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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
other: EC Commission Directive 87/302/EEC of Nov . 18, 1987 ; Part B : Methods for the determination of toxicity : Teratogenicity study (rodent and non-rodent)
Qualifier:
according to guideline
Guideline:
other: U .S . EPA, Pesticide Assessment Guidelines, Subdivision F, § 83-3 : Teratogenicity Study ; NTIS, Revised edition, pp . 126 - 130 (Nov . 1984 )
Qualifier:
according to guideline
Guideline:
other: Japan/MAFF : Testing Guidelines for Toxicology Studies : Teratogenicity Study, pp . 48 - 49 (1995)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Isobutyraldehyde
EC Number:
201-149-6
EC Name:
Isobutyraldehyde
Cas Number:
78-84-2
Molecular formula:
C4H8O
IUPAC Name:
2-methylpropanal
Details on test material:
- Name of test material (as cited in study report): Isobutyraldehyde
- Purity prior to the study: 99.4%
- Purity after the in-life phase of the study (Reanalysis): 98.8%
- Physical state: Liquid
- Batch No: from continuous production out of production column 510 from M 830
- Storage conditions: in closed containers, blanketed with nitrogen, 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, Biberach an der Riss,FRG,
- Age at study initiation: 75 - 84 days
- Weight at study initiation: approx . 231 g (mean)
- Housing: Singly in wire mesh cages type DK III of Becker & Co., Castrop-Rauxel, FRG (floor area about 800 cm2)). Underneath the cages, waste trays were fixed containing bedding material (typ 3/4 dust free embedding, supplied by SSNIFF, Soest, FRG) .
- Diet: Standard diet ad libitum (KLIBA rat/mouse/hamster laboratory diet 24-343-4, 10 mm pellets, Klingentalmühle AG, Kaiseraugst, Switzerland), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 8-11 days

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

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
Animals were housed singly in wire cages located within a glass and steel 1.4 cubic meter inhalation chamber and were exposed to air for 5 days before the exposure period. Food and water was not available during exposure.
Generator system:
- piston metering pump (DESAGA)
- glass vaporizer with glass mixing stage, two component atomizer and thermostat
- two-component atomizer
- thermostat
Generation procedure: The test substance was supplied to the two-component atomizer at a constant rate by means of the metering pump. The generation was carried out using thermostated glass evaporators with downstream mixing units by means of spraying the substance with a two-component atomizer and compressed air into a counter current of ventilation air. Conditioned supply air (about 50% relative humidity, 22°C) and compressed air were used for the exposure in all test groups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The nominal concentration was calculated from the study means of the amounts of substance dosed and the supply air flows.
The concentrations of the inhalation atmospheres were analyzed by gas chromatography. As a rule, daily means were calculated based on two measured samples. From these daily mean values mean concentrations and standard deviations for the entire study and for each replicate were derived. The constancy of concentrations in the inhalation chambers were monitored by means of non-calibrated total hydrocarbon analyzers.
Details on mating procedure:
Two untreated female rats were mated with one untreated male of the same breed . Mating took place from about 4 p.m. to about 7.30 a .m. on the following day. If sperm were detected microscopically in the vaginal smear in the morning, the animals were considered to be fertilized . This day was designated "day 0" (beginning of the study) and the following day "day 1" post coitum (p.c.).
Duration of treatment / exposure:
days 6 through 15 p.c.
Frequency of treatment:
daily, 6 hours per day
Duration of test:
until day 20 p.c.
Doses / concentrationsopen allclose all
Dose / conc.:
3 mg/L air
Dose / conc.:
7.6 mg/L air
Dose / conc.:
12 mg/L air
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on dose-range finding study (see seperate IUCLID entry)

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
A check for dead animals was made daily. The behavior and state of health of the test animals were checked at least 3 times on exposure days and, as a rule, once a day during the preflow period and the post-exposure observation period . During only a groupwise examination was possible .

BODY WEIGHT:
The body weight of the animals was checked on day 0 (= day of detection of sperm) and on days 3, 6, 9, 12, 15, 17 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 the 3 different study period: preflow period (days 0- 6 p.c.); exposure period (days 6- 15 p.c.); post-exposure observation period (days 15 - 20 p.c.). These values were defined as body weight change. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on day 20 p.c. minus weight of the unopened uterus minus body weight on day 6 p.c.).

