Isobutyric anhydride

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: comparable to guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1995

Report date:

1995

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-methylpropan-1-ol

EC Number:

201-148-0

EC Name:

2-methylpropan-1-ol

Cas Number:

78-83-1

IUPAC Name:

2-methylpropan-1-ol

Details on test material:

- Name of test material (as cited in study report): 2-methyl-1-propanol, MEP
- Analytical purity: 99.8%
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: no data
- Stability under test conditions: yes
- Storage condition of test material: no data

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Thomae GmbH, Biberach/Riss, Germany
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: mean body weight approx. 216 g
- Fasting period before study: no data
- Housing: individually in wire-mesh cages, type DK III (EBECO, Becker and Co., Castrop Rauxel, Germany), air conditioned rooms
- Diet (e.g. ad libitum): KLIBA rat/mouse laboratory diet 24-343-4, 10 mm pellets (Klingentalmühle AG, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- 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: horizontal-flow whole-body exposure chamber (glas/steel construction, volume 1.1.m³ (BASF AG, Ludwigshafen, Germany)
- Method of holding animals in test chamber: individually in wire cages
- Source and rate of air: clean air
- Method of conditioning air: no data
- System of generating particulates/aerosols: evaporator maintained at 50-70°C, TS was delivered by a continuously operating pump
- Temperature, humidity, pressure in air chamber: 21-24°C, 49-64%, minimal negative pressure
- Air flow rate: 275 L/Minute
- Air change rate: 15 air changes per hour
- Method of particle size determination: no particle size determination
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography (Hewlett-Packard GC, Model 5840 A with FID detector)
- Samples taken from breathing zone: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the inhalation atmosphere were analyzed hourly during exposure.
Mean concentrations ± SD measured in inhalation chamber: 0.49 ± 0.012 mg/L, 2.50 ± 0.084 mg/L, 10.10 ± 0.330 mg/L

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/4, no further data on mating
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy or postcoitum (pc) respectively

Duration of treatment / exposure:

day 6- 15 day of pregnancy

Frequency of treatment:

6 hours / day

Duration of test:

20 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0.5; 2.5; 10 mg/L
Basis:
nominal conc.

No. of animals per sex per dose:

25 females per group

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: preliminary range-finding study
- Rationale for animal assignment (if not random): random

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 3, 6, an from then on at 3-day intervals until day 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus and ovaries

OTHER: Yes
- gross pathology for all animals

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter; fixation in Bouin's solution according to the method of Barrow and Taylor (1969)
- Skeletal examinations: Yes: half per litter; fixation in ethyl acohol and staining according to a modified method of Dawson (1926)
- Head examinations: No data

Statistics:

The Dunnett test (Dunnett, 1955, 1964) was used to statistically compare body weight, body weight changes, the number of corpora lutea, implants, resorptions, live fetuses, and pre- or postimplantation losses. The Fisher's exact test (Dixon, 1981) was used for evaluating the conception rate, maternal mortality, and all fetal findings.

Historical control data:

Historical control data were used to further evaluate findings of low significance.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No differences in body weight/body weight gain were observed between controls and treated groups.
In addition no clinical signs of toxicity were seen.
Gross-pathologiy examination revealed no effects that could be attributed to the exposure with isobutanol.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

10 mg/L air

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The fetuses did not showed any substance-related effects (no indications of any embryo-/fetotoxicity or any teratogenic effect).

The uterine weights of the treated animals were not significantly different from the controls. Compound related effects occured neither for conception rate, mean number of corpora lutea, and implantation sites nor in the values calculated for the pre- and postimplantation loss and the number of resorptions as well as viable fetuses.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

10 mg/L air

Basis for effect level:

other: embryotoxicity

2

Dose descriptor:

NOAEL

Effect level:

10 mg/L air

Basis for effect level:

other: fetotoxicity

3

Dose descriptor:

NOAEL

Effect level:

10 mg/L air

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

In no test group (0.5 mg/l - 10 mg/l) substance-related effects neither on the dams nor on the fetuses were obtained. There were no indications of substance-related maternal toxicity and embryo-/fetotoxicity or any teratogenic effect.
    

Table 1     Data on Reproduction, Maternal Body Weight Change and Uterine Weight

	Dose [mg/L]
	0 (sham)	0.5	2.5	10.0
Number of animals	25	25	25	25
Number of dams (pregnant animals)	21	23	23	19
Corpora lutea/dam	15.2	15.5	15.3	14.5
Implants/dam	13.6	14.6	13.1	13.8
Live fetuses/dam	12.3 (2.89)	13.7 (1.99)	12.3 (3.35)	13.2 (1.86)
Dead implants/dam	1.2	1.0	0.8	0.6
Sex ratio (male / female	53.3 / 46.7	48.7 / 51.3	53.0 / 47.0	50.8 / 49.2
Mean fetal weight [g]	3.9 (0.29)	3.9 (0.28)	3.9 (0.21)	3.9 (0.15)
Mean placental weight [g]	0.46 (0.07)	0.43 (0.06)	0.45 (0.06)	0.46 (0.05)
Mean corrected maternal body weight change [g] *	47.7 (9.21)	50.3 (6.86)	50.4 (8.25)	50.9 (6.96)
Intact uterine weight [g]	71 (15.28)	78.1 (10.12)	70.0 (17.36)	75.6 (11.59)

Numbers in parentheses indicate standard deviations

* corrected maternal body weight change = (body weight on day 20 of pregnancy) - (body weight on day 6 of pregnancy) - (uterine weight)

Tabelle 2 Fetal Anomalies

	Dose [mg/L]
	0 (sham)	0.5	2.5	10
Number of litters evaluated	21	23	23	19
Number of fetuses evaluated	124	164	149	128
Total fetal external malformations
Fetal incidence	0	0	0	2 (0.8)
Litter incidence	0	0	0	2 (10.5)
Total fetal soft tissue malformation
Fetal incidence	0	0	0	1 (0.8)
Litter incidence	0	0	0	1 (5.3)
Total fetal soft tissue variations
Fetal incidence	60 (48.0)	47** (31.2)	47* (35.1)	49 (40.2)
Litter incidence	19 (90.5)	18 (78.3)	20 (87.0)	17 (89.5)
Total fetal skeletal malformations
Fetal incidence	5 (3.7)	9 (5.5)	2 (1.3)	1 (0.8)
Litter incidence	5 (23.8)	7 (30.4)	2 (8.7)	1 (5.3)
Total fetal skeletal variations
Fetal incidence	59 (44.0)	75 (45.7)	72 (48.3)	51 (39.8)
Litter incidence	19 (90.5)	22 (95.7)	22 (95.7)	18 (94.7)

Numbers in parentheses indicate percentage of fetuses/litters affected

* significantly different from control; p < 0.05

** significantly different from control; p < 0.01

The external changes showed two types of malformations. Anasarca was observed in two fetuses. In addition, one showed a cleft palate.

The only soft tissue malformation was dilatation of both ventricles (globular shaped heart).

Variations were seen in all groups including controls. They occurred independent of exposure or concentration.

The skeletal examination revealed various malformations of the sternebrae and/or the vertrebral column. Variations seen in the ribs and the sternum were found in all groups.

Any observed differences between groups were within the range of biological variation and/or occurred without a clear concentration dependency.

Applicant's summary and conclusion

Conclusions:

No signs of maternal and developmental toxicity or teratogenicity were noted in this inhalation study, where the maximum exposure concentration was 10 mg/L.

Executive summary:

The developmental toxicity of isobutanol (purity 99.8%) was tested in an inhalation study in pregnant wistar rats. 25 female animals were exposed to vapors of isobutanol at concentrations of 0, 0.5, 2.5 and 10 mg/L for 6 hours/day from day 6 through 15 of gestation. All animals were killed on day 20 of gestation. The fetuses were removed and examined for compound-related effects.

There were no treatment-related effects on maternal toxicity (no mortality, no significant differences between controls and treated groups in clinical signs, body weight development, and gross pathology) even at the highest dose. The maternal NOAEL is 10 mg/L . 

The fetuses did not exhibit any signs of treatment-related embryo-/fetotoxicity or teratogenic effects. The developmental NOAEL is 10 mg/L.

(Klimisch 1990).

This inhalation developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirements for a developmental toxicity study (OECD 414).