2,2',2''-nitrilotriethanol

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

For the endpoint toxicity to reproduction a WoE Approach is conducted using results from the registered substance TEA and studies performed with the structurally analogous substance MEA-HCl (CAS 2000-42-7).

No two-generation reproduction toxicity study is available for TEA. Under the conditions of a two-generation reproduction toxicity study with MEA HCl, the NOAEL for systemic toxicity and fertility, reproductive performance in parental F0 and F1 Wistar rats is 300 mg/kg bw/day. The NOAEL for pre-and postnatal developmental toxicity in their offspring is 1000 mg/kg bw/day.
Furthermore, in a screening reproduction/developmental toxicity study (OECD 421) with TEA in rats, the NOAEL for systemic toxicity as well as for reproductive performance and fertility in parental animals was established at 1000 mg/kg bw/day, the highest dose tested. The NOAEL for postnatal toxicity in the offspring was 1000 mg/kg bw/day, whereas the NOAEL for prenatal developmental toxicity was determined to be 300 mg/kg bw/day based on decreased numbers of implants and delivered pups, and an increased postimplantation loss.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 000STD77L0

Species:

rat

Strain:

Wistar

Sex:

male/female

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).

Route of administration:

oral: gavage

Vehicle:

water

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.

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:

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.

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 labor 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. 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.

Oestrous cyclicity (parental animals):

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.

Sperm parameters (parental animals):

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

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):

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.

Postmortem examinations (offspring):

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.

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.

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

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

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.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

> 1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

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

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance and fertility

Effect level:

> 1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

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

Key result

Critical effects observed:

no

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

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

300 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Decreased numbers of implants and delivered pups, and an increased postimplantation loss.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

03 Aug 2006 - 15 Jan 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

Jan 2001

Deviations:

yes

Remarks:

Food consumption was not determined between days 14 and 21 after parturition

Qualifier:

according to guideline

Guideline:

other: Corrigendum to EC Commission Directive 2004/73/EC, Part B: Methods for the determination of toxicity: Two-Generation Reproduction Toxicity Study; Official Journal of the European Comm unities; No. L216, pp. 236–246

Version / remarks:

29 Apr 2004

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

Aug 1998

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- No.of test material: 1) 05/0372-2; 2) 05/03723; 3) 05/0372-4
- Lot/batch No.: ad 1) JB116/2+3 (from 09 Aug – 04 Oct 2006); ad 2) JB116/4 (from 04 Oct – 29 Nov 2006); ad 3) JB116/9-17 (from 29 Nov 2006 until the scheduled termination of the in life part of the study)
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, under N2

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Han)

Details on species / strain selection:

