: approximately 120 days; duration breeding: approximately 70 days; duration pregnancy: approximately 165 days). Animals were treated daily for 2.5 hours (7 days/week) before breeding, during breeding and during pregnancy with doses of 200, 600 and 1800 ppm. The two-cohort study design utilized 48 adult females (24 females/cohort), 4 adult males (2 males/cohort), and their offspring. For each cohort, adult females were initially separated into 6 groups, with 4 animals per group based on known or estimated age, size, and colony parity. Females from each of the 6 groups were then randomly assigned to one of four methanol-exposure groups. For postnatal developmental evaluation, 8 to 9 infants were available per group, in total 34 infants, among them 26 in-utero treated offsprings.

 

P0

The results of 50 (Cohort 2) to 100 (Cohort 1) clinical observations did not indicate the presence of overt toxicity in the adult females. During the entire study, only 6 females failed to respond to the visual task (3 control females and 3 methanol exposed females). Of the 46 females observed, 23 failed the motor coordination task during the study. All of these females failed the task during the baseline period as well as during methanol exposure, and none exhibited a pattern of responses indicative of fine-motor incoordination due to methanol exposure. The number of females who became ill and required medication was low, and unrelated to dose. The weights of all females were stable during the study. Mean weight gain during pregnancy varied from 1.3 to 1.8 kg across all exposure groups. The results did not indicate a significant methanol exposure effect on maternal weight gain during pregnancy (p > 0.12, all tests). There was no overt toxicity in adult females from any of the 4 methanol exposure groups. Lethargy, uncoordinated motor movements, and labored respiration were not observed during the study. No significant differences in the lengths of menstrual cycle of females across the 4 methanol exposure groups during the baseline period were observed There was no statistically significant methanol exposure effect on cycle lengths in the females (p = 0.45). The duration of menstrual cycle remained stable at approximately 30 days. The frequency of conception was approximately the same across the 4 exposure groups (82 % in the control group, 75 % at 200 ppm, 82 % at 600 ppm, and 83 % at 1800 ppm). Conception frequencies were not affected by methanol exposure.

A total of 37 infants were delivered from the 46 females. Two females delivered stillborn infants, 1 infant in the control group and 1 infant at 600 ppm. One female at 1800 ppm required a Caesarean (C) section to deliver a dead fetus.

The rate of complications during pregnancy or labour and delivery were 22 % for the control females and the 200 ppm females, 33 % for the 600 ppm females, and 30 % for the 1800 ppm females. No significant differences across methanol exposure groups were found. The live-birth delivery rates were between 90 and 100 % for all 4 exposure groups. There were no sire related effects on pregnancy length. A significant effect on pregnancy length due to methanol exposure was found. Each of the 3 methanol-exposed groups had significantly shorter durations of pregnancy than did the control group.

 

F1

The offspring characteristics analyzed were birth weight, crown–rump length, head circumference, head length, and head width. No significant effects of methanol exposure on the offspring size at birth were found. Concerning cohort differences, a significant methanol-exposure-group-by-cohort interaction for birth weight and head circumference was found.

Exposure to methanol at concentrations of up to 1800 ppm for over 1 year did not produce overt signs of toxicity (motor incoordination, blindness, and/or respiratory effects) in adult female non-human primates. Chronic methanol exposure did not interfere with the menstrual cycle or the ability of females to conceive. The timed-mating procedures used (3 matings/day between days 11 and 13 of the menstrual cycle) typically produce close to 100 % conception rates in normal groups of M. fascicularis females (Mahoney, 1975). The overall conception rate for this study was lower than expected, at 80 %. This was due to a sire effect: 1 of the males in Cohort 2 successfully impregnated only 4 females.

There was no pregnancy-induced folate deficiency. Fetal and newborn mortality frequencies were low for all of the exposure groups. One female at 1800 ppm had to be C-sectioned to deliver a dead fetus, and 2 females (1 control infant, 1 infant at 600 ppm) vaginally delivered full-term stillborn infants. The autopsy on the fetus delivered by C-section indicated the presence of hydrocephalus with significant autolysis in all of the major organs. Autopsies on the 2 stillborn infants indicated that the lungs were not inflated and that they had died close to or during delivery. No malformations were observed, and the cause of death for both infants was asphyxiation.

