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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
other: Data sharing dispute
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with official test guideline; GLP; read-across applies as test substance was "3-Methylbutan-1-ol" and not isopentyl acetate.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1995
Report date:
1990

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methylbutan-1-ol
EC Number:
204-633-5
EC Name:
3-methylbutan-1-ol
Cas Number:
123-51-3
Molecular formula:
C5H12O
IUPAC Name:
3-methylbutan-1-ol
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): "3-methyl-1-butanol"
- CAS Nr.: 123-51-3
- Test substance No. 88/56 [H 21670]
- Physical state: liquid/colourless
- Analytical purity: 98.6% (acc. to report Feb 2, 1988)
- Stability under test conditions: The stability during the study period was confirmed by reanalysis (see report of Nov 25, 1988)
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Dr. K. Thomae GmbH, D-7950 Biberach/Riss, FRG). The animals were free from any clinically evident signs prior to the beginning of the study.
- Age at study initiation: ca 11 weeks
- Weight at study initiation: ca 216 g
- Housing: singly in wire cages (type D III of Becker & Co, Castrop-Rauxel, FRG)
- Diet (e.g. ad libitum): KLIBA rat/mouse laboratory diet 24-343-4 10 mm pellets, Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland; during the exposure-free observation period
- Water (e.g. ad libitum): tap water; during the exposure-free observation period
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS in fully air-conditioned rooms
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

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: the animals were exposed singly in wire cages (D III) in glass-steel inhalation chambers (manufacturer: BASF Aktiengesellschaft), Volume Vz 1,100 1 (test group 1, 2 and 3), Volume V 2 1,600 1 (test group 0 and parts of the test groups during the preflow period).

- Method of holding animals in test chamber: whole-body exposure system (glass-steel inhalation
chamber) with a volume of about 1.1 m3 (test groups 1 - 3); volume of the inhalation chamber of the control group: 1.6 m3
- Source and rate of air: the test substance was supplied by means of two continuously driven piston pumps (Unita, Braun) in test group 1, a continuously metering pump (Optimat MP) in test group 2, and another continuously metering pump (Desaga) in test group 3 to a vaporizer heated with a circulating thermostat and evaporated. The evaporation temperatures are shown in the following table.

Test group ml/h evaporation temperature [°C] supply air [l/h] exhaust air [l/h]
0 fresh air 30000 29500
1 13.7-14.3 50 21500 22000
2 75.6-82.8 60 21500 22000
3 295-305 70 21500 22000

A stream of fresh air measured with a rotameter took up the vapors. A further stream of fresh air was passed in downstream of the vaporizer. After passing through a mixing device, this mixture of vapors and air was supplied to exposure system

- Temperature, humidity, pressure in air chamber: the pressure in the inhalation chambers was measured continuously (inclined manometer) and recorded, as a rule, 3 times/exposure. Conditioned supply air ( about 50% humidity, 22 °C) was used for the exposure in all test groups. The temperature in the
exposure systems was measured continuously (digital thermometer, Diehl) and recorded, as a rule, 3 times/exposure. The relative humidity in the chambers was checked with a humidity measuring probe (Vaisala) at least once a day and also recorded.
- Air flow rate: all air flows, supply air and exhaust air were adjusted by means of flowmeters (rota meter) for all test groups and recorded, as a rule, 6 times/exposure.

TEST ATMOSPHERE
- Brief description of analytical method used: the concentration in the inhalation chambers was monitored by means of a GC-method. The concentrations of the test groups were analyzed by gas chromatography after absorption of MEB samples in 2-propanol. The gas chromatographs were calibrated with weighed amounts of the test substance.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration in the inhalation chambers was monitored by means of a GC-method. The concentrations of the test groups were analyzed by gas chromatography after absorption of MEB samples in 2-propanol.
The gas chromatographs were calibrated with weighed amounts of the test substance.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/4
- Length of cohabitation: 15.5 hours (overnight)
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
days 6 - 15 of gestation
Frequency of treatment:
6 h/d
Duration of test:
20 d
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/L air (nominal)
Dose / conc.:
0.5 mg/L air (nominal)
Dose / conc.:
2.5 mg/L air (nominal)
Dose / conc.:
10 mg/L air (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent no treatment
Details on study design:
Dose selection rationale
In a pretest no maternal toxic effects could be observed at concentrations up to 5 mg/l, which is the limit test concentration. Because fetotoxic effects were reported with other alcohols at somewhat higher concentrations, the highest concentration selected for the study was 10 mg/l, which is near to the saturated vapor concentration at approx. 20°C (12 - 14 mg/l). In order to determine dose-response relationships, an intermediate (2.5 mg/ml) and a low (0.5 mg/ml) concentration levels were also selected.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: the behavior and state of health of the test animals were checked on workdays at least 3 times on exposure days and, as a rule, once during the post-exposure observation period.