The rat is the preferred animal species for reproduction studies according to test guidelines. This strain was selected since extensive historical control data were available for Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 44 (+/- 1) days
- Weight at study initiation: (P) Males: 162.1 (142.5 – 186.5) g; Females: 126.2 (110.6 – 145.1) g;
- Fasting period before study: none
- Housing: 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:
• overnight mating: male and female mating partners were housed together in type DK III cages
• gestation day 18 – lactation day 21: pregnant animals and their litters were housed in Makrolon type M III cages (floor area of about 800 cm²). The M III cages were also supplied by Becker & Co.
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 in racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA,
Kaiseraugst, Switzerland
- Water: ad libitum
- Acclimation period: 16 days
- Other: 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
The test substance was weighed and thoroughly mixed with a small amount of food. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer. Test diets were prepared at intervals, which guaranteed that the test substance in the diet remained stable throughout the feeding period. During the first week of the premating period, F0 parental animals received dietary Ethanolamine hydrochloride (EAH) concentrations based on the body weight of randomization and historical food consumption data given below:
Food consumption males: 19 g
Food consumption females: 15 g
The dietary concentration of EAH was calculated using the following formula: BWx . D / FCx = ppm
where
BWx = mean body weight on day x [g]
D = desired dose [mg/kg body weight/day]
FCx = mean daily food consumption on day x [g]
ppm = dietary EAH concentration for the week/period following day x
- During the remaining premating period, the dietary concentrations of EAH were adjusted weekly for each group and sex based on body weight and food consumption measurements from the preceding week.
- During the mating period of the F0 parental animals, each group and sex received the concentrations of EAH used during the last week of the premating period. This concentration was maintained throughout the mating period with the following exception: During cohabitation, both sexes received the test substance preparation for females as soon as the male was placed in the cage of its female partner. Both sexes returned to their normal test diet when they were separated the following morning. This test diet cycle remained in effect until there was evidence of successful mating. At that time, the mated animals received the test substance preparations described below at the first opportunity in the specific week.
- During the gestation period, dietary concentrations of EAH for the F0 males were again adjusted weekly on the basis of body weight and food consumption data from the preceding week. The EAH concentrations in the diet of the F0 females were the same as those used during the last week of the premating period.
- During the lactation period, dietary concentrations of EAH for the F0 males continued to be adjusted weekly on the basis of body weight and food consumption data from the preceding week. The EAH concentrations in the diet of the F0 females were 50% of those used during the last week of the premating period. This dietary adjustment, derived from historical body weight and food consumption data, maintained the dams at the desired doses of EAH during this period of increased food intake.
- Post weaning, dietary EAH levels for parental male animals awaiting necropsy were adjusted weekly based on body weight and food consumption data from the preceding week. The EAH concentration of parental female diets was the same as those used during the last week of the preceding premating period.
- Until all litters were weaned (when the last selected F1 pup reached age of day 21 p.p.), the food for the weaned F1 pups selected as F1 parental animals was prepared with EAH concentrations on the basis of historical body weight and food consumption data for rats of similar age.
- During the first week of the premating period of F1 parental animals, dietary EAH concentrations were formulated on the basis of actual body weight on day 0 and historical food consumption data.
Subsequently, dietary EAH levels for each F1 dose group and sex were adjusted as described for F0 parental animals.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: over night
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged (how): individual

In general, male and female animals were mated overnight at a 1 : 1 ratio for a maximum of 2 weeks. Each female animal was paired with a predetermined male animal from the same dose group throughout the entire mating period. Mating was accomplished by placing the male in the cage of the female mating partner from about 4.00 p.m. until 7.00 - 9.00 a.m. of the following morning. Deviations from these 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 was examined for the presence of sperm. If sperm were detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "day 0" and the following day "day 1" post coitum (p.c.).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of EAH in the diet over 32 days at room temperature was investigated analytically before the beginning of the study. Homogeneity and concentration control analyses were carried out at the beginning and toward the end of the premating periods. At least one analysis of test substance preparations for female animals was carried out during the gestation and lactation periods.

The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.

Duration of treatment / exposure:

semichronic duration (> 75 days)

Frequency of treatment:

continuously

Details on study schedule:

F0 PARENTAL ANIMALS
After the acclimatization period, the F0 generation parental animals continuously received the test substance at the appropriate concentrations in the diet up to about 16 hours before they were sacrificed. At least 75 days after the beginning of treatment, males and females from the same dose group were mated. The females were allowed to litter and rear their pups (F1 generation pups) until day 4 (standardization) or 21 after parturition. After weaning of F1 pups the F0 generation parental animals were sacrificed.
F1 PARENTAL ANIMALS
After weaning, 25 males and 25 females of the F1 pups of test groups 00, 01, 02 and 03 (0, 100, 300 and 1000 mg/kg bw/d) were taken per group as the basis of the F1 generation parental animals. These animals were chosen by lot and it was attempted to take each litter into account. If fewer than 25 litters were available in a group or if one sex was missing in a litter, more animals were taken from the other litters of the respective test group to obtain the full number. All selected animals were treated with the test substance at the same dose level as their parents from their growth into adulthood up to about one day before they were sacrificed. At least 75 days after assignment of the F1 generation parental animals, the males and females were generally mated at a ratio of 1:1. Partners were randomly assigned, mating of siblings was excluded. The females were allowed to litter and rear their pups (F2 generation pups) until day 4 (standardization) or 21 after parturition. Shortly after the F2 generation pups had been weaned, the F1 generation parental animals were sacrificed.
STANDARDIZATION OF LITTERS (F1 AND F2 GENERATION PUPS)
On day 4 p.p., individual litters were standardized in such a way that, where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If it was not possible for individual litters to have 4 pups/sex, it was proceeded in such a way that 8 pups per litter were present for further rearing (e.g. 5 male and 3 female pups). Standardization of litters was not performed in litters with ≤ 8 pups.
PUPS AFTER STANDARDIZATION AND AFTER WEANING
After standardization or weaning, all pups were sacrificed by means of CO2 with the exception of those F1 generation pups, which were chosen as F1 generation parental animals. All sacrificed pups, including stillborn pups and those that died during their rearing period, were subject of a macroscopic (external and visceral) examination. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days and once daily on weekends

