Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEC (maternal toxicity) = 1.3 mg/L for rats
NOAEC (teratogenicity = 1.3 mg/L for rats
NOAEC (maternal toxicity) = 2.39 mg/L for monkeys
NOAEC (teratogenicity) = 2.39 mg/L for monkeys
Negative for spermatozoa morphological anomalies: NOAEL (oral) = 1000 mg/kg bw/day

Link to relevant study records

Referenceopen allclose all

Endpoint:
fertility, other
Remarks:
based on test type
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
Guideline:
other: not specified
Principles of method if other than guideline:
Male mice were treated orally with methanol and maintained without exposure for 5 weeks. Sperm morphology was examined.
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory, Portage, MI
- Age at study initiation: 4-month old
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
no data
Details on mating procedure:
not applicable
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
5 consecutive days
Frequency of treatment:
daily
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Statistics:
The effects of exposure and non-exposure for each factor were compared by Student´s t-test.
Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) [Nie et al., 1975].
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
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
Immunological findings:
no effects observed
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
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
In the methanol-treated animals, a slight, but statistically insignificant increase in sperms abnormalities (1.86 +-0.91% vs. 1.12 +-0.39% in the water control) was observed, while the treatment with cyclophoshamide (100 mg/kg) resulted in an about 5-fold increase (5.84 +-1.94).
Reproductive performance:
not examined
Key result
Dose descriptor:
LOAEC
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: sperm morphology
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
other: sperm
Treatment related:
yes
Dose response relationship:
not specified
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEC
Remarks on result:
not measured/tested
Key result
Reproductive effects observed:
no

In the methanol-treated animals, a slight, but statistically insignificant increase in sperms abnormalities (1.86 +-0.91% vs. 1.12 +-0.39% in the water control) was observed, while the treatment with cyclophoshamide (100 mg/kg) resulted in an about 5-fold increase (5.84 +-1.94).

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:
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 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
Gross pathological findings:
no effects observed
Neuropathological findings:
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.

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:
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 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
Gross pathological findings:
no effects observed
Neuropathological findings:
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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
mouse
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
1 300 mg/m³
Study duration:
chronic
Species:
rat
Additional information

No impairment of fertility and reproductive performance was found in male and female rats (parent and daughter generations) exposed to methanol (NEDO, 1987).

In a two-generation reproduction study, rats were exposed to methanol by inhalation for 19-20 hours/day (NEDO, 1987). No treatment-related alterations in general observations and reproductive parameters were found. None of the fertility indices including sexual cycle, days needed for insemination, insemination rate and pregnancy rate showed statistically significant differences. There were no differences for body weight, food consumption and water consumption during gestation and lactation period, either. No abnormalities were observed in findings on delivery and nursing behaviour and necropsy data of F0 animals. No firm conclusions can be drawn about fertility of either sex, as the copulation time of 21 days was comfortably long for successful insemination and gametogenesis was not considered. In the F1 and F2 progeny (both sexes), no histological changes and no effects on testes or ovaries were reported. However, a decrease in brain weights was evident at 1.3 mg/L methanol, but without noticeable histological changes and functional impairments. This phenomenon is believed to represent a change occurring during the prenatal period (Takeda and Katoh, 1988). However, no quantitative data and statistical level were documented for organ weights. 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.

In a one-generation reproduction study in monkeys (Burbacher et al., 1999), adult female monkeys were exposed to methanol vapour (2.5 hours/day; 0, 200, 600, 1800 ppm) during prebreeding, breeding and pregnancy. No signs of overt maternal toxicity were noted during the study in any of the dose groups. Methanol exposure had no effects on the tested reproductive performance, including menstrual cycles, conception rate, and live-birth delivery rate. However, all methanol-exposed animals had a decrease of about 6 to 8 days in duration of pregnancy compared to control animals. It is not clear whether this decrease in duration of pregnancy was related to methanol exposure, since there was no dose-response and no differences among offspring groups in body weight, size or other physical parameters (head size, crown rump length). Moreover, the duration of pregnancy was within the reported normal range for this species (NTP, 2003). Prenatal exposure to methanol had no effect on infant growth and physical development for the first 9 months. However, results of infant assessments during the first 9 months of life were confounded by the normal variance and the low number of animals. The NOAEC for reproductive effects can be determined to be at the highest concentration tested of 2.39 mg/L (1800 ppm).

In another study which investigated reproductive effects, there was an insignificant increase in morphological anomalies in spermatozoa in male mice at 1000 mg/kg bw/day after oral dosing for five weeks (Ward et al., 1984).

It is also pointed out that in repeated dose toxicity studies ovarian and testicular tissues have not been adversely affected by methanol administration.


Short description of key information:
NOAEC (maternal toxicity) = 1.3 mg/L for rats
NOAEC (teratogenicity = 1.3 mg/L for rats
NOAEC (maternal toxicity) = 2.39 mg/L for monkeys
NOAEC (teratogenicity) = 2.39 mg/L for monkeys
Negative for spermatozoa morphological anomalies: NOAEL (oral) = 1000 mg/kg bw/day

Effects on developmental toxicity

Description of key information
NOAEC(maternal toxicity) = 1.33 mg/L for rats
NOAEC(teratogenicity) = 1.33 mg/L for rats and mice
LOAEL (maternal toxicity) = 1700 mg/kg bw for mice
LOAEL( teratogenicity) = 5000 mg/kg bw for mice
NOAEC(maternal toxicity) = 2.39 mg/L for monkeys
NOAEC(teratogenicity) = 2.39 mg/L for monkeys
Link to relevant study records

Referenceopen allclose all

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:
Treatment of pregnant female mice on GD 6-10 with test substance by oral administration twice daily, investigation of the influence of folate in the diet on incidence of developmental defects in the offspring.
GLP compliance:
not specified
Species:
mouse
Strain:
CD-1
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles Rivers, Inc., Gilroy, CA
- Age at study initiation: 8 weeks
- Weight at study initiation: group weights on GD0 (mean ± SEM): 25.72 ± 0.48 to 27.19 ± 0.73 g
- Fasting period before study: no data
- Housing: stainless steel wire-bottomed cages
- Diet: amino-acid based, folic-acid free diet supplemented with either 400 or 1200 nmol folic acid/kg diet and 1 % succinylsulfathiazole for 5 weeks prior to mating and throughout breeding and gestation
- Water (e.g. ad libitum): no data
- Acclimation period: 5 weeks (already fed with special diet during this period)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 50
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: not applicable
Vehicle:
water
Details on exposure:
VEHICLE
- Concentration in vehicle: 15.65 % in deionized water
- Amount of vehicle (if gavage): no data
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2 or 1/3
- Length of cohabitation: 12 h
- Further matings after two unsuccessful attempts: no (second breeding period was continued for up to 10 days)
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
GD 6-10
Frequency of treatment:
twice daily 2500 mg/kg bw
Duration of test:
until GD 18
Dose / conc.:
5 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
21 to 24 dams per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: knowledge from previous experiments
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 5, 10, 12, 15, 18
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 18
- Organs examined: liver, kidney, gravid uterus, blood (plasma and hematocrit, folate concentrations, MN formation in maternal reticulocytes)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: fetal weights, crown-rump lengths, fetal liver folate concentrations, fetal hematocrit, MN formation in fetal reticulocytes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
The pregnant dam and the litter were considered the units for comparisons. Continuous variables were analyzed using the two-way analysis of variance procedure and the Fisher PLSD for multiple comparisons of means. These analyses were carried out on STATVIEW SE+ (Abacus, Berkeley, CA). Incidences of fetal malformations and frequency of micronuclei, based on affected litters, were analyzed using binomial statistics.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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:
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:
not examined
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Details on maternal toxic effects:
Maternal toxic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
5 000 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced mean fetal weight and reduced mean crown-rump length
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
not examined
Changes in sex ratio:
not examined
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
not specified
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
skeletal abnormalities (cleft palate, exencephaly)
Visceral malformations:
not examined
Key result
Dose descriptor:
LOAEL
Effect level:
5 000 mg/kg bw/day (nominal)
Basis for effect level:
other: teratogenicity
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
external: cranium
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
50 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
not specified

