Registration Dossier

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:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 3-Methyl-2-butenal; CAS107-86-8; Substance No. 00/0680-2
- Physical state: Liquid/colorless to yellowish-clear
- Analytical purity: 97.7 area-% (method: GC, analytical report : 00L00514)
- Lot/batch No.: 00/12 from November 16, 2000
- Storage condition of test material: Refrigerator, under N2 without light

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: sexually mature, virgin
- Housing: singly from day 0 - 20 p.c. in type DK III stainless steel wire mesh cages
- Diet: Kliba maintenance diet rat/mouse/hamster meal, supplied by PROVIMI KLIBA SA, Kaiseraugst, Switzerland ad libitum
- Water: ad libitum

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
The aqueous test substance suspensions were prepared at the beginning of the administration period and thereafter at intervals which took into account the analytical results of the stability verification. For the preparation of the suspensions, an appropriate amount of the test substance was weighed depending on the dose group, in calibrated beakers and subsequently suspended in 0.5% Carboxymethylcellulose CB 30.000 in doubly distilled water using a high-speed homogenizer (Ultra Turrax, JANKE & KUNKEL KG, Germany). A magnetic stirrer was used to keep the suspensions homogeneous during treatment of the animals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Period of at least 4 days at room temperature were carried out before the study was initiated.
Samples of the test substance suspension were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations. The samples which were taken for the first concentration control analyses at the beginning of the administration period were also used to verify the homogeneity of the concentration (300 mg/kg body weight/day). 3 samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running.
Details on mating procedure:
The animals were mated by the breeder ("time-mated") and supplied on day 0 post coitum (= detection of vaginal plug / sperm). The animals arrived on the same day (i.e. day 0 p.c.) at the experimental laboratory. The following day was designed "day 1" post coitum (p.c.). Between start of the study (beginning of the experimental phase) and first administration (day 6 p.c.) the animals were acclimated to the laboratory conditions.
Duration of treatment / exposure:
day 6 to 19 p.c.

Frequency of treatment:
7 administrations per week
Duration of test:
until day 20 p.c.
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 150 and 450 mg/kg (first study); 300 mg/kg (second study)
Basis:
actual ingested
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
The test substance suspensions were administered to the animals orally (by gavage) once a day from implantation to one day prior to the expected day of parturition (day 6 to day 19 p.c.) always at approx. the same time of day (in the morning). The animals of the control group were treated in the same way with the vehicle (0.5 % carboxymethylcellulose CB 30.000 in doubly distilled water). The volume administered each day was 10 ml/kg body weight. The calculation of the volume administered was based on the last individual body weight.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, twice a day on working days and once on weekends and public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited (days 0- 20 p.c.).

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 p.c.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- With the exception of day 0, the consumption of food was determined on the same days as was body weight.

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 20 p.c.
- Organs examined: - Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
• live fetuses
• dead implantations:
a) early resorptions (only decidual or placental tissues visible or according to SALEWSKI (Salewski, 1964) from uteri from app arently 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)

OTHER: Mortality
A check was made twice a day on working days or once a day (Saturday, Sunday or on public holidays) (days 0- 20 p.c.)
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
- Other: dead fetuses
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
Statistics:
DUNNETT-test (two-sided) for the hypothesis of equal means [Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight]
FISHER'S EXACT test (onesided) for the hypothesis of equal proportions [Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings]
WILCOXON-test (one-sided) for the hypothesis of equal medians: [Proportions of fetuses with malformations, variations and/or unclassified observations in each litter]
Indices:
Furthermore, calculations of conception rate and pre- and postimplantation losses were carried out:
- The conception rate (in %) was calculated according to the following formula:
(number of pregnant animals / number of fertilized animals)x 100

- The preimplantation loss (in %) was calculated according to the following formula:
[(number of corpora lutea - number of implantations) / number of corpora lutea] x 100

- The postimplantation loss (in %) was calculated from the following formula:
[(number of implantations - number of live fetuses)/ number of implantations]x 100
Historical control data:
The historical control data used for interpretation of findings refer to the same test facility, the same rat strain and supplier of the animals and cover a period of about 5 months.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
Maternal toxicity was not exclusively after oral administration of 450 mg/kg bw/d to pregnant rats.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
First study:
There were no substance-related influences on the gestational parameters up to and including 150 mg/kg body weight/day. Conception rate, mean number of corpora lutea, total implantations, resorptions and live fetuses, fetal sex ratio or the values calculated for the pre- and the postimplantation losses were unaffected by treatment. The test substance administration evoked no signs of developmental toxicity and in particular no indications for teratogenicity at the two dose levels, where fetuses for further assessment were available. Placental and fetal body weights were unaffected and the external, soft tissue and/or skeletal (including cartilage) examinations of the fetuses revealed no biologically relevant differences between the control and the mid and the low dose groups (i .e. 50 and 150 mg/kg body weight/day).

Second study:
The daily oral administration of 300 mg 3-Methyl-2-buten-1-al (Prenal)/kg body weight to the dams did not impair their food consumption or body weight development, did not cause any substance-induced necropsy findings and had no influence on the gestational parameters. Uterus weight, conception rate, mean number of corpora lutea, total implantations, resorptions and live fetuses, fetal sex ratio or in the values calculated for the pre- and the postimplantation losses were unaffected by treatment. The administration of 300 mg/kg body weight/day of the test substance to pregnant rats evoked no signs of prenatal developmental toxicity and in particular no indications for teratogenicity.
These results fit quite well to the results from the previous (first) prenatal developmental toxicity study in Wistar rats with 3-Methyl-2-buten-1-al (Prenal) performed under comparable conditions, but with dose levels of 50, 150 and 450 mg/kg body weight/day (BASF AG, 2002).

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Basis for effect level:
other: fetotoxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
If both studies are assessed together, the test substance was already lethal for the dams at 450 mg/kg body weight/day, evoked only transient salivation in nearly all 300 mg/kg females and in a few 150 mg/kg dams, and did not induce any effect on the females at 50 mg/kg body weight/day. The test substance administration had no effects on the gestational parameters and induced no signs of prenatal developmental toxicity, in particular no indications for teratogenicity at dose levels, where fetuses for further assessment were available (i.e. 50, 150 and 300 mg/kg body weight/day).