BODY WEIGHT: Yes
- Time schedule for examinations: the body weight of the animals was checked on day 0 day of dectection of sperm) and on days 3, 6, 9, 12, 15, 18 and 20 p.c. As a rule, the animals were weighed at the same time of the day. The difference between the body weight on the day of weighing and the
body weight on the previous weighing was calculated. Moreover the same was done for 3 different study periods:
* preflow period (day 0- 6 p.c.)
* exposure period (days 6 - 15 p.c.)
* observation period (days 15 - 20 p.c.).

These values are defined as body weight change. Moreover, the corrected body weight gain (body weight on day 20 p.c. minus the body weight on day 6 p.c. minus weight of the uterus before it was opened) was determined after the dams had been sacrificed at the end of the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: on day 20 p.c. the dams (as well as moribund dams) were sacrificed by cercical dislocation and the fetuses removed by cesarean section. These animals and dams which died intercurrently as well as theof uterus before it was opened, number of corpora lutea, number and distribution of implantation sites classified as live fetuses or dead implantations: early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy); late resorptions (embryonic or fetal tissue in addition to placental tissue visible); dead fetuses (hypoxemic fetuses which did not breath spontaneously after the uterus had been opened).
- Furthermore, calculations of conception rate and pre and postimplantation losses were carried out.

OTHER: a check for dead animals was made daily.
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: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
The statistical evaluation of the data was carried out on the computer systems of the Department of Toxicology (responsible person: Dr. Hoffmann). Examinations of the dams and fetuses Dunnett's Test was used for statistical evaluation of body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, weight of fetuses, weight of placentae, corpora lutea, implantations, pre and postimplantation loss, resorptions and live fetuses. Fisher's Exact Test was used for statistical evaluation of conception rate, mortality (of the dams) and all fetal findings. Significances resulting from these tests were indicated as p < 0.05 or p < 0.01.
Indices:
- The conception rate was calculated according to the following formula: (number of pregnant animals/ number of fertilized animals)*100
- The preimplantation loss (in %) was calculated according to the following formula: (number of corpora lutea - number of implantations/number of corpora lutea)*100
- The postimplantation loss (in %) was calculated from the following formula: (number of implantations - number of live fetuses/number of implantations)*100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no substance-induced clinical signs or findings in all test groups (0 - 3) at any time of the study period (preflow, exposure, post-exposure observation).
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No deaths were recorded throughout the study period in test groups 0, 2 and 3. One animal of test group 1, which was not pregnant, was found dead in cage on day 12 p.c.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Only pregnant dams were used for the calculations of mean maternal body weights and body weight change. Only pregnant dams with scheduled sacrifice (day 20 p.c.) were taken for the calculation of mean gravid uterine weights, mean net maternal body weight change (corrected body weight gain) and summary of reproduction data. In this study 3, 4 and 2 females (respectively in test group 1, 4 and 3) were excluded from the above mentioned calculations since they did not conceive (while one animal died in the test group 2).

The body weights of all animals in test group 1 compared to the control (0) were not statistically significant. In test group 2, the body weight change was statistically significantly increased (p < 0.05) between days 12 -15 p.c. In test group 3, compared to the control (group 0), the body weight change was statistically significantly decreased (p < 0.05) between days 6 -9 p.c. and statistically significantly increased (p < 0.01) between day 12-15 p.c. Over the total exposure period (days 6-15 p.c.) no substance-related effects were observed. It cannot be decided clearly, whether there was a slight effect on body weight retardation in the first days of exposure (6 - 9 p.c.) followed by an adaptation/recovery phase in the further days of exposure (12 - 15 p.c.) or whether this is an incidental finding. In case this effect may be considered as a very slight indication of maternal toxicity only during the first phase of exposure to a very high concentration of 10 mg/l of test substance.

The results of the corrected body weight gain (body weight on day 20 p.c. minus body weight on day 6 p.c. minus weight of the uterus before it was opened) do not show any differences of biological relevance between the groups.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Uterus weight: the uterus weights of the animals were not influenced by the exposure to the test substance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy findings: there were no substance-related observations at necropsy in any of the dams.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Pre- and post-implantation loss:
effects observed, non-treatment-related
Total litter losses by resorption:
effects observed, non-treatment-related
Early or late resorptions:
effects observed, non-treatment-related
Dead fetuses:
effects observed, non-treatment-related
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
effects observed, non-treatment-related
Other effects:
not examined
Details on maternal toxic effects:
Reproduction data of dams: the conception rate varied between 80 and 100%. There were no substance-related and/or statistically significant differences between the groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age (also compared to the lab's historical control data).

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEC
Effect level:
2.5 mg/L air (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights were not influenced by the test substance exposure (all values are within the range of biological variation (also comparable to the historical control values).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1- 3 (0.5, 2.5 and 10 mg/l) was comparable with the control fetuses (the differences observed in comparison being without any biological relevance).
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean placental weights in test groups 1-3 (0.5, 2.5 and 10 mg/l) were not influenced by the administration of the test substance to the dams (the differences observed being without any biological relevance).
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The external examination of the fetuses revealed one malformation (polydactyly) in one fetus out of 326 fetuses in the highest dose group (10 mg/l) and no variations in any group. So-called unclassified observations were recorded for 4 fetuses of the control group (blood coagulum around placenta), 9 fetuses (from one litter) in test group 2 (2.5 mg/l) (placentae necrobiotic) and 1 fetus of the highest dose group (10 mg/l) (placentae fused).
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Various malformations of the sternebrae (sternebra(e) bipartite, ossification centers dislocated, cleft sternum) and/or the vertebral column (e.g. thoracic vertebral body/bodies dumbbell-shaped (asym.) or bipartite (asym.)) were seen in very few fetuses (4 - 8) in all test groups, the differences not being statistically significant. The variations elicited were related to the ribs shortened or missing 13th, accessory 14th ribs or rudimentary cervical ribs) and the sternum (sternebra(e) of irregular shape, bipartite or accessory sternebra) and were found in all groups to about the same extent with the exception of a lower incidence of shortened 13thrib(s) in the highest dose group. In all groups signs of retardations (incomplete or missing ossification of hyoid, skull, metacarpal or metatarsal bones, vertebral bodies and/or sternebra(e)) were found without any clear differences of biological relevance between the groups.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The examination of the organs of the fetuses revealed two malformations in test group 2 (2.5 mg/l). For one fetus a globular shaped heart, for another one dextrocardia was recorded. Variations were detected in all groups. The very common finding (dilated renal pelvis) in the rat strain used in this study occurred without any dose-response relationship. The occurrence of the other variation (hydroureter) did not show any statistically significant difference between the groups. The examination of the organs of the fetuses revealed no so-called unclassified observations (like blood coagulum around the bladder) in any test group.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Reproduction data of dams: the conception rate varied between 80 and 100%. There were no substance-related and/or statistically significant differences between the groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age (also compared to the lab's historical control data).

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEC
Effect level:
10 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The fetuses did not show any substance-related effects (neither embryo-/fetotoxic nor teratogenic effects).

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Upon inhalation exposure of 3-methyl-1-butanol to pregnant rats, no teratogenic effects were found up to the highest test concentration. The NOAEC (teratogenicity) is 10 mg/L air (nominal) (= highest concentration tested). The NOEC for maternal toxicit) is 2.5 mg/L air (nominal) (this was the second highest concentration tested; at the highest concentration of 10 mg/L air a transient body weight change was observed at the beginning of the exposure).
Executive summary:

Upon inhalation exposure of 3-methyl-1-butanol to pregnant rats, no teratogenic effects were found up to the highest test concentration. The NOAEC (teratogenicity) is 10 mg/L air (nominal) (= highest concentration tested). The NOEC for maternal toxicity is 2.5 mg/L air (nominal) (this was the second highest concentration tested; at the highest concentration of 10 mg/L air a transient body weight change was observed at the beginning of the exposure).

Based on the available and adequate data on the metabolism of isopentyl acetate in the body (see under 7.1 Toxicokinetics, metabolism and distribution) it can be assumed that the inhalative expo

ure towards a certain concentration of isopentyl acetate would finally lead to an equal molar exposure towards 3-methyl-1-butanol and acetic acid in the body. Acetic acid is not regarded as toxicologically relelevant compared to 3-methyl-1-butanol. Developmental toxicity effects of isopentyl acetate should therefore be found to be the same or similar to the effects of 3-methyl-1-butanol. This study on 3-methyl-1-butanol is therefore regarded as meaningful for the description of developmental toxicity effects of isopentyl acetate. All available and adequate data on developmental toxicity was generated by the use of 3-methyl-1-butanol as test substance. These data are regarded together in a weight of evidence approach (see purpose flags under "7.8.2.") to evaluate possible developmental toxicity effects of isopentyl acetate.