A. Folate levels (measured on gd 18, 8 d after the final methanol treatment):

Low-folate diet produced a decline in folate in the maternal liver (total folate approx. -30%), in maternal plasma (approx. -30%), in maternal erythrocytes (approx. -30%) vs. normal-folate diet and in the fetal liver (approx. -60 to -70%) (Fu et al., 1996).

Methanol had no marked influence on maternal and folate levels irrespective of folate supplementation, except in maternal plasma where there was some evidence of a reduction of about 20 % . Methanol treatment was slighty fetotoxic (reduced mean fetal weight and reduced mean crown-rump length), but had no impact other reproductive parameters. It showed some evidence of a teratogenic effect (increased incidences of cleft palate and exencephaly) under folate-adequate supply, but this was hardly statistically significant: cleft palate (2/222 vs. 0/282) and execephaly (5/222 vs. 1/282 in the respective high-folate control).

Likewise, folate deficiency failed to produce significant malformations (in accordance with previous reports: e.g. Heid et al., 1992): cleft palate (5/215 vs. 0/282) and execephaly (2/215 vs. 1/282 in the respective high-folate control). However, cleft palate, but not exencephaly was significantly increased in the presence of methanol: 39/235 vs. 5/215 and 8/235 vs. 2/215 of folate-poor control, respectively. Methanol did not induce micronuclei in maternal or fetal blood.

B. In pregnant CD-1 mice given methanol (5 g/kg/d) from gestation day 6 - 15, one group receiving folate deficient, the other folate supplemented diet, there were no differences in the formate-blood levels between both groups: 5.13 +-0.68 mmol/L (folate-def.) and 3.90 +-0.94 mmol/L (folate-suppl.) vs. 0.36 +-0.13 mmol/L (untreated control). But developmental toxicity was significantly higher in folate-deficient dams (Hong et al. 1997). The results indicate that increased plasma formate levels in dams do not underlie the increased developmental toxicity of methanol in mice fed low dietary folate (Hong et al. 1997).

CONCLUSION:

Folate deficiency enhanced the teratogenic effects of methanol in mice.

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:
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 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.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
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:
mortality
maternal abnormalities
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.

CONCLUSION:

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
1 700 mg/kg bw/day
Study duration:
subchronic
Species:
mouse
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
1 330 mg/m³
Study duration:
subacute
Species:
rat
Additional information

A. Animal data

Developmental toxicity has been observed in many rodent studies which resulted in a variety of effects in offspring due to prenatal and/or postnatal dosing.

In a developmental study, rats were exposed to 270, 1330 and 6650 mg/m³ methanol by whole-body inhalation from gestation days 7 through 17 for 23 hours/day (NEDO, 1987). In the top dose, maternal toxicity was recorded. In the progeny, there was fetal malformation, increased perinatal mortality and developmental delay. Teratogenic effects occurred only at the maternally toxic exposure concentration. Exposure levels of 1.33 mg/L 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 NOAEC for maternal and developmental toxicity is considered to be 1.33 mg/L.

In a second whole-body inhalation developmental study rats were exposed in chambers from gestation days 1 through 19 at 6650 and 13300 mg/m³ and from gestation days 7 through 15 towards 26.6 mg/L for 7 hours/day (Nelson et al., 1985). In the high dose group, significantly reduced food consumption without adverse effect on body weight gain was noted in maternal animals. No signs of maternal toxicity were observed in the lower dose groups. No influence on the number of corpora lutea and of implantations was reported. No effects on fetal lethality and resorption were found. There was no evidence of embryotoxic/teratogenic activity of methanol at 6.65 mg/L. At the highest concentration, an increased number of litters with skeletal and visceral malformations was noted. These included in particular rudimentary and extra cervical ribs and exencephaly and encephalocele, and, to minor extent, cardiovascular and urinary-tract defects. In this study a NOAEC for maternal and developmental toxicity of 6.65 mg/L was obtained.

In a developmental whole-body inhalation study, mice were exposed to methanol (1330, 2660, 6650, 9970, 13300, 19940 mg/m³) on gestation days 6 to 15 for 7 hours/day. Additionally, an orally exposed group was included for comparison (Rogers et al., 1991, 1993). There were no signs of maternal toxicity. Developmental effects occurred after inhalation of 2660 mg/m³ (Rogers et al., 1991, 1993). The dose related increase in cervical ribs or ossification sites lateral to the seventh cervical vertebra was significant at 2.66 mg/L. Significant increases in the incidence of exencephaly and cleft palate were observed at 6650 mg/m³. At the highest dose, almost complete resorption of embryos in most litters occurred. Reduced fetal weight was noted at 13300 mg/m³ and above. In this study, NOAECs for maternal and developmental toxicity were 19940 and 1330 mg/m³, respectively.

A study employing a single intraperitoneal injection of mice on gestation day 7 resulted in craniofacial malformations and malformations of the holoprosencephaly spectrum at 1700 mg/kg bw (LOAEL), no NOAEL could be identified (Fu et al., 1995).

In another study of Fu et al. (1996) pregnant female mice were orally administered 5000 mg/kg methanol on gestation days 6 to 10. The influence of folate in the diet on the incidence of developmental defects in the offspring was investigated. Methanol had no marked influence on maternal folate levels, irrespective of folate supplementation, except in maternal plasma where there was some evidence of a reduction of about 20 %. Methanol treatment was slighty fetotoxic (reduced mean fetal weight and reduced mean crown-rump length), but had no impact on other reproductive parameters. There was some evidence of a teratogenic effect (increased incidences of cleft palate and exencephaly) under folate-adequate supply, but this was hardly statistically significant: cleft palate (2/222 vs. 0/282) and exencephaly (5/222 vs. 1/282 in the respective high-folate control). Likewise, folate deficiency failed to produce significant malformations (in accordance with Heid et al., 1992): cleft palate (5/215 vs. 0/282) and exencephaly (2/215 vs. 1/282 in the respective high-folate control). However, cleft palate, but not exencephaly was significantly increased in the presence of methanol: 39/235 vs. 5/215 and 8/235 vs. 2/215 of folate-poor control, respectively.

In pregnant CD-1 mice given methanol (5000 mg/kg bw/day) from gestation days 6 to 15, one group receiving folate-deficient and the other folate-supplemented diet, there were no pronounced differences in the formate blood levels between both groups: 5.13 ± 0.68 mmol/L (folate-def.) and 3.90 ± 0.94 mmol/L (folate-suppl.) vs. 0.36 ± 0.13 mmol/L (untreated control). Developmental toxicity, however, was significantly higher in folate-deficient dams (Hong et al., 1997). On balance, the results indicate that developmental toxicity of methanol in mice on low dietary folate is not linked to increased formic acid levels (Hong et al., 1997), however, folate deficiency may enhance the teratogenic effects of methanol in mice.

All these rodent studies on the endpoint developmental toxicity, though of scientific interest, are of limited relevance for a classification to humans (see also attachment) since methanol follows a different kinetic pattern in humans (see also chapter 7.1) and causes severe acute toxicity at rather low doses. In contrast, rodents do not respond to methanol in the same sense. Rodents tolerate high methanol doses without signs of toxicity and high doses are also required in order to achieve developmental effects. The experimental species that appears to be much closer to humans in terms of kinetics and acute toxicity is the rabbit. In terms of developmental toxicity to rabbits, so far, only screening experiments are available which have confirmed that rabbits are apparrently much less sensitive than rats or mice for developmental effects: rabbits received on gestation day 7 or 8 a dose of 4000 mg/kg bw (in two portions; Sweeting et al., 2010; Sweeting and Wells, 2015; DeSesso et al., 2015) via i.p. injection and there was no clear evidence of a developmental effect.

