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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference Type:
study report

Materials and methods

Test guidelineopen allclose all
according to guideline
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
according to guideline
other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
Purity: 99.5 area-%
Test subtance No.: 09/0386-1
Batch identification: 000STD77L0

Test animals

Details on test animals or test system and environmental conditions:
Male and female Wistar rats, strain Crl:WI(Han), supplied by Charles River Laboratories, Research Models and Services, Germany GmbH, which were free from any clinical signs of disease, were used for the investigations. 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 used as F0 generation parental animals. All other animals used in this study (F1 generation pups) were derived from the supplier-provided animals.

During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
- During overnight matings, male and female mating partners were housed together in Makrolon type M III cages.
- Pregnant animals and their litters were housed together until PND 4.

Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment wooden gnawing blocks (Typ NGM E-022; supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria) were added. 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 housed 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 air change rate was 15 times per hour. There were no or only minimal deviations from these limits. The day/night cycle was 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h.

The animal room was completely disinfected using a disinfector ("AUTEX" fully automatic, formalin-ammonia-based terminal disinfection) before use. Walls and floor were cleaned each week with water containing about 0.5% Incidin Extra N (supplied by Ecolab Deutschland GmbH, Hanau, Germany) and 0.5% Mikro-Quat (supplied by Ecolab GmbH & Co. OHG, Düsseldorf, Germany). The food used was ground Kliba maintenance diet mouse/rat “GLP” 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 was supplied from water bottles (ad libitum). The bedding used was Lignocel FS 14 fibres, dustfree bedding, supplied by SSNIFF, Soest, Germany.

The 45 male and 45 female rats were 9 weeks old when they arrived from the breeder. During an acclimatization period of about 6 days, animals with lowest and highest body weights were eliminated from the study and used for other purposes. The 40 male and 40 female animals included in the study were 10 weeks old at the beginning of treatment, and their body weights varied between:
- male animals: 357.7 g - 301.4 g
- female animals: 173.2 g - 205.8 g

The assignment of the animals to the different test groups was carried out using a randomization program, according to their weight four days before the beginning of the administration period (day -4).

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
The test substance solutions in drinking water were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification. The maximum period for which each preparation was used was 7 days. For the preparation of the administration solutions the test item was weighed in a graduated measuring flask depending on the dose group, topped up with drinking water and subsequently thoroughly shaken until completely dissolved.
The volume administered each day was 10 ml/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.

Analyses of the test substance preparations
The analyses were carried out at at Competence Center Analytics, BASF SE, Ludwigshafen, Germany. Analytical verifications of the stability of the test substance in drinking water for a period of 7 days at room temperature were carried out prior to the start of the study. Given that Triethanolamin rein is completely miscible with drinking water, solutions were considered to be homogenous without further analysis. Samples of the test substance solutions were sent to the analytical laboratory twice during the study period for verification of the concentrations. Of each sample, one additional reserve sample was retained. Details of the sampling schedule were recorded with the raw data.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The concentration of the test substance in the vehicle (drinking water) was checked by capillary electrophoresis (CE) with internal standard quantification, using a Beckman P/ACE MDQ automated capillary electrophoresis system including capillary oven and UV-detector.
Details on mating procedure:
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group.

The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of 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 the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "GD 0" and the following day "GD 1".
Duration of treatment / exposure:
Premating period of 2 weeks and a mating period (max. 2 weeks) in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females.
Frequency of treatment:
Doses / concentrations
Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
actual ingested
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle


Maternal examinations:
A check for moribund or dead animals was made twice daily on working days or once daily on Saturdays, Sundays or public holidays. If animals were in a moribund state, they were sacrificed and necropsied. The examinations of these animals were carried out according to the methods established at the pathology laboratory.

Clinical observations
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal. For technical reasons, however, the clinical observations recorded during the premating period were printed out on a weekly basis. Individual data of daily observations can be found in the raw data.

Food consumption
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter was determined on PND 1 and 4.

Body weight
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals: 1) During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20; 2) females with litter were weighed on the day of parturition (PND 0 ) and on PND 4.
Females waiting for necropsy were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day. The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.

For the males, mating and fertility indices (male mating index and male fertility index) were calculated for F1 litters.

Postmortem examinations
Parental animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Special attention was given to the reproductive organs.
The following weights were determined in all animals sacrificed on schedule: anesthetized animals, epididymides, testes, ovaries.
The following organs or tissues of parental animals were fixed in 4% buffered formaldehyde or in modified Davidson’s solution: all gross lesions, adrenal glands, pituitary gland, testis (fixed in modified Davidson’s solution), epididymides (fixed in modified Davidson’s solution), prostate gland, seminal vesicles, coagulation glands, ovaries (fixed in modified Davidson’s solution), uterus, oviducts, vagina.
The uteri of all cohabited female F0 parental animals will be examined for the presence and number of implantation sites.
The uteri of apparently nonpregnant animals or empty uterus horns will be placed in 10% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations. Then the uteri will be rinsed carefully under running water. When the examinations are completed, the uteri will be transferred to the Pathology Laboratory for further processing.
Fetal examinations:
Pup number and status at delivery
The status (sex, liveborn or stillborn) and number of all delivered pups were determined as soon as possible on the day of birth. At the same time, the pups were also examined for macroscopically evident changes. Pups that die before this initial examination are defined as stillborn pups.

Pup viability/mortality
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 (PND 0) and of pups dying between PND 1-4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4.

Sex ratio
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle. The sex of the pups was finally confirmed at necropsy.

Clinical observations
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned.

Body weight
The pups were weighed on the day after birth (PND 1) and on PND 4. 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). “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

Postmortem examinations
All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia by means of CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically. All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding.
- Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter: simultaneous com-parison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
- Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions.
- Proportions of affected pups per litter with necropsy observations: pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Weight parameters (pathology): Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians.
Parental animals: male mating index, male fertility index, female mating index, female fertility index, gestation index, live birth index, postimplantation loss.
Offspring: pup viability index, sex ratio

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Most high-dose animals and one low-dose animal showed transient salivation for a few minutes immediately after each treatment. This was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. The slightly lower body weight gain of the 1000 mg/kg females during gestation was likely caused by the increased postimplantation loss rather than a systemic toxic effect of the test compound.
No other test substance-related adverse effects were observed.

Effect levels (maternal animals)

Dose descriptor:
Effect level:
> 1 000 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The following test substance-related adverse effects/findings were noted:

1000 mg/kg bw/day
- Lower mean number of implantation sites (about 20% below control)
- Increased postimplantation loss (19.4%* [*=p≤0.05] vs. 3.7% in control)
- Lower average litter size (about 33% below control).

300 mg/kg bw/day
- No test substance-related adverse effects

100 mg/kg bw/day
- No test substance-related adverse effects

Effect levels (fetuses)

Dose descriptor:
Effect level:
300 mg/kg bw/day
Basis for effect level:
reduction in number of live offspring
changes in litter size and weights
other: Decreased numbers of implants and delivered pups, and an increased postimplantation loss.

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion