3-methylbut-2-en-1-ol

Developmental toxicity / teratogenicity

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 - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-methylbut-2-en-1-ol

EC Number:

209-141-4

EC Name:

3-methylbut-2-en-1-ol

Cas Number:

556-82-1

Molecular formula:

C5H10O

IUPAC Name:

3-methylbut-2-en-1-ol

Details on test material:

- Name of test material (as cited in study report): 3-Methylbut-2-en-1-ol (Prenol)
- Test substance No.: 00/0274-1
- Lot/batch No.: Ch. 00/18, Abl. No. 56-1706
- Date of production: 10 Apr 2000
- Physical state/appearance: Liquid/colorless, white
- Analytical purity: 99.1 area % (method: GC, analytical report: 00L00155)
- Homogeneity: Homogeneous (analytical report: 00L00155)
- Stability: The stability under storage conditions was confirmed by reanalysis
- Storage conditions: Room temperature
- Stability under test conditions: The stability of aqueous test substance suspensions over a period of 7 days at room temperature could be demonstrated.
Analytical laboratory: Analytical Department of BASF Aktiengesellschaft

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sexually mature, virgin Wistar rats (CrIGIxBrIHan:Wl);
- Source: Charles River Laboratories, Germany
- Age at study initiation: 70 - 84 days
- Weight at study initiation: 143.3 - 182.5 g.
- Housing: singly
- Diet (e.g. ad libitum): ground Kliba maintenance diet rat/mouse/hamster meal, supplied by PROVIMI KLIBA SA, Kaiseraugst, Switzerland. Food was available to the animals ad libitum throughout the study (from the day of supply to the day of necropsy).
- Water (e.g. ad libitum): tap water quality from water bottles, available ad libitum.
- Acclimation period: 5 days (from day 1 p.c. to day 6 p.c.)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%.
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light):12 hours / 12 hours

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
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 30000 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.

VEHICLE
- Concentration in vehicle: 500; 2000; and 6000 mg/100ml
- Amount of vehicle (if gavage): 10 ml/kg body weight

TREATMENT
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).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in doubly distilled water for a period of at least 7 days at room temperature were carried out before the study was initiated.
Samples of the test substance suspensions 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 for the samples of the low and the high concentrations (50 and 600 mg/kg body weight/day). 3 samples (one from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running.
Subsequently, the test substance suspensions were analyzed by GC.
The results of the analyses of aqueous test substance suspensions confirmed the correctness of the prepared concentrations. The analytical values of the samples corresponded generally to the expected values within the limits of the analytical method. The mean values were found to be in the range of 91.5 - 99.0% of the nominal concentration.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant: the animals were mated by the breeder ("time-mated") and supplied on day 0 post coitum
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post coitum.

Duration of treatment / exposure:

day 6 - 19 post coitum (p.c.)

Frequency of treatment:

once per day

Duration of test:

until day 20 post conception

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 200 and 600 mg/kg body weight
Basis:
actual ingested

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on findings of a maternal toxicity pretest (BASF AG, 10R0274/00103)

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
• 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.).
• Clinical symptoms: The animals were examined 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: All animals were weighed on days 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 p.c.. The body weight change of the animals was calculated from these results.

CORRECTED BODY WEIGHT GAIN (NET MATERNAL 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.).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
With the exception of day 0, the consumption of food was determined on the same days as body weight was measured.
WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice by cervical dislocation on gestation day 20 p.c.
Due to technical reasons, the study was carried out in 2 sections. Each dose group was represented in each section. A treatment interval of one day elapsed before the next section.

The high dose animal that was found dead was examined according to the same procedures as the females killed on schedule (exception: no weight determinations of uterus).
- Necropsy was performed and dams were assessed by gross pathology in randomized order to minimize bias.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes, on day 20 post coitum, all surviving females were sacrificed and assessed by gross pathology
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of dead fetuses: Yes, hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened
- Number of live and dead fetuses: Yes, (dead fetuses: hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)
- Number of early resorptions: Yes, only decidual or placental tissues visible or according to SALEWSKI (Salewski, 1964) from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy.
- Number of late resorptions: Yes, embryonic or fetal tissue in addition to placental tissue visible.