The developmental effects of methanol in rats and mice occur at dose levels at which also other alcohols (e.g. Propanol) are prone to developmental effects in rats and mice. Methanol only has a specifically high toxicity in humans (which is quite exceptional, different from other alcohols and not displayed in rodents) and it is assumed that the classification of methanol as acutely toxic upon all exposure routes would practically preclude an uptake of such doses unless in cases of accidents or fraudulent misuse.

On balance, considering the apparent absence of developmental effects of methanol in rabbits and in monkeys (see C., below) and its pronounced acute toxicity in humans which practically precludes an uptake of doses known to be required for developmental effects in rodents, methanol has not to be classified for developmental toxicity in humans. Overall, the rodent data are not sufficient to presume similar effects in humans.

 

B. Human data

There are no relevant epidemiological studies or case reports which describe an increase in the incidence of malformations in children of mothers exposed to methanol during pregnancy.

The limited data available on methanol exposure on reproductive and developmental effects do not show an association (NTP, 2003).

In an epidemiological study, the reproductive effects of various occupations and associated exposures to complex mixtures were examined in women who gave birth to infants with and without cleft lip or cleft palate (Lorente et al., 2000). No association was found between methanol exposure and oral clefts. The small number of subjects exposed to methanol, the lack of individual exposure data, and confounding factors by other chemical exposures did not allow to draw firm conclusions as to the role of methanol on these outcomes.

NTP (2003) reviewed several studies that investigated the association between the periconceptional use of multivitamins containing folic acid and birth defects (e. g. neural tube defects and orafacial clefts). These studies suggest that folate deficiency in humans may lead to greater susceptibility to such effects. However, in all of these reviewed studies, the association between methanol and these effects was not directly investigated. Therefore, no conclusion can be drawn regarding causality between methanol and birth defects based on human data.

NTP (2003) stated that the rodent data on reproductive and developmental toxicity are of relevance for an assessment of the situation in humans even in the light of the known differences in methanol metabolism between rodents and humans. Rodents are adequate models for human exposure as long as formate levels do not accumulate. However, the blood methanol concentrations associated with serious teratogenic effects and reproductive toxicity occur in a dose range which is associated with formate accumulation in humans, metabolic acidosis and visual and clinical effects in humans (NTP, 2003) and far above the European OEL (260 mg/m³).

In humans, transient central nervous system effects generally appear at blood methanol levels higher than 200 mg/L, ocular symptoms appear at blood levels of > 500 mg/L and fatalities haven often occurred in untreated patients with initial blood methanol concentrations in the range of 1500 – 2000 mg/L (see also acute toxicity). Other effects (e.g. marginal, not yet definitive neurological effects observed in primates) may be exhibited at lower inhalation doses and lower methanol blood levels.

C. Overall assessment with special regard to a reproduction toxicity study in nonhuman primates (Burbacher et al., 1999)

C.1 The significance of the study by Burbacher et al. (1999)

In the study by Burbacher et al. (1999; published in a scientific journal in 2004), the health effects of whole body inhalation exposure to methanol for 2.5 hours/day on 7 days/week during prebreeding, breeding and pregnancy (approximately 120, 70, 165 days, respectively) on 11-12 female monkeys (Macaca fascicularis) per dose group and on their offspring (exposed in utero) at 0, 200, 600 and 1800 ppm (0, 0.27, 0.8, 2.39 mg/L) were investigated. The major strenght of this study and its significance for the assessment of the reproductive toxicity of methanol is based on the use of an animal model with high relevance to humans, the inclusion of toxicokinetics evaluations which confirmed established species differences (rodents vs. rabbits, monkeys and humans), the selection of exposure levels known to result in blood concentrations just below that causing nonlinear clearance kinetics in primates, and the conduct of repeated follow-up assessments (developmental/neurobehavioural) of the infants during their first 9 months of life.

C.2 Synopsis of the study by Burbacher et al. (1999)

C.2.1 Blood levels (Toxicokinetics, Maternal health effects)

 

Exposure to methanol vapours did not affect the health of the adult monkeys prior to or during pregnancy. Daily single 2.5-hour exposures to methanol vapours caused short term elevations in blood methanol concentrations of approx. 1- to 2-fold in the 200 ppm exposure group, 3- to 4-fold in the 600 ppm group and 13- to 16-fold in the 1800 ppm group. The more-than-proportionate increase observed in the 1800 ppm exposure group was accompanied by the appearance of nonlinear elimination kinetics at 1800 ppm, a finding that most likely reflects saturation of methanol metabolism (presumably by hepatic alcohol dehydrogenase). After long-term exposure, peak blood methanol concentrations declined slightly over the first month and remained constant thereafter. The concentration of plasma formate remained at baseline levels during the entire course of the study in all exposure groups. Pregnancy had no effect on methanol disposition. Serum folate concentrations were not affected by pregnancy and methanol exposure.

  

At 1800 ppm, after 5 hour elimination, the residual methanol level was near baseline (max. 2-fold higher). The mean estimated elimination half-lives (for 600 and 1800 ppm) ranged between about 60 to 90 minutes.

 

C.2.2 Toxicity to reproduction (Reproductive (fertility) effects, Developmental effects/Offspring assessments)

 

Methanol exposure had no effect on most measures of reproductive performance, including menstrual cycles, conception rate, and live-birth delivery rate. However, all methanol-exposed animals had a decrease of about 6 to 8 days in duration of pregnancy compared to control animals. It is not clear whether this decrease was related to methanol exposure, as there was no dose response and no difference among offspring groups in body weight, size or other physical parameters (head size, crown rump length). However, the duration of pregnancy was within the reported normal range for this species (NTP, 2003). Prenatal exposure to methanol had no effect on infant growth and physical development for the first 9 months. The results of behavioural assessments of offspring did not indicate methanol exposure effects on most domains of early behavioural development. No consistent effects due to methanol exposure were observed on early reflex responses, gross motor development, spatial and concept learning and memory, and social behaviour. Methanol exposure was associated with ratings of "low arousal" on the Neonatal Behavioural Scale. The effects were observed when all of the methanol-exposed infants were compared with controls. Further comparisons, however did not indicate that the effect was dose-dependent. In addition, many of the methanol-exposed infants that were the last to receive optimal scores for the arousal items had been delivered via C-section. Thus, this effect may not be directly related to methanol exposure independent of mode of delivery. Methanol exposure was also associated with a delay in early sensorimotor development of male infants only (Visually Directed Reaching Test). The effect was noted after controlling for the shortened gestation length observed for the 3 methanol-exposed groups. The delay was dose-dependent and ranged from 9 days for the 200 ppm exposure group to over 2 weeks for the 600 and 1800 ppm exposure groups. This observation is based on results from group sizes of 8 to 9 infant animals, with 2 to 5 males and 4 to 7 females per group. The statistical significances (linear contrast test based on ANOVA) of p = or < 0.04: This test indicated a significant difference between control infants compared with all methanol-exposed offsprings combined, as well as with the 600 and 1800 ppm groups for males (p < 0.04). The comparison between the control group and the 200 ppm group was nearly significant (p=0.09).

 The results of the Fagan-Test of Infant Intelligence indicated a possible effect of methanol exposure on visual recognition memory when testing complex stimuli (social problems). Although there were no mean group differences in the novelty scores across the 4 exposure groups, only the control group exhibited a significant novelty preference for social stimuli.

 

C.2.3 Other observations (Offspring assessments)

 

Prenatal methanol exposure was associated with the occurrence of a wasting syndrome in 2/7 female offsprings in the 1800 ppm exposure group after approximately 1 year of age, both living in the same cohort. Those females began to show growth retardation with 12 resp. 17 months of age. They became very weak and had to be euthanized at the age of 20 resp. 36 months. Assay results for viral infection, blood chemistry, complete blood count, and liver, kidney, thyroid, and pancreatic function were within the normal range. Necropsies showed signs of severe malnutrition and gastroenteritis. This symptom was not observed in any of the offsprings of the other cohort.