Two methanol-exposed females each at 200 ppm and 600 ppm were C-sectioned following observations of uterine bleeding without productive labour, presumably due to placental detachment. All 4 infants were delivered alive and without complications. Given the small number of animals exhibiting this condition and the lack of a response at the highest exposure concentration, conclusions concerning methanol exposure as a causative factor in uterine bleeding are not warranted.

It was not clear whether the decrease of about 6 to 8 days in duration of pregnancy noted as compared to controls was related to methanol exposure, since there was no dose-response and no differences among offspring groups in body weight or other physical parameters.

Although the average gestation period of the methanol exposed offspring was significantly shorter than that of the controls, methanol exposure did not affect the size of the offspring at birth. The average birth weight, crown–rump length, and head size of infants in the methanol exposure groups were comparable to those of the control infants. These results do not indicate that reduced offspring size at birth is associated with methanol exposure concentrations insufficient to cause overt maternal toxicity.

Prenatal exposure to methanol had no effect on infant growth and physical development for the first 9 months.

 

Chronic methanol inhalation exposures (0, 200, 600, 1800 ppm) for up to 1 year did not cause overt maternal toxicity in Macaca fascicularis females. The menstrual cycles and the ability of females to conceive and give birth to healthy live-born infants were also unaffected. Methanol exposures, however, were associated with a reduction in the length of pregnancy in this animal model, thus shortening the gestation length of the offspring. The reduction in the length of pregnancy was not dose dependent. Exposures from 200 to 1800 ppm methanol vapour were associated with an average 6 to 8-day (approximately 5 %) reduction in the length of pregnancy. No concomitant decrease in offspring birth size was observed. Based on the study results, the NOAEC for maternal toxicity/reproductive performance and the NOAEC for growth and physical development of the offspring were considered both to be 1800 ppm (2390 mg/m³). No NOAEC was derived by the study authors.

 

Reference:

Mahoney C. (1975.) Practical aspects of determining early pregnancy, stage of foetal development, and imminent parturition in the monkey (Macaca fascicularis). Lab Anim 6: 261–274.

 

Conclusion: No profound effects on fertility were observed.

Effects on developmental toxicity

Description of key information

The reaction mass of methyl acetate and methanol is assessed on the basis of these individual substances methyl acetate and methanol.

 

Methyl acetate

There are no data on developmental toxicicity of methyl acetate. However, due to the rapid hydrolysis of this compound it is justified to base hazard assessment with respect to developmental toxicity on the toxicological properties of the immediate metabolites. Concerning the metabolites of methyl acetate, acetic acid appears to be of less significance, since there are no indications of a fetotoxic or teratogenic potential, whereas for methanol some embryo-/fetotoxic and teratogenic effects were demonstrated in rodents, however at relatively high concentrations, respectively maternal toxic concentrations only. A NOAEC/developmental toxicity for methanol of 1000 ppm (1330 mg methanol/m³) was derived from a study in rats (NEDO, 1987) from >20 hours/day inhalatory exposure, which can be converted to a NOAEC/developmental toxicity of about 3,000 mg methyl acetate/m³. A NOAEC of >2395 mg/m³ after methanol treatment was derived from a teratogenicty study in monkeys, which can be converted into 5525 mg/m³ methyl acetate.

 

Methanol

In a prenatal developmental toxicity study similar to OECD guideline 414, inhalative exposure to 6655 mg/m³ methanol to pregnant rats during gestation (>20 h/day) produced maternal toxicity, fetal malformation, increased perinatal mortality and developmental delay in surviving progeny. Teratogenic effects occurred only at maternally toxic exposure concentration. Therefore, the NOEC for maternal and developmental toxicity is considered to be 1330 mg/m³.

In a teratogenicity study, monkeys were exposed to methanol by inhalation (2.5 hours/day, 7 days/week) prior to breeding and throughout pregnancy (approx. 12 months). No overt materal or fetal toxicity was observed. Therefore, the NOAEC derived in this study was >2395 mg/m³ for maternal and fetal toxicity.