Fetal examinations:

All fetal analyses were conducted without knowledge of treatment group in order to minimize bias.
- Individual placental weights were recorded.
- External examinations: Yes, all animals
• The fetuses were removed from the uterus, sexed, weighed and examined macroscopically for any external findings.
• The sex was determined by observing the distance between the anus and the base of the genital tubercle and was later confirmed in all fetuses, fixed in BOUIN'S solution by internal examination. If there were discrepancies between the "external" and the "internal" sex of a fetus, the fetus was finally sexed according to the appearance of its gonads.
• The viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes and fluids were examined.
• Thereafter, the fetuses were sacrificed by subcutaneous injection of a pentobarbital
• After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and placed in ethyl alcohol, the other half was placed in BOUIN's solution for fixation and further evaluation.

- Soft tissue examinations: Yes: half per litter.
• The fetuses fixed in BOUIN's solution were examined for any visceral findings according to the method of BARROW and TAYLOR (Barrow and Taylor, 1969). After this examination these fetuses were discarded.

- Skeletal examinations: Yes: half per litter, (incl. cartilage)
• The skeletons of the fetuses fixed in ethyl alcohol were stained according to a modified method of KIMMEL and TRAMMELL (Kimmel, CA and Trammell C, 1981). Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually.

Statistics:

DUNNETT-test (two-sided) for the hypothesis of equal means (JASA, Vol. 50, 1096 - 1121, 1955; Biometrics, Vol. 20, 482 - 491): Food consumption, body weight, body weight change, corrected body weight gain (net matemal 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 (one-sided) for the hypothesis of equal proportions (Pairwise comparison of each dose group with the control group): Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings.
WILCOXON-test (one-sided) for the hypothesis of equal medians (Pairwise comparison of each dose group with the control group): Proportions of fetuses with malformations, variations and/or unclassified observations in each litter.

Indices:

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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Details on maternal toxic effects:
The following substance-related findings were obtained:

MORTALITY:
One high dose dam was found dead on day 19 p.c.; it could not be excluded with certainty, that the intercurrent death of this rat shortly before the scheduled termination of the study had a substance-related background.

CLINICAL SYMPTOMS:
600 mg/kg body weight/day:
• Transient salivation in all dams immediately after treatment between days 6 - 19 p.c. However, the observed salivation persisted in the respective females only for a short time (1 to 2 hours) after the actual gavage had taken place. After cessation of treatment on day 19 p.c., salivation did not occur any longer in these rats.
• Transient occurrence of abdominal position (in total 20 out of 25 rats), lacrimation (in total 24 out of 25 rats) and/or piloerection (in total 17 out of 25 rats) shortly after treatment and predominantly at initiation of dosing (days 6 and 7 p.c.) and occurred thereafter only sporadically.
Moreover, one high dose dam showed diarrhea on day 10 p.c.. Thus, all high dose rats showed adverse clinical findings during the administration period, which are considered to be substance-induced.

50 and 200 mg/kg body weight/day:
• No substance-related effects on dams and gestational parameters

- FOOD CONSUMPTION:
600 mg/kg body weight/day:
• Statistically significantly decreased mean food consumption on the first days (6 - 8 p.c.) of the treatment period (about 9% less food intake as in the concurrent control group), but was similar to or even exceeded control values on the following days until termination.
• Food consumption values on days 0 - 20 p.c. of the rats were similar to control values: 20.3 ± 3.00 g for controls and 19.9 ± 3.31 g for high dose groups.

50 and 200 mg/kg body weight/day:
• Food consumption of the mid and low dose rats was not affected by the test substance administration.
• Food consumption values on days 0 - 20 p.c. of the rats were similar to control values. Food consumption values were for controls 20.3 ± 3.00 g, for low dose 20.1 ± 3.13 g and for mid dose 20.4 ± 2.87.

- BODY WEIGHT DATA / CORRECTED BODY WEIGHT GAIN (net maternal body weight change):
• Statistically significantly slightly lowered mean body weights (3.9% below controls) on day 8 p.c. and statistically significantly impaired body weight gain on the first treatment days 6 - 8 p.c. (about 38% below the concurrent control value).
• On the other treatment days, weight gains of the 600 mg/kg bw females were sometimes below and sometimes above those of the corresponding controls without attaining statistical significance.
• Lower corrected body weight gain (about 14% below controls) without attaining statistical significance and statistically significantly lowered mean carcass weight ( 5% below controls).

50 and 200 mg/kg body weight/day:
• Body weight gains of the dams of these test groups were similar to those of the concurrent controls (weight gains days 0 - 20: 98% and 97% of controls for low and mid dose groups, respectively).
• All observable differences in these two groups in comparison to the controls during the treatment period are without any biological relevance.
• The corrected body weight gains of the dams revealed no differences of any biological relevance to the corresponding control group (102% and 103% of controls for low and mid dose groups).

POST-MORTEM EXAMINATIONS:
• Uterus weight: The mean gravid uterus weights of the animals of all test groups were not influenced by the administration of the test substance (96%, 89% and 102% of controls for low, high and mid dose groups).

• Necropsy findings: There were no substance-related observations at necropsy in any of the dams. Only one spontaneous finding, i.e. edema of the lungs occurred in two control females and one low dose dam. Lung edema was also observed in one high dose dam which died intercurrently on day 19 p.c..

• Reproduction data of dams: The conception rate reached 92% at 200 mg/kg bw and 96% at 0, 50 and 600 mg/kg bw. As all rats, which became pregnant had implantation sites at necropsy, a sufficient number of females for the purpose of the study was available. There were no substance-related and/or biologically relevant differences between the different test 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. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.
• A summary of reproduction data is given in table 1 (see remarks on results including tables and figures)

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
• Sex distribution of fetuses:
The sex distribution of the fetuses in all test groups was comparable with that of the control fetuses. The differences observed in comparison to the control were without any biological relevance.

• Weight of placentae:
The mean placental weights in all test groups were not influenced by the test substance administration and were similar to the control values (109%, 118% and 102% of controls for low, mid and high dose animals). All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age. This includes the statistically significantly increased mean placental weights (for male, female and male + female fetuses) in the mid dosed group (200 mg/kg body weight/day). As this observation did not show any relation to dosing it is considered to be spontaneous in nature.

• Weight of fetuses:
The mean fetal body weights in all test groups were not influenced by the test substance administration and were nearly identical with the corresponding control values (100%, 100% and 100% of controls for low, mid and high dose animals).

• Fetal external, soft tissue and skeletal observations:
None of the examined rat fetuses showed external or soft tissue malformations. The scattered occurrence of the few observed skeletal malformations in single fetuses of all test groups including the controls without a consistent pattern, without a clear dose-response relationship and/or at incidences, which are similar to historical control rates did not indicate any substance-induced origin of these findings. The malformations which occurred affected the vertebral column (misshapen sacral vertebra) and the sternum (cleft sternum or malpositioned and bipartite sternebra).

• If all different types of malformations were summarized, in total 6 of the 222 examined control fetuses (2.7%) in 6 out of 24 litters (25%), 2 of the 211 examined low dose fetuses (0.9%) in 2 out of 24 litters (8.3%), one out of 155 mid dose fetuses (0.6%) in one out of 19 litters (5.3%) and 3 out of 220 high dose fetuses (1.4%) in 3 out of 23 litters (13%) showed malformations. The mean percentages of affected fetuses/litter with total malformations amounted to 2.8, 1.1, 0.4 and 1.2% at 0; 50; 200 or 600 mg/kg bw, respectively. These incidences did not suggest any treatment-relationship.