 

C.2.4 No observed adverse effect concentrations (NOAEC)

 

Based on the study results, the NOAEC for maternal toxicity/reproductive performance and the NOAEC for fetotoxicity/teratogenicity were considered both to be 1800 ppm (2.39 mg/L). Further data would be required in order to derive a NOAEC for offspring developmental effects (neurobehavioural development). No NOAEC was derived by the study authors.

C.3 Effective assessments of the results of the study by Burbacher et al. (1999)

Considering the assessments below, there is general agreement that no clear robust methanol-related effects on reproduction and/or development were identified up to and including the highest exposure concentration of 1800 ppm in exposed female monkeys and their offspring (exposed in utero).

HEI Statement, in Burbacher et al. (1999)), peer-reviewed conclusion: "The investigators' findings suggest that repeated inhalation exposure to methanol vapours as high as 1800 ppm would not result in accumulation of blood formate above baseline levels. With the exception of an unexpected shortening of gestation, methanol exposure had no effect on reproductive performance. The most significant result to emerge from this study was the wasting observed in two monkeys exposed in utero to 1800 ppm methanol. Although this observation raises concern for prenatal exposures of this magnitude, pregnant women are unlikely to be exposed to such extremely high concentrations of methanol for prolonged periods of time. Overall, the results provide no evidence of a robust effect of prenatal methanol exposure on the neurobehavioural development of nonhuman primate infants during the first nine months of life (...)".

With regard to the study by Burbacher et al. (1999), shorter gestation lengths, higher incidences of Caesarean (C) sections and neurobehavioural findings noted in methanol-exposed monkeys as compared with controls are not considered to represent toxicologically relevant methanol-related effects for the following reasons: Although there was a statistically significant shorter gestation length in methanol-treated groups as compared to the control group (not dose-dependent), gestation lengths for the treatment groups were consistently within the normal range for this species (Chellman et al., 1999). While five C-sections were performed in the treatment groups (2 females at 200 ppm, 2 females at 600 ppm, 1 female at 1800 ppm), there were no C-sections in the control animals. These surgical procedures were performed in response to signs of possible difficulty in maintenance of the pregnancy (e.g. vaginal bleeding) and thus suggest late reproductive dysfunction in the exposed females. However, in the NTP-CERHR Expert Panel review (NTP, 2004), it was noted that there does not appear to be sufficient evidence to support the claim of increased pregnancy complications following methanol exposure, and that the C-sections may not have been necessary. As vaginal bleeding sometimes occurs in macaques 1 to 4 days prior to delivery of a healthy infant, an ultrasound examination should have been performed prior to C-section conduct in order to confirm fetal or placental problems. The reported neurobehavioural findings are not considered as robust (e.g. NTP, 2004). Overall, most neurobehavioural parameters assessed were not affected by the treatment with methanol and there was no strong evidence of a dose-response relationship. Furthermore, there was a lack of proper controls for multiple comparisons in the statistical analyses; pair-wise tests were conducted, even when the overall ANOVA was not significant.

RAC (2014): "An overall assessment of the monkey studies indicated that methanol may have affected the infants, but that the data were not very robust and clearly not sufficient for classification. Furthermore, there were minimal similarities between the very clear effects noted in rodents and those possibly observed in the monkeys. It is acknowledged that the monkey exposure levels (1800 ppm) and exposure time per day (2.5 hours in monkey vs 7 hours in mice), were lower than the LOAEC of 2000 ppm in mice, and the blood methanol concentration was 35 mg/L at the top dose in monkeys when compared to 537 mg/L in mice at the LOAEC. Therefore, developmental toxicity also in monkeys at higher exposure levels cannot be ruled out. The RAC concludes that there is robust evidence of developmental toxicity of methanol in rodents, but very limited indications of developmental toxicity from non-rodent species which have metabolic pathways more similar to humans."