 

Conclusion:

No experimental data are available for the target substance itself. Thus, the assessment is based on a read-across and weight of evidence approach, using the source substances methyl acetate and methanol. As methanol is the more critical substance, the assessment of the target substance is based on results of source substance 1 (methanol), following a worst case scenario. The NOAEC for developmental toxicity of the reaction mass of methyl acetate and methanol is thus found to be 1330 mg/m³.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental 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

Remarks:

documentation limited

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

Not all parameters mentioned in the guideline were investigated, limited documentation

Principles of method if other than guideline:

According to national standards. Comprehensive study programme on three species (rat, mouse, monkey) including metabolic, pharmacokinetic, short-, long-term, reproduction and carcinogenicity studies.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Housing: individually

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: H 1000 multi-tiered inhalation chambers, Hazleton Systems Inc., USA (volume 2.5 m³)
- Method of holding animals in test chamber: pregnant females were individually housed in wire mash stainless steel cages with 24 rooms placed in the inhalation chamber (whole body exposure)
- Source and rate of air: filtered external air, ventilation rate of 30 (not further specified)
- Method of conditioning air: passed through a medium performance filter, a high performance filter and an activated carbon filter
- System of generating vapours: total vaporizer supplied with liquid methanol by a microprecision pump, vaporization into the filtered air
- Temperature, humidity, pressure in air chamber: 23-26°C, 50-65%, atmospheric pressure
- Air flow rate: 1250 L/min
- Air change rate: 30/h
- Method of particle size determination: not applicable
- Treatment of exhaust air: not specified

TEST ATMOSPHERE
- Brief description of analytical method used: air from the inhalation chamber was extracted at a rate of 1.0 L/min by a sampling apparatus and the methanol concentration measured by a methanol vapor analyzer incorporating an infra-red spectrophotometer. The concentration signal was transmitted to the microprecision pump and used to regulate the methanol concentration in the chamber by adjusting liquid flow.
- Samples taken from breathing zone: no

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.

Details on mating procedure:

- Impregnation procedure: pregnant females, not further specified

Duration of treatment / exposure:

GD 7-17

Frequency of treatment:

continuously, approx. 22.7 h/d

Duration of test:

various durations: until Cesarian section, the age of 8 weeks, and reproduction of F1, respectively

Dose / conc.:

270 mg/m³ air

Dose / conc.:

1 330 mg/m³ air

Dose / conc.:

6 650 mg/m³ air

No. of animals per sex per dose:

36 dams per test and control group, including 12 dams allowed for natural delivery.

Control animals:

yes, concurrent no treatment

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
BODY WEIGHT: Yes
FOOD CONSUMPTION: Yes
WATER CONSUMPTION: Yes
POST-MORTEM EXAMINATIONS: Yes

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Other: embryolethality

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes

Clinical signs:

no effects observed

Mortality:

mortality observed, treatment-related

Description (incidence):

One dam died, another one had to be killed before delivery.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

a decrease in body-weight gain at 5000 ppm

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was reduced during gd 7 through 12 at 5000ppm

Food efficiency:

no effects observed

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

effects observed, treatment-related

Description (incidence and severity):

Drinking water consumption was reduced during gd 7 through 12 at 5000ppm

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

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):

late resorptions: 10.4 % vs. 0.6 % in control, p<0.05 at 5000 ppm, ), but the variance between single litters was high

Dead fetuses:

no effects observed

Changes in pregnancy duration:

effects observed, treatment-related

Description (incidence and severity):

After 5000 ppm, mean gestation time was prolonged by 0.7 days.

Changes in number of pregnant:

not examined

Key result

Dose descriptor:

NOAEC

Effect level:

1.33 mg/L air

Basis for effect level:

maternal abnormalities

Key result

Dose descriptor:

LOAEC

Effect level:

6.65 mg/L air

Basis for effect level:

maternal abnormalities

mortality

other: body weight gain, food and drinking water consumption

Key result

Abnormalities:

effects observed, treatment-related

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight of live fetuses after Cesarian section was reduced (about -20 %, p<0.001) at 5000 ppm

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

Among descendants from the high-dose group, mortality was prominent during the first 4 d after birth with live fetuses showing poor vitality in this time (ca. 10% = 2/12 pups per litter died as compared with 1 to 2% fatal cases in the other groups).

