Trimethyl orthoacetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Trimethyl orthoacetate is rapidly hydrolysed to methanol (CAS no. 67 -56 -1) and methyl acetate, which further hydrolyses to acetic acid (CAS no. 64 -19 -7) as final reaction product in the presence of water or moisture at pH 4, 7 and 9 (< 2.4 h at 50°C). Under neutral (pH 7) and acidic (pH 4) conditions, the half-life was < 1 h. Accordingly, reliable data of the hydrolysis products methanol and acetic acid are used for the assessment of the toxic potential to reproduction, which is entirely appropriate to draw conclusions on the toxicity to reproduction of Trimethyl orthoacetate.

As the endpoint toxicity to reproduction could be waived for the degradation product acetic acid, data on toxicity to reproduction are only presented for the hydrolysis product methanol. In addition data for developmental toxicity are presented for the hydrolysis product acetic acid.

1. Methanol

- Toxicity to reproduction: two-generation study, 3 concentrations, inhalation (vapour, whole body), rat (Sprague-Dawley) m/f (OECD TG 416): NOAEC (develop) = 1.3 mg/L air (= 1000 ppm) (nominal, m/f) (P0: NOAEC (16 weeks) = 1.3 mg/L air (= 1000 ppm) (nominal, m/f); F1: NOAEC (14 weeks) = 1.3 mg/L air (= 100 ppm); F2: NOAEC (8 weeks) = 1.3 mg/L air (= 100 ppm))

- toxicity to reproduction: one-generation study, 3 concentrations, inhalation (vapour, whole body), monkey (Macaca fascicularis) m/f (OECD TG 415): NOAEC = 2.39 mg/L (= 1800 ppm) P0 (nominal, female) and F1 (nominal)

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

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 °C
- 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
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)

Sex:

male/female

Basis for effect level:

other: reproductive parameter

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

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)

Sex:

male/female

Basis for effect level:

other: reproductive parameter, brain weight

Key result

Dose descriptor:

LOAEC

Generation:

F1

Effect level:

1.3 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

other: reproductive parameter, brain weight

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)

Sex:

male/female

Basis for effect level:

other: reproductive parameter, brain weight

Key result

Dose descriptor:

LOAEC

Generation:

F2

Effect level:

1.3 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

other: reproductive parameter, brain weight

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:

read-across from supporting substance (structural analogue or surrogate)

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.

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

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
- Fasting period before study:
- 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:

During 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:

subchronic

Species:

rat

Effects on developmental toxicity

Description of key information

Trimethyl orthoacetate is rapidly hydrolyzed to methanol and methyl acetae, which further hydrolyses to acetic acid as final reaction product in the presence of water or moisture at pH 4, 7 and 9 (< 2.4 h at 50°C). Reliable data of the hydrolysis products methanol and acetic acid are used for the assessment of the genotoxic potential, which is entirely appropriate to draw conclusions on the toxicity to reproduction of Trimethyl orthoacetate.

2. Acetic acid

- Developmental toxicity: oral (gavage), rat (Wistar), (equivalent or similar to EU Method B.31): NOAEL (developmental toxicity) = 1600 mg/kg bw/day (nominal)

- Developmental toxicity: oral (gavage), rabbit ( Dutch), (equivalent or similar to EU Method B.31): NOAEL (developmental toxicity) = 1600 mg/kg bw/day (nominal)

- Developmental toxicity: oral (gavage), mouse (CD-1), (equivalent or similar to EU Method B.31): NOAEL (maternal toxicity) = 74.3 mg/kg bw/day (nominal), NOAEL (developmental toxicity) = 345 mg/kg bw/day (nominal)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP status not known, near guideline study, published in peer reviewed literature, limitations in design and/or reporting but otherwise adequate for assessment.

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Virgin adult female albino rats (Wistar derived stock) were individually housed in mesh bottom cages in temperature and humidity controlled quarters with free access to food and fresh tap water.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The females were dosed with the indicated dosages by oral intubations. The controls were sham treated with the vehicle at a level equivalent to the group receiving the highest test dose. The test material was prepared and doses calculated according to the following table:

Dosage Dose Concentration
(mg/kg) (ml/kg) (mg/ml)
250 1 250
251 - 540 2 125 - 250
501 - 750 3 133 - 250
751 - 1000 4 187 - 250
1001 - 1250 5 200 - 250
1251 - 1500 6 208 - 250
1501 - 1600 6.4 235 - 250

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

The females were mated with young adult males, and observation of the vaginal sperm plug was considered Day 0 of gestation.

Duration of treatment / exposure:

Days 6 - 15 of gestation

Frequency of treatment:

Daily

Duration of test:

Days 0-20 of gestation

No. of animals per sex per dose:

25 mated females

Control animals:

yes, sham-exposed

Details on study design:

An additional group of mated females was dosed with 150 mg/kg aspirin and served as a positive control

Maternal examinations:

Body weights were recorded on Days 0, 6, 11, 15, and 20 of gestation.
All animals were observed daily for appearance and behaviour with particular attention to food consumption and weight, in order to rule out any abnormalities which may have occurred as a result of anorexic effects in the pregnant female animal

Ovaries and uterine content:

On Day 20 all dams were subjected to Caesarean section under surgical anesthesia, and the numbers of implantation sites, resorption sites, and live and dead fetuses were recorded. The urogenital tract of each dam was examined in detail for anatomical normality.