Referring to the assessment by RAC (2014), it has to be recognized that the effects noted in mice at the LOAEC of 2000 ppm (corresponding to a plasma methanol concentration of 537 mg/L; Rogers et al., 1993) were confined to increases in cervical ribs or ossification sites lateral to the seventh cervical vertebra, i.e. to findings (variations, retardations) raising a low level of concern. Only at 5000 ppm (plasma methanol concentration of 1650 mg/L) and above, i.e. at concentrations clearly lethal to humans, an increase in malformations such as exencephaly and cleft palate was noted. Similarly, relevant developmental (teratogenic) effects in rats were only evident at very high exposure levels. In addition, comparing exposure durations, in the study by Burbacher et al. (1999) the actual exposure duration for the monkeys was mostly considerably longer than 2.5 hours, particularly at the high dose, as indicated by toxicokinetics data (continuing exposure longer per day is not appropriate in monkey as it can elicit stress). Comparisons in exposure durations however have to account for interspecies differences in organogenesis, critical periods of development and overall length of gestation. When such considerations are made, then the monkey study can be regarded as comparable to (and far more comprehensive than) the mouse study in terms of exposures relevant for developmental toxicity (teratogenicity) evaluations. Specifically in the monkey study by Burbacher et al. (1999), exposure started prior to conception and continued through all three trimesters of pregnancy.

C.4 The relevance of species differences for the assessment of developmental toxicity

Based on the available evidence, humans are not susceptible to the methanol-related developmental toxicity observed in rats and mice, due to the substantial species differences in metabolism. Different enzymes involved in methanol metabolism provide the basis

for the kinetic differences between rodents and humans, which result in the formation of different metabolites at different metabolic rates. Moreover, it is clear from available animal data that, based on differences in metabolism and the formation/accumulation of formic acid in humans, which leads to maternal toxicity at much lower concentrations, the developmental effects observed in rats and mice in the absence of maternal toxicity are not relevant to humans. Methanol is presented in the ECHA Guidance on CLP (2015) as an example for not using rodent toxicity data to classify methanol for acute toxicity and specific organ toxicity on the basis of the non-relevance of rodent toxicity data to humans. This is due to species differences between humans and rodents, rendering the rodent data on methanol irrelevant to humans. The same approach should be applied for developmental toxicity assessment. Methanol has a uniquely high acute toxicity for humans with target organ toxicity for the ophthalmic nerve, which is different to toxicity seen in rodents. There is a large database on the toxicity of methanol and more recent data further support the previous EU decision not to classify methanol for developmental toxicity.

C.5 Comparison of the weight of evidence to the CLP requirements

For a detailed assessment, please refer to the attached document, which provides an argumentation why classification of methanol for developmental toxicity is not required according to the CLP criteria. Of note, the RAC (2014) presented recently an evaluation on the same issue and concluded that, based on the available information, there is not sufficient evidence to classify methanol for developmental toxicity in humans.

C.6 Conclusion

The weight of evidence does not indicate that a classification of methanol for developmental toxicity is required. Even though developmental effects at very high concentrations, exceeding those tested in the Burbacher study (1999), i.e. >1800 ppm, cannot be fully excluded for humans, blood concentrations similar to those observed in rodents at the LOAEC for developmental toxicity would result in severe acute toxicity or lethality in humans. This would require methanol vapour concentrations exceeding the Indicative Occupational Exposure Limit Value (IOELV) of 200 ppm by at least an order of magnitude. Please refer also to the attached document(s).

Toxicity to reproduction: other studies

Additional information

see endpoint summary and attached document.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Conclusive in rodents, but not employed for classification to humans (see endpoint summary and attached documents). Based on major species differences between humans and rodents (metabolic pathway/enzymes, mode of action, toxicokinetics), considering the overall weight of evidence, and in line with the evaluation of reproductive toxicity provided by the Committee for Risk Assessment (RAC, 2014), methanol does not appear to be toxic to reproduction. As a result the substance is not considered to be classified for toxicity to reproduction under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.