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Only after intra-uterine exposure to 5000 ppm: "atresia of cervical arch/vertebra foramen costotransversarium" (45 %), " bifurcated vertebral center" (14 %) and "cervical rib" (65 %) as well as "excessive sublingual neuropore" (50 %), all of which malformations having no or little relevance in the other group except of "atresia foramen" with about 25 % in the control and about 4 to 8 % in the other exposure groups

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Only after intra-uterine exposure to 5000 ppm: About 50 % of the fetuses with ventricular septal defects (visceral malformation in 16/20 litters or 64/131 fetuses) vs. 0% or near 0% in all other groups, and residual thymus (variation in all 20 litter or 70/131 fetuses) vs. about 2.4 to 2.9 % in 4 litters each of all other groups.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

After 5000 ppm, food and drinking water consumption of dams was reduced also after birth during lactation. Slight retardation of growth was still significant at weaning. Water consumption was slightly reduced, in particular for females.
Early indicators for post-natal development:
In pups from the 5000-ppm group, eruption of upper incisor and opening of eyelid for both sexes and descensus testis for males were significantly earlier than in the controls in relation to term of delivery, but not in relation to the whole gestation time which was prolonged for this group. There were no differences in behavioral and functional tests as compared to control and other test groups. At the age of 8 weeks, brain, thyroid (males), thymus and testis (males) weights were lower (p<0.01), and pituitary-gland weight of males was higher (p<0.05). But histological examination revealed no treatment-related changes. 16.5 % of the offsprings (15/91 in 8/12 litters) had hemilateral thyroprivia (missing thyroid lobe, mostly left). There was no histopathological lesion in the tissue. The defect was attributed to an impairment of organogenesis.
Reproductive performances of F1 (from 5000 ppm):
No significant effects on sexual cycle, genital function and reproductive performance of the F1 progeny were noted.

Key result

Dose descriptor:

NOAEC

Effect level:

1.33 mg/L air

Basis for effect level:

other: teratogenicity

Key result

Dose descriptor:

LOAEC

Effect level:

6.65 mg/L air

Basis for effect level:

other: teratogenicity

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: visceral and skeletal malformations, postnatal growth and survival

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

6.65 mg/m³ air

Treatment related:

yes

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.

Executive summary:

The exposure to 5000 ppm of pregnant rats during gestation (>20 h/d) produces maternal toxicity, fetal malformation, increased perinatal mortality and developmental delay in surviving progeny. Teratogenic effects occurred only at maternally toxic exposure concentration. Exposure levels of 1000 ppm or less did not induce toxic symptoms in maternal animals, structural abnormalities or delay in growth or functional development in the F1-generation. Therefore, the NOEC for maternal and developmental toxicity is considered to be 1000 ppm.

Reference 2

Endpoint:

developmental 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

Principles of method if other than guideline:

The present study was designed to characterize maternal reproductive performance and early offspring effects following exposure to methanol vapor in a nonhuman primate model.

GLP compliance:

not specified

Species:

monkey

Strain:

Macaca fascicularis

Route of administration:

inhalation: vapour

Duration of treatment / exposure:

prior to breeding and throughout pregnancy

Frequency of treatment:

ca. 2.5 h/d, 7 d/wk

No. of animals per sex per dose:

48 adult animals

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

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:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

One female each in the control and 600-ppm group delivered a stillborn infant and a cesarean section (C-section) was required to deliver a hydrocephalic infant who died in utero in the maternal 1800-ppm group.

Changes in pregnancy duration:

effects observed, treatment-related

Description (incidence and severity):

The mean length of pregnancy in the MeOH-exposed groups was significantly decreased by 6 to 8 days when compared to controls.

Changes in number of pregnant:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Although not statistically significant, five MeOH-exposed females were C-sectioned due to pregnancy complications such as uterine bleeding and prolonged unproductive labor

Key result

Dose descriptor:

NOAEC

Basis for effect level:

other: effect type not specified

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

uterus

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no MeOH-related effects on offspring birth weight.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

A total of 34 live-born infants were delivered (control=8, 200 ppm=9, 600 ppm=8, 1800 ppm=9)

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no MeOH-related effects on newborn health status

Key result

Dose descriptor:

NOAEC

Basis for effect level:

other: effect type not specified

Remarks on result:

not determinable

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Executive summary:

The results of the present study indicate that, for this nonhuman primate model, daily 2 1/2 h exposures to MeOH vapor from 200 to 1800 ppm for nearly 1year do not cause overt maternal toxicity in M. fascicularis females. The menstrual cycle and the ability of females to conceive were unaffected by these exposures. The incidence of maternal complication during pregnancy and delivery was high in the methanol-exposed females. The increase in complications however, was not statistically significant when compared to controls. The health status of live-born offspring was unaffected by maternal MeOH exposure. MeOH exposures were associated, however, with a reduction in the length of pregnancy. The reduced pregnancy lengths of the MeOH-exposed females may reflect the premature activation of the fetal HPA axis that controls timing of birth. Whether this represents a direct (fetal) or indirect maternal) treatment effect is unknown. Independent of the specific biological mechanism, the reduced pregnancy durations of MeOH-exposed dams suggest a subtle but systematic disturbance in the timing of labor and delivery.
The authors suggest that future studies on the public health consequences of MeOH exposure during pregnancy should examine the neuroendocrine control of parturition.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

1 330 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

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

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Methyl acetate

Methyl acetate is rapidly hydrolysed after intake. Due to the rapid hydrolysis of the substance, no relevant systemic exposure to the parent compound was observed (see toxicokinetics chapter) Accordingly, the toxicological properties of the immediate metabolites are taken into consideration for evaluation of methyl acetate toxicity with respect to reproductive and developmental toxicity. The cleavage products relevant for the assessment are methanol and acetic acid.  The same conclusion was drawn in the EU risk assessment on methyl acetate (EU RAR 2008).

 

Assessment of metabolites: Acetic acid

The available data on acetic acid indicates that acetic acid is not teratogenic in vivo. Teratogenic evaluation of apple cider vinegar was made in rabbits. The administration of up to 1.6 g per kg body weight of the test material daily to pregnant rabbits (days 6 through day 18 of gestation) had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls. Sodium acetate displayed no teratogenicity in the developing chicken embryo at levels up to 200 mg per kg of egg when injected into the air cell or yolk of unincubated eggs, or at levels up to 100 mg per kg egg when injected into the air cell or yolk of eggs after 96 hours of incubation (cited from NTIS, 1977).

 

Supporting Developmental toxicity data on acetic acid as released from methyl acetate can further be gathered from tert-butyl acetate (TBAc). Similar to methyl acetate TBAc is rapidly hydrolyzed to acetic acid (Groth and Freundt, 1994; MAK, 2016). A developmental toxicity study of tert-butyl acetate (TBAc) in rats shows embryotoxicity in form of visceral and skeletal variations at a maternally toxic dose in excess of the limit dose (i.e., 1,600 mg/kg/ day) and is minimally embryotoxic, showing the same variations, at  adose (i.e., 800 mg/kg/day) that did not induce signs of maternal toxity (Yang et al., 2007). However, no evidence for the teratogenicity of TBAc was noted in rats. The NOAEL for maternal toxicity was 800 mg/kg b.w. per day. The NOAEL for developmental toxicity was 400 mg/kg b.w. per day. The MAK commission (MAK, 2016) provided a plausible rationale on the observed embryotoxicity at high doses: " On the basis of the consistent findings with tert-butyl alcohol in developmental toxicity studies (skeletal variations and the absence of teratogenicity), it may be assumed that the foetotoxic effects that were observed in the studies with tert-butyl acetate were induced by the tert-butyl alcohol metabolite or its secondary metabolites." Based on the available data on tert-butyl acetate and on acetate and tert-butanol, its major metabolites, EFSA (2012) concluded that tert-butyl acetate do not give rise to concerns regarding systemic toxicity, developmental toxicity or genotoxicity. It can be concluded that there are no indications that the hydrolysis product acetic acid has a relevant contribution to the developmental toxicity potential of methyl acetate.

 

Assessment of metabolites: Methanol

In a developmental toxicity study, after continuous inhalatory exposure of rats, impaired development of the offspring (reduced numbers of live fetuses, resp. of live pups per litter, increased number of late resorptions, reduced fetal body weight, fetal visceral and skeletal abnormalities, postnatal mortality) was observed at concentrations of 5000 ppm (6655 mg/m³) together with signs of maternal toxicity (reduced body weight gain), whereas at the next lower exposure level (1000 ppm/ 1330 mg/m³) such effects were no longer demonstrated.

 

In another developmental toxicity study, daily 2 1/2 h exposures to MeOH vapor from 200 to 1800 ppm for nearly 1 year did not cause overt maternal toxicity in M. fascicularis females. The menstrual cycle and the ability of females to conceive were unaffected by these exposures. The incidence of maternal complication during pregnancy and delivery was high in the methanol-exposed females. The increase in complications however, was not statistically significant when compared to controls. The health status of live-born offspring was unaffected by maternal MeOH exposure. MeOH exposures were associated, however, with a reduction in the length of pregnancy. The reduced pregnancy lengths of the MeOH-exposed females may reflect the premature activation of the fetal HPA axis that controls timing of birth. Whether this represents a direct (fetal) or indirect maternal) treatment effect is unknown. Independent of the specific biological mechanism, the reduced pregnancy durations of MeOH-exposed dams suggest a subtle but systematic disturbance in the timing of labor and delivery. The NOAEC derived in this study was >1800 ppm (>2395 mg/m³) for maternal and fetal toxicity.

 

Conclusion on developental toxicity/teratogenicity for methyl acetate:

There are no data on developmental toxicity of methyl acetate. However, due to the rapid hydrolysis of this compound it is justified to base hazard assessment with respect to reproduction on the toxicological properties of the immediate metabolites. Concerning the metabolites of methyl acetate, acetic acid appears to be of less significance, since there are no indications of a fetotoxic or teratogenic potential, whereas for methanol some embryo-/fetotoxic and teratogenic effects were demonstrated in rodents, however at relatively high concentrations, respectively maternal toxic concentrations only. A NOAEC/developmental toxicity for methanol of 1000 ppm (1330 mg/m³) was derived from the inhalation study by NEDO (1987) in rats.

 

Literature

 

NEDO (1987). Toxicological Research of methanol as a fuel for Power Station, Summary Report on Tests with Monkeys, Rats and Mice. New Energy Development Organization. Tokyo, Japan.

 

MAK Collection for Occupational Health and Safety (2016), Vol 1, No 2: tert-Butyl acetate p. 578-596

 

EFSA (European Food Safety Authority) (2012): SCIENTIFIC OPINION Scientific Opinion on the evaluation of the substances currently on the list in the annex to Commission Directive 96/3/EC as acceptable previous cargoes for edible fats and oils – Part II of III. EFSA Journal 2012; 10 85): 2703

 

Methanol

Teratogenicity, monkeys, RL2

In a teratogenicity study, monkeys were exposed to methanol by inhalation (2.5 hours/day, 7 days/week) prior to breeding and throughout pregnancy. In maternal animals, no mortality, no clinical signs and no gross pathological findings were observed. There were no hematological, neuropathological or histopathological findings There occurred no pre-/post-implantation losses, no total litter losses by resorption, no early or late resorptions and no treatment-related increases in the number of dead fetuses. One female each in the control and 600-ppm group delivered a stillborn infant and a cesarean section (C-section) was required to deliver a hydrocephalic infant who died in utero in the maternal 1800-ppm group. The mean length of pregnancy in the MeOH-exposed groups was significantly decreased by 6 to 8 days when compared to controls. Although not statistically significant, five MeOH-exposed females were C-sectioned due to pregnancy complications such as uterine bleeding and prolonged unproductive labor.

For fetuses, no body weight changes, no changes in sex ratio, no changes in litter sizes+ weights and no external, skeletal or visceral malformations were observed. There were no MeOH-related effects on newborn health status.

The results of the present study indicate that, for this non-human primate model, daily 2 1/2 h exposures to MeOH vapor from 200 to 1800 ppm for nearly 1 year do not cause overt maternal toxicity in M. fascicularis females. The menstrual cycle and the ability of females to conceive were unaffected by these exposures. The incidence of maternal complication during pregnancy and delivery was high in the methanol-exposed females. The increase in complications however, was not statistically significant when compared to controls. The health status of live-born offspring was unaffected by maternal MeOH exposure. MeOH exposures were associated, however, with a reduction in the length of pregnancy. The reduced pregnancy lengths of the MeOH-exposed females may reflect the premature activation of the fetal HPA axis that controls timing of birth. Whether this represents a direct (fetal) or indirect maternal) treatment effect is unknown. Independent of the specific biological mechanism, the reduced pregnancy durations of MeOH-exposed dams suggest a subtle but systematic disturbance in the timing of labor and delivery. The authors suggest that future studies on the public health consequences of MeOH exposure during pregnancy should examine the neuroendocrine control of parturition. The NOAEC derived in this study was >1800 ppm (>2395 mg/m³) for maternal and fetal toxicity.

 

 

Teratogenicity, rats, RL2

In a prenatal developmental toxicity study similar to OECD guideline 414, inhalative exposure (>20 h/day) to 200, 1000 or 5000 ppm methanol of pregnant rats during gestation (gestation day 7-17) was investigated. The study duration was based on time point of cesarian section, the age of 8 weeks, and reproduction of F1, respectively. 36 dams per test and control group, including 12 dams allowed for natural delivery were used. 5000 ppm produced maternal toxicity, fetal malformation, increased perinatal mortality and developmental delay in surviving progeny. Teratogenic effects occurred only at maternally toxic exposure concentration. Exposure levels of 1000 ppm or less did not induce toxic symptoms in maternal animals, structural abnormalities or delay in growth or functional development in the F1-generation. Therefore, the NOEC for maternal and developmental toxicity is considered to be 1000 ppm (1330 mg/m³).

 

 

Teratogenicity, mice, RL4

In a teratogenicity study in mice, 2000 or 2500 mg/kg bw methanol was administered to mice by gavage on gestation days 6-15. Methanol, which is detoxified via a folic acid-dependent pathway, has been shown to be teratogenic in mice. Given recent observations that the level of dietary folic acid intake may be inversely related to the occurrence of select birth defects in humans, the authors tested the hypothesis that dietary folic acid intake would influence the developmental toxicity of methanol. Virgin female mice were fed one of three diets containing 400 (low), 600 (marginal), or 1,200 (adequate) nmol folic acid/kg diet for 5 weeks prior to and following mating. On gestation days 6-15, dams were administered by gavage either vehicle (distilled, deionized water) or methanol at 2.0 or 2.5 g/kg body weight, twice daily. On GD 18, mice were weighed and killed and the liver, kidneys, and gravid uteri removed and weighed. Implantation sites, live and dead fetuses, and resorptions were counted; fetuses were weighed individually and examined for cleft palate and exencephaly. One third of the fetuses in each litter were examined for skeletal morphology. Both methanol and low dietary folic acid increased the incidence of cleft palate, with the highest number of affected litters in the low dietary folic acid group. These results support the concept that maternal folate status can modulate the developmental toxicity of methanol. The LOAEC for fetal toxicity was found to be 2000 mg/kg bw.in absence of maternal toxicity.

 

 

Teratogenicity, rats, RL 3

In a prenatal developmental toxicity study similar to OECD guideline 414, rats were exposed daily (7 hours/day) to methanol from gestation day 7- to day 19. The following parameters were recorded: maternal bodyweight, maternal blood methanol levels, offspring bodyweight, offspring mortality, motor activity (day 31-21, 30, 60), olfactory learning (day18), behavioural thermoregulation (day 20-21), T-maze learning (day 23-24), acoustic startle response (day 24, 60), reflex modification audiometry (day 60), pubertal landmarks (day 31-56), passive avoidance (day 72), visual-evoked potentials (day 160). 5 or 6 dams were examined with 10 - 11 offspring. Behavioural tests were performed on 4 - 5 offspring animals. Maternal blood methanol levels were between 3.8 and 3.1 mg/mL. The treatment led to reduced maternal body weight (significantly), the only effect noted in dams. No increase in post-implantation loss was observed. Postnatal body weights were modestly but statistically lower after treatment. No increase in postnatal mortality was observed. The behavioral tests revealed no significant effects of methanol treatment. The number of pups available and tested (mostly, 1/sex/litter) was probably too small to draw firm conclusions about postnatal developmental effects on behavior.

 

Overall conclusion: Based on the results for methanol, the more critical of the two source substances methanol and methylacetate, the the NOAEC for developmental toxicity of the reaction mass of methyl acetate and methanol is found to be 1330 mg/m³.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The reaction mass of methyl acetate and methanol is assessed on the basis of the individual constituents methyl acetate and methanol using a read-across approach. The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Following a worst scenario, methanol, the more critical of both substances, is used for classification. Based on the very high doses of methanol which were needed to induce signs of developmental toxicity, the reaction mass of methyl acetate and methanol is not considered to be classified for developmental toxicity according to EU Regulation (EC) No 1272/2008 (CLP), as amended for fifteenth time in Regulation (EU) No 2020/217.