DETAILED CLINICAL OBSERVATIONS: Yes
All parental animals were checked daily for clinically evident signs of toxicity. For technical reasons, however, the clinical observations recorded during the premating periods were printed out on a weekly basis (the daily observations can be found in the raw data). The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams.

BODY WEIGHT: Yes
- Time schedule for examinations: body weights of F0 and F1 parents were determined once weekly; during gestation and lactation F0 and F1 females were weighed on days 0, 7, 14 and 20 of gestation, and on days 1, 4, 7, 14 and 21 after birth.

FOOD CONSUMPTION: Yes
In general, food consumption was determined once a week (each time for a period of at least 6 days) for the male and female F0 and F1 parental animals. For the females during pregnancy (animals with evidence of sperm), food consumption was determined weekly 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 will begin to consume considerable amounts of solid food offered, and therefore, there was no point in such measurement. Furthermore, food consumption was not determined for females without positive evidence of sperm and for females without litter.

COMPOUND INTAKE: Yes
The intake of test substance was calculated from the amount of food consumed and is expressed as mg/kg body weight/day (mg/kg bw/d). The calculation of the group values/day was carried out according to the following formula: intake of test substance on day x in mg/kg bw/d = (daily food consumption on day x in grams) x (concentration in ppm) / (body weight on day y in grams (last weighing before day x))

MALE REPRODUCTION DATA:
For every F0 and F1 breeding pair, following parameters were recorded: mating partners, number of mating days until positive evidence of sperm, and gestational status of the female.

FEMALE REPRODUCTION AND DELIVERY DATA:
For F0 and F1 females, following parameters were noted: mating partners, number of mating days until positive evidence of sperm, and gestational status.

BLOOD SAMPLINGS:
Blood samples were taken from all F0 and F1 parental animals of each sex and test group during week 10 of premating treatment and the plasma was analyzed for the concentration of the test substance.

Oestrous cyclicity (parental animals):

Estrous cycle data were evaluated for F0 and F1 generation females over a three week period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.

Sperm parameters (parental animals):

Immediately after necropsy and organ weight determination, the right testis and cauda epididymidis were taken from the F0 and F1 males of all dose groups. The following parameters were determined:
- sperm head count in testis
- sperm head count in cauda epididymidis
- sperm morphology
- sperm motility
Preparation of specimens for sperm morphology and sperm motility examinations were carried out in a randomized sequence. To evaluate a possible effect on sperm parameters, sperm head count and sperm morphology were evaluated for the control and highest dose group (1000 mg/kg bw/d). Only in case of significant differences between these groups, additional sperm head counts were made for the mid-dose (300 mg/kg bw/d) and, if necessary, for the low-dose group (100 mg/kg bw/d) to reveal the dose level affecting these parameters.