Fetal examinations:

The body weights of the live pups were recorded. All fetuses were examined grossly for the presence of external congenital abnormalities. One-third of the fetuses of each litter underwent detailed visceral examinations employing the Wilson technique. The remaining two-thirds were cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects.

Details on maternal toxic effects:

Details on maternal toxic effects:
The administration of up to 1600 mg/kg (body weight) of the test material to pregnant rats for 10 consecutive days had no clearly discernible effect on nidation or on maternal survival.

Dose descriptor:

NOAEL

Effect level:

1 600 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The administration of up to 1600 mg/kg (body weight) of the test material to pregnant rats for 10 consecutive days had no clearly discernible effect on 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.

Dose descriptor:

NOAEL

Effect level:

1 600 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The administration of up to 1600 mg/kg (body weight) to pregnant rats for 10 consecutive days 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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP status not known, near guideline study, published in peer reviewed literature, limitations in design and/or reporting but otherwise adequate for assessment.

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

Dutch

Details on test animals or test system and environmental conditions:

Virgin, adult, Dutch-belted female rabbits were individually housed in mesh bottom cages in temperature and humidity-controlled quarters with free access to food and fresh tap water.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The females were dosed with the indicated dosages by oral intubations. The controls were sham treated with the vehicle at a level equivalent to the group receiving the highest test dose. The test material was prepared and doses calculated according to the following table:

Dosage Dose Concentration
(mg/kg) (ml/kg) (mg/ml)
250 1 250
251 - 500 2 125 - 250
501 - 750 3 133 - 250
751 - 1000 4 187 - 250
1001 - 1250 5 200 - 250
1251 - 1500 6 208 - 250
1501 - 1600 6.4 235 - 250

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

On Day 0, each doe was given an injection of 0.4 ml of human chorionic gonadotropin (400 IU) via the marginal ear vein. Three hours later, each doe was inseminated artificially with 0.3 ml of diluted semen from a proven donor buck using approximately 20 x 106 motile sperm.

Duration of treatment / exposure:

Days 6 - 18 of gestation

Frequency of treatment:

Daily

Duration of test:

Days 0-29 of gestation

No. of animals per sex per dose:

Approximately 12 pregnant females.

Control animals:

yes, sham-exposed

Details on study design:

An additional group of mated females was dosed with 2.5mg/kg 6-aminonicotinamide on day 9 and served as a positive control

Maternal examinations:

Body weights were recorded on Days 0, 6, 12, 18, and 29 of gestation.
All animals were observed daily for appearance and behaviour with particular attention to food consumption and weight, in order to rule out any abnormalities which may have occurred as a result of anorexic effects in the pregnant female animal

Ovaries and uterine content:

On Day 29 all does were subjected to Caesarean section under surgical anaesthesia, and the numbers of corpora lutea, implantation sites, resorption sites and live and dead foetuses were recorded. The urogenital tract of each animal was examined in detail for normality.

Fetal examinations:

The body weights of the live pups were recorded. all foetuses underwent a detailed gross examination for the presence of external congenital abnormalities. The live foetuses of each litter were then placed in an incubator for 24 hours for the evaluation of neonatal survival. All surviving pups were sacrificed, and all pups examined for visceral abnormalities (by dissection). All foetuses were then cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects.

Details on maternal toxic effects:

Details on maternal toxic effects:
The administration of up to 1600 mg/kg (body weight) of the test material to pregnant rabbits for 13 consecutive days had no clearly discernible effect on nidation or on maternal survival.

Dose descriptor:

NOAEL

Effect level:

1 600 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The administration of up to 1600 mg/kg (body weight) of the test material to pregnant rabbits for 13 consecutive days had no clearly discernible effect on 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.

Dose descriptor:

NOAEL

Effect level:

1 600 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The administration of up to 1600 mg/kg (body weight) to pregnant rabbits for 13 consecutive days 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.

Executive summary:

In a developmental toxicity study in rabbits, apple cider vinegar, that contains 5% acetic acid, was administered by gavage at dose levels of 0, 16, 74.3, 345 and 1600 mg/kg/day for 13 consecutive days. The authors reported that administration of up to 1600 mg/kg bodyweight of the test material to dams for 13 days revealed no discernible effect on maternal or foetal survival, or on soft of skeletal tissues.

There was a reduction in pregnancy rate at the high dose and from 74.3 mg/kg/day, a dose-dependent decrease in maternal bodyweight gain in dams. Some deaths or abortions occurred in all treated groups and some litter losses were reported at 345 and 1600 mg/kg/day. Maternal effects were much more noticeable than effects on foetal development. It has been concluded that these findings are a consequence of the bactericidal properties of, orally administered, acetic acid within the gastrointestinal tract of female rabbits (EU, 2008). These effects are considered not to be a direct effect on embryonic implantation and development of acetic acid (EU, 2008). It is likely that this accounts for the apparent increased sensitivity of this species to oral administration of acetic acid.

Reference 3

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP status not known, near guideline study, published in peer reviewed literature, limitations in design and/or reporting but otherwise adequate for assessment.

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

Virgin adult female albino CD-1 outbred mice were gang-housed in disposable plastic cages in temperature and humidity-controlled quarters with free access to food and fresh tap water.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Dose volume 10 ml/kg body weight

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

The females were mated with young adult males, and observation of the vaginal sperm plug was considered Day 0 of gestation.

Duration of treatment / exposure:

Days 6 - 15 of gestation

Frequency of treatment:

Daily

Duration of test:

Days 0-17 of gestation

No. of animals per sex per dose:

25 mated females

Control animals:

yes, sham-exposed

Details on study design:

An additional group of mated females was dosed with 150 mg/kg aspirin and served as a positive control

Maternal examinations:

Body weights were recorded on Days 0, 6, 11, 15, and 17 of gestation.
All animals were observed daily for appearance and behaviour with particular attention to food consumption and weight, in order to rule out any abnormalities which may have occurred as a result of anorexic effects in the pregnant female animal

Ovaries and uterine content:

On Day 17 all dams were subjected to Caesarean section under surgical anesthesia, and the numbers of implantation sites, resorption sites, and live and dead fetuses were recorded. The urogenital tract of each dam was examined in detail for anatomical normality.

Fetal examinations:

The body weights of the live pups were recorded. All fetuses were examined grossly for the presence of external congenital abnormalities. One-third of the fetuses of each litter underwent detailed visceral examinations employing the Wilson technique. The remaining two-thirds were cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects.

Details on maternal toxic effects:

Details on maternal toxic effects:
The administration of up to 1600 mg/kg (body weight) of the test material to pregnant mice for 10 consecutive days had no clearly discernible effect on nidation or on maternal survival.

Dose descriptor:

NOAEL

Effect level:

345 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

74.3 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The administration of up to 1600 mg/kg (body weight) of the test material to pregnant mice for 10 consecutive days had no clearly discernible effect on 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.

Dose descriptor:

NOAEL

Effect level:

345 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: dvelopmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The administration of up to 1600 mg/kg (body weight) to pregnant rats for 10 consecutive days 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.

Executive summary:

In a developmental toxicity study in mice, apple cider vinegar, that contains 5% acetic acid, was administered by gavage at dose levels of 0, 16, 74.3, 345 and 1600 mg/kg/day for 10m consecutive days. The authors reported that administration of up to 1600 mg/kg bodyweight of the test material to dams for 10 days revealed no discernible effect on maternal or foetal survival, or on soft of skeletal tissues.

There was no effect on the foetal development, in the presence of slight maternal toxicity (reduced bodyweight gain) at 345 mg/kg/day. At the highest dose tested, 1600 mg/kg/day, there was an increase in the number of litters containing a dead foetus and some reductions in ossification. The NOAEL for maternal and developmental toxicity in mice were 74.3 mg/kg/day and 345 mg/kg/day, respectively. 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

345 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Based on the lack of any relevant observed adverse effects and the absence of any other test item related effects, it is rather impossible to hypothesize a concrete mode of action.

There were no adverse effects noted with regard to systemic toxicity on the parental generation in the most relevant 2 -generation study on a suitable read-across substance (hydrolysis product, methanol), which is consistently supported by all other available studies.

With regard to reproductive performance and offspring development, there were no treatment-related effects on mating for treated animals or in conception rates for treated animals. There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

Regarding Litter responses, no toxicologically relevant signs of toxicity were detected for offspring from all treatment groups.

The ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive toxicity was hence considered to be 1300 mg/m³.

No definitive human relevance framework can be described due to the lack of any other effects securing any postulation, and no conclusion on biological plausibility can be drawn.

Despite the fact that no mode of action analysis can be performed, no data gap was identified here. The tonnage-driven data requirements under REACH were fully met, and the lack of relevance of the observed effects does also not indicate any high hazard for humans and so does not trigger any further examinations.

Justification for classification or non-classification

On the basis of the results of the 2 -generation reproductive toxicity study of a suitable read-across substance to the registered one (hydrolysis product methanol), the no observed adverse effect level (NOAEL) of the test item was determined. The administration of the test item to rats via inhalation, at all dose levels, did not result in any toxicologically relevant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic, reproductive and developmental toxicity was therefore considered to be 1300 mg/m³. According to Regulation 1272/2008, Table 3.7.1(a) Hazard categories for reproductive toxicants, a substance must be considered as reproductive toxicant under the following conditions: Suspected human reproductive toxicant: Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification. Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects.No relevant effects on fertility or development were noted up to the top dose of 1300 mg/m³ and no systemic toxicity was noted in the study considered most relevant, which was supported by all other available studies. This study was considered most relevant due to the at least subchronic study duration, and other studies with shorter exposure durations were hence disregarded, as in this study the animals were also dosed diring the whole pregnancy period, covering hence also all possible developmental effects. Consequently, the criteria for classification as reproductive toxicant (Cat. 2) are not met, the substance does not need to be classified according to Regulation 1272/2008.