Methyl acetate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

publication

Title:

Toxicological Research of Methanol as a Fuel for Power Station, Summary Report on Tests with Monkeys, Rats and Mice

Author:

NEDO

Year:

1987

Bibliographic source:

New Energy Development Organization, Tokyo, 1987

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

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

Principles of method if other than guideline:

According to national standards. Comprehensive study programme including metabolic, pharmacokinetic, short-, long-term, reproduction and carcinogenicity studies.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methanol

EC Number:

200-659-6

EC Name:

Methanol

Cas Number:

67-56-1

Molecular formula:

CH4O

IUPAC Name:

methanol

Details on test material:

TS-Freetext:
Methanol, reagent special grade from Junsei Chemicals Co.,
<1 ppm vinyl chloride, <3 ppm formaldehyde
------------------

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Inc.
- Age at study initiation: 12 weeks
- Diet: Solid Chow for rat (CRF-1, Charles River Japn Inc.)
- Water: sterilised and filtrated water (ad libitum)
- Acclimation period: 2 weeks

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

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 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:

gestation day 7 - 17

Frequency of treatment:

Animals were exposed to methanol gas continuously except during routine procedures such as observations and examinations.
Care was taken to ensure equal exposure time for all groups including the control.

Duration of test:

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

270 mg/m³ air

Doses / concentrations 2

Dose / conc.:

1 330 mg/m³ air

Doses / concentrations 3

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

Examinations

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

Statistics:

All the data obtained were analyzed by t-test, U-test of Mann-Whitney, Fisher's exact test or Armitage's chi2 - test.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Mortality:

mortality observed, treatment-related

Description (incidence):

In the high-dose group, 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

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

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

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

Dead fetuses:

no effects observed

Changes in pregnancy duration:

effects observed, treatment-related

Description (incidence and severity):

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

Changes in number of pregnant:

not examined

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEC

Effect level:

1.33 mg/L air

Based on:

test mat.

Basis for effect level:

early or late resorptions

2

Key result

Dose descriptor:

LOAEC

Effect level:

6.65 mg/L air

Based on:

test mat.

Basis for effect level:

body weight and weight gain

early or late resorptions

food consumption and compound intake

mortality

water consumption and compound intake

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

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.
Also after birth, body weight gain values of high-dose group (in both males and females) tended to be lower than control values.
Particularly in females, significant differences were noted throughout the period after weaning and obvious depression of body weight gain was present. In males, however, it was of smaller extent significant differences were noted up to Week 5 of birth (after weaning) but not thereafter.
These F1 animals were all small in size and showed decrease in milk consumption and depressed vitality.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

in the high-dose group (5,000 ppm), cases of death occurred in many litters during Days 0 - 4 of birth, which resulted in significantly lower survival rate of pups for this interval.

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

In the high-dose group (5,000 ppm), cases of asthenia and death occurred in many litters during Days 0 - 4 of birth, which resulted in significantly lower survival rate of pups for this interval. ln addition, all cases of death occurred within the day of birth in the other groups, while it continued to occur on and after Day 1 of birth in this group and there were cases, though very limited in number, showing a depression of vitality even on Day 4 of birth.
On and after Day 4 of birth (the day of litter size adjustment), however, there was no more case of death even in the high-dose (5,000 ppm) group, and no case which had abnormal appearance, either.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

One fetus each of the mid-dose (1,000 ppm) and high-dose group (5,000 ppm) had systemic edema. There was no other fetus having any external malformation.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

The high-dose group (5,000 ppm) showed a delay for almost all ossification parameters. Delay with significant difference was noted for body of cervical vertebrae, upper incisor, metacarpals and proximal phalanx of front limbs: "atresia of cervical vertebrae foramen costotransversarium" (45 %), " bifurcated vertebral centra" (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):

In the high-dose group (S,000 ppm), about one-half of the fetuses (visceral malformation in 16/20 litters or 64/131 fetuses) showed ventricular septal defect. This malformation was observed in 80% (16/20) of litters. Except for 5 fetuses (3 litters) having exeessive left subclavian artery, no other significant difference was noted.
As for variation, fetuses having residual thymus increased (variation in all 20 litter or 70/131 fetuses) and fetuses having serpenginous urinary duct decreased. The differences were significant for both the variations.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Post-natal morphological differentiation:
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. Reflex reaction, emotional (open field test), learning ability (pole-climbing test) and movement coordination (rotor rod test) did not show any treatment-related change.
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.

Effect levels (fetuses)

1

Key result

Dose descriptor:

NOAEC

Effect level:

1.33 mg/L air

Based on:

test mat.

Sex:

not specified

Basis for effect level:

changes in postnatal survival

skeletal malformations

visceral malformations

Remarks on result:

other: Teratogenic effects occurred only at maternally toxic exposure concentration.

2

Dose descriptor:

LOAEC

Effect level:

6.65 mg/L air

Based on:

test mat.

Sex:

not specified

Basis for effect level:

changes in postnatal survival

skeletal malformations

visceral malformations

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

other: visceral and skeletal malformations, postnatal growth and survival

Description (incidence and severity):

Teratogenic effects occurred only at maternally toxic exposure concentration

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

6.65 mg/L air

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Applicant's summary and conclusion

Conclusions:

Pregnant rats were exposed to 0, 200, 1000 and 5000 ppm of methanol by inhalation during fetal organogenesis (days 7 - 17 of gestation). The exposure to 5000 ppm 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.