POST-MORTEM EXAMINATIONS:
On day 20 p.c., the dams were sacrificed in randomized order by cervical dislocation and the fetuses dissected from the uterus. After the dams had been sacrificed, they were necropsied and assessed by gross pathology. The uterus and the ovaries were removed and the following data were recorded: Live fetuses, dead implantations: a) early resorptions (only decidual or placental tissues visible or according to Salewski (1964) from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy) b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible) c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened) Furthermore, calculations of conception rate and pre- and postimplantation losses were carried out.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Fetal examinations:
Fetuses were weighed, sexed, and examined macroscopically for external findings. Viability of each fetus, and the condition of the placenta, umbilical cord, and fetal membranes and fluids were also examined. Individual placental weights were also recorded. After fixation, approximately one half of the fetuses from all groups was subject to soft tissue examination; the skeletons of approximately half the fetuses were stained and examined microscopically for abnormalities.
Statistics:
DUNNETT's Test [Dunnett ; 1955, 1964] was used for a simultaneous comparison of several dose groups with the control. This test was performed two-sided and was used for the statistical evaluation of the following parameters: Body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the unopened uterus, number of corpora lutea, number of implantations, number of resorptions and number of live fetuses ; proportion of preimplantation loss, post-implantation loss, resorptions and live fetuses in each litter ; litter mean fetal body weight and litter mean placental weight .
FISHER's Exact Test [Siegel, 1956] was used for a pairwise comparison of each dose group with the control for the hypothesis of equal proportions. This test was performed one-sided and was used for female mortality, females pregnant at terminal sacrifice and the number of litters with fetal findings.
The WILCOXON-Test [Nijenhuis, 1978a ; Hettmansperger, 1984] was used for a comparison of each dose group. with the control for the hypothesis of equal medians. This test was performed one-sided and was used for the proportion of fetuses with malformations, variations, retardations and/or unclassified observations
in each litter.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No abnormal signs and findings were detected throughout the study.
Mortality:
no mortality observed
Description (incidence):
No deaths were recorded throughout the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight of the animals of test group 1, compared to the control group, was statistically significantly decreased on day 0 p.c. The body weight of the animals of test group 2, compared to the control group, was statistically significantly decreased on day 9 p.c. and 17 p.c. . The body weight of the animals of test group 3, compared to the control group, was statistically significantly decreased on day 0 p.c. and from day 9 p.c. until the end of the study (day 20 p.c.). The body weight change of the animals of test group 1, compared to the control group, was not statistically significantly different. The body weight changes of the animals of test groups 2 and 3, compared to the control group, were statistically significantly decreased from day 6 p.c. to day 9 p.c. and also if the entire exposure period (day 6 to day 15) was considered. This depression of body weight development in test groups 2 and 3 is considered to be substance related. The corrected body weight gain (terminal body weight on day 20 p.c. minus weight of the uterus before it was opened minus body weight on day 6 p.c.) was statistically significantly lower in the high and in the intermediate dose group (about 85% of the value of the concurrent control group). Furthermore the carcass weight (terminal body weight minus uterine weight) of the high dose group was statistically significantly decreased (95%), which is related to the test substance administration . All other differences between the groups are without any biological relevance.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The uterus weights of the animals were not influenced by the administration of the test substance. The differences between the groups are without biological relevance and do not show any dose-response relationship.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
At necropsy, single animals of the control and test groups 1 and 2(3 or 7.5 mg/L) showed lungs with edema and/or marginal emphysema; these findings, which showed no relation to dosing, have to be related to the sacrifice of the animals . Moreover, one control dam and one female of the 7.5 mg/L group had a hydrometra; both animals did not become pregnant. The occurrence of hydrometra in these two animals is considered to be spontaneous in nature.
Histopathological findings: neoplastic:
no effects observed

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:
no effects observed
Dead fetuses:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The conception rate varied between 92% (test group 2: 7 .5 mg/L) and 100% (test group 1- 3 mg/L and test group 3 - 12 mg/L).
There were no substance-related and/or biologically relevant 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 for historical control data.

Effect levels (maternal animals)

Dose descriptor:
NOAEC
Effect level:
3 mg/L air (analytical)
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 placental weights in test groups 1, 2 and 3 were not influenced by the test substance exposure and were similar to the control values. The mean fetal weights in test groups 1, 2 and 3 were not influenced by the test substance exposure and there were no biologically relevant differences concerning the means of the control group and of the substance-exposed groups.
Changes in sex ratio:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
The external examination of the fetuses revealed three types of malformations in single fetuses of test groups 2 and 3. Anasarca was recorded for two fetuses of the intermediate dose group. The latter fetus showed also a kinky tail. Anasarca was also recorded for one high dose fetus which showed in addition a shortened tail. All three aforementioned external malformations were found at low incidences and without a clear dose-response relationship; moreover, the differences between the groups occurred without statistical significance and these malformations or very similar ones are also present at comparable ranges in the historical control data. Therefore, the observed external malformations at the intermediate and high dose level in the present study are considered coincidental and not biologically significant. No external variations were found in any group. So-called unclassified observations (placentae fused or necrobiotic) were recorded without a clear relation to exposure levels for single fetuses in all groups including the controls.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Malformations of the fetal skeletons were seen in 5 out of 184 (= 2.7%) fetuses (in 5 out of 24 litters (= 21%)) of the control, in 10 out of 173 (= 5.8%) fetuses (in 8 out of 25 litters (= 36%)) of the 3 mg/L group, in 7 out of 162 (= 4.3%) fetuses (in 6 out of 23 litters (= 26%)) of the 7.5 mg/L group and in 13 out of 175 (= 7.4%) fetuses (in 9 out of 25 litters (= 36%)) of the 12 mg/L group. All skeletal malformations occurred without any statistically significant differences between the substance-exposed groups and the concurrent control group. Skeletal malformations, that were not correlates of external findings were related to the sternum (sternebra(e) bipartite, ossification centers dislocated or cleft sternum), the vertebral column (vertebral body/bodies dumbbell-shaped (asymmetrical) or bipartite (asymmetrical), vertebrae absent or vertebral arches fused), the skull (exoccipital bone(s) fused with atlas) and the ribs (fused ribs). These skeletal malformations or very similar ones can be found at comparable or even higher fetal/litter incidences in the historical control data. The other observed skeletal malformations (i.e. scapula(e) shortened or deformed; humerus, femur or radius/ulna shortened and bent); os ilium deformed) occurred exclusively in two fetuses of the 7 .5 mg/L group and high dose fetus. These fetuses showed a generalised edema (anasarca) at the external examination. As a consequence of the generalised accumulation of serum in the fetal bodies, the described skeletal findings occurred and thus are correlates of the external finding. The skeletal variations elicited were related to the ribs (shortened or absent 13th, accessory 14th, rudimentary cervical or wavy rib(s)), the sternum (sternebra(e) of irregular shape or bipartite), the skull (epactal bone between parietal and interparietal bones or splitting of skull bones) and the clavicula (deformed). All skeletal variations recorded appeared without a clear dose-response relationship, can be found in a similar frequency in the historical control data and/or the differences between the groups are without biological relevance. This includes the statistically significantly increased occurrence of affected fetuses/litter with rudimentary cervical rib(s) in test group 3. In all groups signs of skeletal retardations occurred substantiated by incomplete or missing ossification of skull (incl . hyoid) bones, vertebral column, sternebra(e), tibia/fibula and metatarsal bones. In total, the number of affected fetuses/litter with skeletal retardations was statistically significantly increased in test group 3. Since this value (61 .3%) is fully within the historical control range (41 .7 - 71 .5%) and thus is in the degree of variability for skeletal retardations, which has been observed in other studies using this strain of rats, it is not considered to be treatment-related. All other differences between the groups in respect to skeletal retardations appeared without statistical significance and/or without a clear dose-response relationship.
Visceral malformations:
no effects observed
Description (incidence and severity):
The examination of the organs of the fetuses revealed two different malformations. Hydrocephaly was recorded for one fetus of test group 2 and dilatation of both heart ventricles (globular shaped heart) occurred in one fetus of test group 0 (control). Both malformations, which were not dose-related, are also present at a low incidence in the historical control data. Therefore, these malformations are considered to be spontaneous in nature. Soft tissue variations (dilated renal pelvis and/or hydroureter) were detected in all groups without any statistically significant and/or biologically relevant differences between the groups. Both findings are very common ones in the rat strain used and all respective values are fully in the range of biological variation. No so-called unclassified observations (like bloody imbibition of kidney(s)) were recorded during the soft tissue examination.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEC
Effect level:
>= 12 mg/L air (analytical)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion