Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
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
Cross-reference
Reason / purpose:
reference to same 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 415 (One-Generation Reproduction Toxicity Study)
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch No.: 00/12
Physical state/appearance: Clear liquid, colorless to yellowish
Purity: 97.7 area-%
Homogeneous
The stability under storage conditions was proven by reanalysis.
Storage conditions: Refrigerator, under N2 without light)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
The animals were free from clinical signs of disease. The females were nulliparous and non-pregnant at the beginning of the study.
According to a written statement from the breeder, male and female animals were derived from different litters. This was necessary to rule out the possibility of sibling mating. These animals were taken to form the F0 generation parental animals. All other animals used in this study were derived from these animals.
The rats of the FO parental generation were identified uniquely by ear tattoo. The unit digit of the animal number was tattooed on the outside of a rat's left ear, the ten digit on the inside of the left ear and the hundred digit was tattooed on the inside of the right ear.
All live pups were identified by skin tattoo on day 1 post partum (p .p.) and with picric acid between days 10 and 15 after birth.
During the study period, the rats were housed individually in type DK III stainless steel wire mesh cages supplied; by BECKER & CO., Castrop-Rauxel, Germany (floor area of about 800 cm2), with the following exceptions: from day 18 of gestation until day 21 after birth, the pregnant animals and their litters were also housed in Makrolon type M III cages. The M III cages were again supplied by BECKER & CO. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
The animals were accommodated in fully air-conditioned rooms in which central air conditioning guaranteed a range of temperature of 20 - 24°C and a range of relative humidity of 30 - 70%.
The day/night rhythm was 12 hours (12 hours light from 6.00 a.m. to 6.00 p.m. and 12 hours darkness from 6 .00 p.m : to 6 .00 a .m .) in general.
Before use each room was completely disinfected using a disinfector ("AUTEX" fully automatic, formalin-ammonia-based terminal disinfection). Usually, each week the walls and the floor were cleaned with water containing about 0 .1 % Incidin (supplied by HENKEL-Ecolab, Düsseldorf, Germany).
The food used was ground Kliba maintenance diet rat/mouse /hamster meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, which was available to the animals ad libitum throughout the study (from the day of supply to the day of or the day before necropsy).
Drinking water used to prepare aqueous 3-Methyl-2-buten-1-al (Prenal) solutions was of tap water quality. Water was supplied to the animals in the control group, and aqueous 3-Methyl-2-buten-1-al (Prenal) solutions were supplied to the animals in test groups 1-3 (50; 200 and 800 ppm), using Makrolon drinking bottles supplied by BECKER & CO., Castrop-Rauxel, FRG, with a capacity of 300 ml. The drinking water bottles were filled or changed twice times a week. The bottles had a stainless steel cap with a silicone sealing ring and a hole of diameter 0.6 mm, and each was placed in a recess in the cage top. The animals obtained water or aqueous 3-Methyl-2-buten-1-al ( Prenal) solutions by licking the drops from the hole in the cap. The special design of the drinking bottle substantially prevented uncontrolled emptying or leakage. The water drunk during the study was obtained from the central supply of the Experimental Toxicology and Ecology of BASF Aktiengesellschaft and was taken from a tap in the laboratory at the appropriate times.
The bedding used throughout the study was Ssni ff (type 3/4) supplied by SSNIFF SPEZIALDIÄTEN GmbH, Soest, Germany.