• External variations did not occur in any of the fetuses in this study. Soft tissue variations, exclusively in the form of dilated renal pelvis and ureters, and a broad range of skeletal variations occurred in all test groups including the controls. All fetal and litter incidences for these variations and the corresponding mean percentages of affected fetuses/litter did not show a clear relation to dosing, were not considered to be of any toxicological relevance and/or can be found at a comparable frequency in the historical control data. This includes the statistically significantly increased rates of one soft tissue variation (dilated renal pelvis) in the 200 mg/kg bw group, of one skeletal variation (cervical rib) at the high dose and of total skeletal variations at the mid dose.
The mean percentages of affected fetuses/litter with total soft tissue variations amounted to 4.4 ± 8.98% (control), 10.7 ± 19.51% (50 mg/kg body weight/day), 21.0 ± 23.76% (200 mg/kg body weight/day) and 17.3 ± 28.05% (600 mg/kg body weight/day). If these values are compared with the respective historical control data (affected fetuses/litter range: 3.2 - 22.2%; mean value 8.1 %) it becomes obvious, that this quite common soft tissue finding in rat fetuses appeared by chance at a very low incidence in the concurrent control group. Moreover, all respective values from the substance-treated groups fall fully into this range and do not show a clear relation to dosing. Thus, a substance-induced effect concerning the occurrence of soft tissue variations in the different groups can be excluded with certainty.
In all groups, signs of skeletal variations with or without involvement of corresponding cartilaginous structures elicited. The mean percentages of affected fetuses/litter with skeletal variations amounted to 91.4, 87.5, 95.2 and 90.9% at 0; 50; 200 or 600 mg/kg body weight/day. The vast majority of the noted skeletal variations appeared without a clear relation to dosing, without biologically relevant differences between the groups and/or can be found at a comparable frequency in the historical control data. Only the finding "cervical rib" (cartilage not present) occurred at a statistically significantly increased rate in the high dose group (*= p< 0.05 ) and the rate of "total skeletal variations" was statistically significantly increased at 200 mg/kg (*= p<0.05 ). These marginal fluctuations in comparison to the concurrent control group are considered to be without any biological relevance as they are fully within the respective historical control ranges, are devoid of a dose-response relationship and/or did not affect the overall rate of skeletal variations.
• If all variations were summarized, in total 111 of the 222 examined control fetuses (50%) in all 24 litters (100%), 106 of the 211 examined low dose fetuses (50%) in all 24 litters (100%), 92 out of 155 mid dose fetuses (59%) in all 19 litters (100%) and 122 out of 220 high dose fetuses (55%) in all 23 litters (100%) showed variations. Calculated using the Wilcoxon test for, the mean percentages (± S.D) of affected fetuses/litter with total variations amounted to 50.3 ± 8.2, 53.3 ± 18.27, 62.2 ± 15.06 (p ≤ 0.01) and 55.6 ± 14.87 % at 0; 50; 200 or 600 mg/kg bw respectively. These incidences did not suggest a treatment-relationship, but reflected the usual biological variation inherent in the strain of rats used for this experiment. The statistically significantly increased percentage of fetuses/litter with total variations at 200 mg/kg is fully included in this assessment as it was predominantly caused by a randomly increased rate of a very common soft tissue variation (dilated renal pelvis) in the mid dose group.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

>= 600 mg/kg bw/day

Basis for effect level:

other: No indications for developmental toxicity were observed.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: Summary of reproduction data

	Prenol [mg/kg body weight]
	0	50	200	600
Conception rate (%)	96	96	92	96
Corpora lutea (mean± SD)	10.6±1.64	10.8±1.67	10.8±1.96	10.9±1.63
Implantation sites (mean± SD)	9.6±1.69	9.6±2.22	8.8±2.67	10.0±1.58
Preimplanation loss (mean± SD)	9.2±10.32	10.2±17.7	18.4±20.36	8.2±7.98
Postimplanation loss (mean± SD)	4.2±7.85	10.6±17.87	10.1±16.69	4.3±7.32
Total resorptions (mean± SD)	0.4±0.71	0.8±1.14	0.7±0.82	0.4±0.66
Live fetuses (mean± SD)	9.3±1.89	8.8±2.77	8.2±2.95	9.6±1.88