2,2',2''-nitrilotriethanol citrate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Nov 2009 to 29 Jul 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Purity: 99.5 area-%
Test subtance No.: 09/0386-1
Batch identification: 000STD77L0

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Other: Rats were free from any clinical signs of disease. 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.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: The 40 male and 40 female animals included in the study were 10 weeks old at the beginning of treatment.
- Weight at study initiation: male animals, 357.7 g - 301.4 g; female animals, 173.2 g - 205.8 g.
- Housing: 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 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.
- Enrichment: 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 bedding used was Lignocel FS 14 fibres, dustfree bedding, supplied by SSNIFF, Soest, Germany.
- Diet (e.g. ad libitum): 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)
- Water (e.g. ad libitum): Drinking water was supplied from water bottles (ad libitum).
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%):30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION AND ADMINISTRATION
- 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.

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".

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSES OF THE TEST SUBSTANCE PREPARATIONS
- 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.
- The analyses were carried out 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 the test substance 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.

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:

Daily

Details on study schedule:

- After the acclimatization period, the test substance was administered to the parental animals orally by gavage, once daily at approximately the same time in the mornings.
- Females in labour were not treated.
- The treatment lasted up to one day prior to sacrifice.
- The animals of the control group were treated with the vehicle (drinking water), in the same way.
- Males and females from the same dose group were mated after a 14 days premating period, overnight in a ratio of 1:1. The females were allowed to deliver and rear their pups until day 4 after parturition. Shortly after PND 4 the parental females were sacrificed and examined. Pups were sacrificed on PND 4 and gross necropsied. The male animals were sacrificed 36 days after the beginning of the administration, and examined.

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control:

None

Parental animals: Observations and examinations:

MORTALITY
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.

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.

Oestrous cyclicity (parental animals):

- The parturition and lactation behaviour 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 behaviour 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.

Litter observations:

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 (parental animals):

SACRIFICE / GROSS NECROPSY
Parental animals were sacrificed by decapitation under Isoflurane anaesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Special attention was given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
- 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.

Postmortem examinations (offspring):

SACRIFICE
All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia by means of CO2.

GROSS NECROPSY AND HISTOPATHOLOGY / ORGAN WEIGTHS
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.

Statistics:

- 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 comparison 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.

Reproductive indices:

Male mating index, male fertility index, female mating index, female fertility index, gestation index, live birth index, postimplantation loss

Offspring viability indices:

Pup viability index, sex ratio

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Only at 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).

All test groups
- No effects on male mating index, male fertility index, female mating index, fertility index

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse systemic effects were observed up to the highest dose tested

Dose descriptor:

NOAEL

Remarks:

reproductive performance and fertility

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was lack of a dose-response relationship for the observed mortality suggesting that there is no association to treatment.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

No test substance-related adverse findings were observed in F1 pups.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Decreased numbers of idelivered pups.

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

no

Relevant for humans:

not specified

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2',2''-nitrilotriethanol

EC Number:

203-049-8

EC Name:

2,2',2''-nitrilotriethanol

Cas Number:

102-71-6

Molecular formula:

C6H15NO3

IUPAC Name:

2,2',2''-nitrilotriethanol

Test material form:

liquid: viscous

Specific details on test material used for the study:

Purity: 99.5 area-%
Test subtance No.: 09/0386-1
Batch identification: 000STD77L0

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Other: Rats were free from any clinical signs of disease. 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.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: The 40 male and 40 female animals included in the study were 10 weeks old at the beginning of treatment.
- Weight at study initiation: male animals, 357.7 g - 301.4 g; female animals, 173.2 g - 205.8 g.
- Housing: 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 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.
- Enrichment: 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 bedding used was Lignocel FS 14 fibres, dustfree bedding, supplied by SSNIFF, Soest, Germany.
- Diet: 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)
- Water: Drinking water was supplied from water bottles (ad libitum).
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%):30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION AND ADMINISTRATION
- 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSES OF THE TEST SUBSTANCE PREPARATIONS
- 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.
- The analyses were carried out 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 the test substance 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 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".

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:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- After the acclimatization period, the test substance was administered to the parental animals orally by gavage, once daily at approximately the same time in the mornings.
- Females in labour were not treated.
- The treatment lasted up to one day prior to sacrifice.
- The animals of the control group were treated with the vehicle (drinking water), in the same way.
- Males and females from the same dose group were mated after a 14 days premating period, overnight in a ratio of 1:1. The females were allowed to deliver and rear their pups until day 4 after parturition. Shortly after PND 4 the parental females were sacrificed and examined. Pups were sacrificed on PND 4 and gross necropsied. The male animals were sacrificed 36 days after the beginning of the administration, and examined.

Examinations

Maternal examinations:

MORTALITY
- Time schedule: 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.

CLINICAL OBSERVATIONS
- Time schedule: at least once daily
- Observations: Any signs of morbidity, pertinent behavioural changes and signs of overt toxicity.

FOOD CONSUMPTION
- Time schedule: 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
- Time schedule: 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 were weighed weekly. Body weight data of females waiting for necropsy were solely used for the calculations of the dose volume.
- Time schedule, exceptions 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.

ESTROUS CYCLICITY
- The parturition and lactation behaviour 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 behaviour 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.

POSTMORTEM EXAMINATIONS
SACRIFICE / GROSS NECROPSY
Parental animals were sacrificed by decapitation under Isoflurane anaesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Special attention was given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
- 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.

Ovaries and uterine content:

- The uteri of all cohabited female F0 parental animals were examined for the presence and number of implantation sites.
- The uteri of apparently nonpregnant animals or empty uterus horns were placed in 10% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations. Then the uteri were rinsed carefully under running water. When the examinations are completed, the uteri were 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.

SACRIFICE
All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia by means of CO2.

GROSS NECROPSY AND HISTOPATHOLOGY / ORGAN WEIGTHS
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.
g.

Statistics:

- 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.

Indices:

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

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Maternal developmental toxicity

Number of abortions:

not examined

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Only at 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)

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Remarks:

Only at 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)

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No adverse systemic effects were observed up to the highest dose tested.

2

Dose descriptor:

NOAEL

Remarks:

Developmental

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

pre and post implantation loss

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

100 mg/kg bw/day:
- liveborn index of 87%, which was due to three out of ten low-dose litters
- the isolated occurrence in the low-dose group and consequentially the lack of a dose-response relationship for these findings suggests that there is no association to treatment.

300 and 1000 mg/kg bw/day::
- The rate of liveborn pups was not affected by the test substance.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

slight differences were regarded to be spontaneous in nature.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Only at 1000 mg/kg bw/day:
- Lower average litter size (about 33% below control).

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

100 mg/kg bw/day:
- The viability index indicating pup mortality during lactation (PND 0 - 4) was reduced (81%). The reason for this were three out of ten low-dose litters which consisted either of stillborn pups or where all remaining liveborn pups died shortly after birth.
- The isolated occurrence in the low-dose group and consequentially the lack of a dose-response relationship for these findings suggests that there is no association to treatment.

300 and 1000 mg/kg bw/day:
- No treatment-related effects observed

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

no effects observed

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:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in litter size and weights

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

not specified