Administration / exposure

Route of administration:
oral: drinking water
Details on exposure:
Drinking water solutions were prepared once or twice a week.
The required weight of test substance was added to the appropriate amount of drinking water and agitated with a magnetic stirrer until the test substance was completely dissolved (about 3 minutes).
Details on mating procedure:
In general, each of the male and female animals was mated overnight at a 1 : 1 ratio for a maximum of 3 weeks or until there was evidence of copulation. Generally, throughout the mating period, each male animal was mated with a predetermined female animal from the same dose group. Matings occurred by placing the female in the cage of a male mating partner from about 4.00 p.m. until 7.00 - 9 .00 .m. the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data.
A vaginal smear was prepared after each mating and examined for sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "day 0" and the following day "day 1" p.c. (post coitum).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verification of the stability of the test substance in the drinking water for a period of 4 days at room temperature was carried out. The homogeneity of the test substance in the drinking water was guaranteed due to the fact that 3-Methyl-2-buten-1-al (Prenal) is completely soluble in water.
Samples of each concentration were drawn for concentration control analyses twice at the start of the administration period for the FO generation parental animals, after about 2 months and at the end of the study.
Duration of treatment / exposure:
Premating exposure period (males and females): at least 76 days.
The F1 pups were raised up until day 4 (culling) or 21 post partum (p.p.).
Frequency of treatment:
continuously
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 6, 21 and 77 mg/kg body weight/day (0, 50, 200, 800 mg/l)
Basis:
nominal in water
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
The objective of this study was to determine the possible adverse effects of 3-Methyl-2-buten-1 -al (Prenal) on the integrity and performance of the male and female reproductive systems, including gonadal function, mating behavior, conception, gestation, parturition, lactation and weaning, and on growth and development of offspring from one successive generation of Wistar rats continuously administered to the test substance in the drinking water. The study should also provide information about the effects of 3-Methyl-2-buten-1-al (Prenal) on neonatal morbidity, mortality (possible target organs in the offspring), and data on prenatal and postnatal developmental toxicity.
The following concentrations in drinking water were selected:
50 ppm: as an expected "no observed adverse effect level"
200 ppm: as intermediate dose level
800 ppm: as highest dose level
Positive control:
-

Examinations

Parental animals: Observations and examinations:
At least twice daily a check was made for dead or moribund animals. If animals were in a moribund state, they were sacrificed and necropsied. The examinations of these animals were carried out according to the methods in the laboratory of Pathology.
All parental animals were checked daily for clinical signs of toxicity. For technical reasons, however, the clinical observations recorded during the premating periods were printed out on a weekly basis.
The littering and lactation behavior of the dams was generally evaluated in the mornings in connection with the daily clinical inspection of the dams. Only special findings (e.g., animal could not litter, umbilical cord not cut), were documented on an individual dam basis.
The littering behavior of the dams was also inspected on weekdays (except holidays) in the afternoons in addition to the evaluations in the mornings.
The day of littering was considered the 24 hour period from about 3.00 p.m. of one day until about 3.00 p.m. of the following day. Deviations from this procedure were possible on Saturdays, Sundays and on public holidays.
Animals in a moribund state were sacrificed and examined in the laboratory of Pathology.
During the premating period of the FO generation parental animals water consumption was determined once a week (each time for a period of 3 days).
After the 10th week, water consumption of the females during gestation (animals with evidence of sperm) was determined for days 0-1, 6-7, 13-14 and 19-20 p.c.
During the lactation period (animals with litter) water consumption was determined for days 1-2, 4-5, 7-8, 14-15 p.p.
Water consumption was not determined for days 20-21 after parturition, since during this time the pups will begin to consume considerable amounts of water and therefore there was no point in such a measurement.
Water consumption of the FO males was not determined after the 10th study week until sacrifice. Furthermore, there was no determination of water consumption in the females during the mating periods, in the females without positive evidence of sperm during the programmed gestation phase, or in the females without litters during the lactation phase.
During the premating period of the FO generation parental animals food consumption was determined once a week (each time for a period of 7 days).
After the 10th week food consumption of the females during pregnancy (animals with evidence of sperm) was determined for days 0-7, 7-14 and 14-20 p.c. During the lactation period (animals with litter) food consumption was determined for days 1-4, 4-7, and 7-14 p.p. Food consumption was not determined between days 14 and 21 after parturition as required in the test guidelines, since during this time pups begin to consume considerable amounts of solid food offered, and therefore there was no point in such a measurement.
Food consumption of the FO males was not determined after the 10th test week through sacrifice. Furthermore, there was no determination of food consumption in the females during the mating periods, in the females without positive evidence of sperm during the programmed gestation phase, or in the females without li tters during the lactation phase.
In general, the body weight of the FO male and female parental animals was determined once a week at the same time of the day (in the morning); if possible, the weightings were carried out until nearly the end of the respective study period. The body weight change of the animals was calculated from these results.
The following exceptions are notable for the FO female animals: a) During each mating period (mating of the FO generation parental animals) females
were weighed on the day of positive evidence of sperm (day 0 p .c.) and on days 7, 14 and 20 post coitum. b) Females showing no positive evidence of sperm in vaginal smears were not weighed during the mating interval. c) Females with litter were weighed on the day after parturition (day 1 p.p.) and on days 4, 7, 14 and 21 post partum. d) Females without litter were not weighed during the lactation phase.
The intake of test substance was calculated from the amount of drinking water consumed and expressed in mg/kg body weight per day.
The mating partners, the number of mating days until vaginal sperm could be detected in the female, and the gestational status of the female were noted for FO breeding pairs.
Estrous cyclicity (parental animals):
-
Sperm parameters (parental animals):
-
Litter observations:
All pups derived from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn members of each litter. Pups which died before the first determination of their status on the day of birth were designated as stillborn pups.
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (day 0) and of pups dying between days 1-4, 5-7, 8-14 and 15-21 of the lactation period were determined; however, pups which died accidentally or had to be sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day of birth, and on lactation days 4, 7, 14 and 21.
On the day of birth (day 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Subsequently the sex of the pups was assessed by the external appearance of the anogenital region and/or the mammary line of the animals and was finally confirmed at necropsy.
The pups were weighed on the day after birth (day 1 p .p .) and on days 4 (before standardization), 7, 14 and 21 after birth. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning) and on day 4 p.p. immediately before standardization of the litters. In the relevant summary tables pup body weights and pup body weight gains are listed for males, females and males + females.
All live pups were examined each day for clinical symptoms (including grossmorphological findings).
Postmortem examinations (parental animals):
After fertility had been reevaluated, the animals were sacrificed and subjected to gross-pathological assessments.
The uteri of the females reevaluated for fertility were examined at the reproduction toxicology laboratory for live and dead implantations. In the case of an apparently non-pregnant animal or of an empty uterus horn in the case of single-horn pregnancy, the uterus was stained with sodium sulfide and assessed for early implantations according to the method of SALEWSKI (Salewski, E. 1964) . Then the uteri were rinsed carefully under running water. After these examinations were completed, the uteri were transferred to the pathology lab for further fixation.
Postmortem examinations (offspring):
All pups with scheduled sacrifice (i .e . pups which were culled on day 4 p.p. and pups which were sacrificed on day 21 p.p.) were killed by means of CO2. These pups were examined externally and eviscerated, their organs were assessed macroscopically. Thereafter the pups were discarded .
Statistics:
Dunnett's test wass used to compare treated and control groups (food intake, body weight data, number of pups/liter, etc.).
Fisher's Exact test for pairwise comparison of each dose group with control group (mating and fertility indices, pub viability data).
Two-sided Wilcoxon test (proportion of affected pups/litter with necropsy observations).

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

800 ppm groups (approx. 77 mg/kg body weight/day)
CLINICAL EXAMINATIONS: statistically significantly reduced water consumption during premating (males: about 11 % less, females: about 18% less than the concurrent controls if calculated for entire premating), gestation (approx. 17% below controls) and - without attaining statistical significance - during lactation (approx. 10% below controls); statistically significantly reduced food consumption of the F0 males during the first premating weeks
REPRODUCTIVE PERFORMANCE/ GROSS AND HISTOPATHOLOGICAL FINDINGS: no substance-related adverse effects in FO males and FO females

50 and 200 ppm groups (approx. 6 or 21 mg/kg body weight/day)
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ GROSS AND HISTOPATHOLOGICAL FINDINGS: no substance-related adverse effects in F0 males and F0 females

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
800 ppm
Sex:
male/female
Basis for effect level:
other: = about 77 mg/kg bw/day NOAEL for reproductive performance and fertility, systemic toxicity and developmental toxicity (growth and development of the offspring) for the F0 parental rats and their progeny.
Remarks on result:
other: Generation: F0 and F1 (migrated information)
Dose descriptor:
NOEL
Effect level:
200 ppm
Sex:
male/female
Basis for effect level:
other: = about 21 mg/kg bw/d due to the reductions in water and/or food consumption at the top dose level in both genders

Results: F1 generation

Details on results (F1)

no substance-related adverse effects in all three tested doses

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study the NOAEL (no observed adverse effect level) for reproductive performance and fertility, systemic toxicity and developmental toxicity (growth and development of the offspring) is 800 ppm (about 77 mg/kg body weight/day) for the F0 parental rats and their progeny.
The NOEL for the FO parental rats was fixed at 200 ppm (about 21 mg/kg body weight/day) due to the reductions in water and/or food consumption at the top dose level in both genders.