Litter observations:

PUP NUMBER AND STATUS AT DELIVERY:
On the day of birth, all pups derived from the F0 parents (F1 litter) and the F1 parents (F2 litter) were examined as soon as possible to determine the total number of pups, and the number of liveborn as well as stillborn pups of each litter. Pups, which died before examination, were designated as stillborn pups.
PUP VIABILITY/MORTALITY:
- In general, a check was made for dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) and once in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated as dexribed in the section 'Postmortem examinations (offspring)'
- 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.
SEX RATIO:
- On the day of birth (day 0 p.p.), 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 and was finally confirmed at necropsy.
- The sex ratio was calculated at day 0 and day 21 p.p. according to the following formula:
Sex ratio = (number of live male or female pups on day 0/day 21 p.p. / number of live male and fema
le pups on day 0/day 21 p.p.) X 100
PUP 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 any special findings, these were documented with the dam concerned.
PUP BODY WEIGHT DATA:
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 based on 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.
SEXUAL MATURATION:
- Vaginal opening: All female F1 pups selected to become the F1 parental generation females (25/group) were examined daily for vaginal opening beginning on day 27 p.p. On the day of vaginal opening, the body weights of the respective animals were additionally determined.
- Preputial separation: All male F1 pups selected to become the F1 parental generation males (25/group) were examined daily for preputial separation beginning on day 40 p.p. On the day of preputial separation, the body weights of the respective animals were additionally determined.

Postmortem examinations (parental animals):

All F0 and F1 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. As soon as possible after termination, one portion of the liver (lobus medialis) of each 10 dams per group was sampled to be analyzed for choline concentration.
ORGAN WEIGHTS:
Weight assessment was carried out on all animals sacrificed at scheduled dates. The following weights were determined:
1. Anesthetized animals, 2. Liver, 3. Kidneys, 4. Adrenal glands, 5. Testes, 6. Epididymides, 7. Cauda epididymis, 8. Prostate, 9. Seminal vesicles including coagulation glands, 10. Ovaries, 11. Uterus, 12. Spleen, 3. Brain, 14. Pituitary gland, 15. Thyroid glands (with parathyroid glands).
ORGAN/TISSUE FIXATION:
The following organs or tissues of the F0 and F1 generation parental animals were fixed in 4% neutral buffered formaldehyde solution or in BOUIN’s solution, respectively:
1. Vagina, 2. Cervix uteri, 3. Uterus, 4. Ovaries (fixed in BOUIN´s solution), 5. Oviducts, 6. Left testis (fixed in BOUIN´s solution), 7. Left epididymis (fixed in BOUIN´s solution), 8. Seminal vesicles, 9. C
oagulation glands, 10. Prostate, 11. Pituitary gland, 12. Adrenal glands, , 3. Liver, 14. Kidneys, 15. Spleen, 16. Brain, 17. Thyroids (with parathyroids), 18. All gross lesions. After fixation, the organs fixed in BOUIN´s solution were embedded in Paraplast. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings. All gross lesions were examined. Of the fixated organs the organs of all animals in the control group and the high dose group were evaluated. Additionally, the organs for mating pair suspected of reduced fertility were evaluated.
DIFFERENTIAL OVARIAN FOLLICLE COUNT (DOFC) IN F1 GENERATION:
From both ovaries (”ovary 1” and “ovary 2”) of F1 female animals (control and top dose), five sections were taken from the proximal and the distal part of the ovaries, respectively, at least 100 μm apart from the inner third of the ovary. All ovarian sections were prepared and evaluated. Primordial follicles and growing follicles were counted by light microscope (magnification: 100x) on each of these slides, – according to the definitions given by Plowchalk et al. (PLOWCHALK, D. R., B. J. SMITH, and D. R. MATTISON: Assessment of Toxicity to the Ovary Using Follicle Quantitation and Morphometrics. In: Methods in Toxicology, Vol. 3, Part B: Female Reproductive Toxicology (J. J. HEINDEL and R. E. CHAPIN, Editors), p. 57-68, 1993, Academic Press). To prevent multiple counting on serial slides – especially of the growing follicles – only follicles with an oocyte with visible chromatin on the slide were counted. The number of each type of follicle was recorded individually for ovary 1 and ovary 2 of every animal on any of the slide levels (level 1-10), giving in summary the incidence of each type of the follicles by using EXCEL sheets for the reporting of the results. Finally, the results of all types of follicles were summarized for all animals per group in dose groups 10 and 13. As primordial follicles continuously develop into growing follicles, the assessment of the follicles was extended to the combined incidence of primordial plus growing follicles. In general, the fifth slide of the left and right ovary was evaluated for histological findings. A correlation between gross lesions and histopathological findings was performed.

Postmortem examinations (offspring):

PUP ORGAN WEIGHTS:
After scheduled sacrifice brain, spleen and thymus of 1 pup/sex and litter from the F1 and F2 pups were weighed. Normally, the first male and the first female pups/litter were taken for these examinations. For the calculation of the respective relative organ weights, pup body weights were taken, which were determined routinely during the in-life phase on day 21 p.p.
PUP NECROPSY OBSERVATIONS:
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. or subsequent days) were killed by means of CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically. All stillborn pups and all pups that died up to weaning were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.

Statistics:

see in 'Any other information on materials and methods incl. tables'

Reproductive indices:

For the males, mating and fertility indices were calculated for F1 and F2 litters according to the following formulas:
- Male mating index (%) = (number of males with confirmed mating / number of males placed with females) x 100
- Male fertility index (%) = (number of males proving their fertility / number of males placed with females) x 100
The number of males with confirmed mating was defined by a femal with vaginal sperm or with implants in utero.
The number of males proving their fertility was defined by a female with implants in utero.
For females, mating, fertility and gestation indices were calculated for F1 and F2 litters according to
the following formulas:
- Female mating index (%) = (number of females mated / total number of pups born) x 100
- Female fertility index (%) = (number of females pregnant / number of females mated) x 100
- Gestation inex (%) = (number of females with live pups on the day of birth / number of females
pregnant) x 100
The number of females mated was defined as the number of females with vaginal sperm or with implants in utero.
The number of females pregnant was defined as the number of females with implants in utero.
The total amount of delivered pups/dam was recorded and the number of liveborn and stillborn pups noted. The live birth index was calcualted for F1 and F2 litters according to the following formula:
- Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100
The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:
Postimplantation loss (%) = ((number of implantations – number of pups delivered) / number of implantations) x100

Offspring viability indices:

Viability and lactation indices were calculated according to the following formulas:
- Viability index (%) = (number of live pups on day 4 (before standardization of litters) after birth /number of live pups on the day of birth) x 100
- Lactation index (%) = (number of live pups on day 21 after birth / number of live pups on day 4(after standardization of litters) after birth) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

F0: No clinical signs or changes of the general behavior, which may be attributed to the test substance, were detected in F0 male or F0 female parental animals of the test groups 01 and 02 (100 and 300 mg/kg bw/day). Intensively yellow discolored urine was recorded in all F0 males and females of test group 03 (1000 mg/kg bw/day) from study week 3 onwards until the end of the treatment period. This urine discoloration mirrored the systemic availability of the test substance rather than being an adverse effect and was most likely caused by the excreted test compound and/or its metabolites.
Clinical observations for females during gestation of F1 litters: All F0 females of test group 03 showed intensively yellow discolored urine during the entire gestation period for F1 litter. No other clinical findings were observed in the test groups 00-03 (0, 100, 300 and 1000 mg/kg bw/day). One sperm positive female of test group 02 (300 mg/kg bw/day) and one of test group 03 (1000 mg/kg bw/d) did not deliver F1 pups. This observation was not considered to be associated to the test compound.
Clinical observations for females during lactation of F1 litters:
All F0 females of test group 03 showed intensively yellow discolored urine during the entire lactation period for F1 litter. One high-dose female (1000 mg/kg bw/day) had just one pup (female), which was cannibalized by its mother on lactation day 8. No other clinical findings were observed in the test groups 00-03 (0, 100, 300 and 1000 mg/kg bw/day).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled mortalities of male and female parental animals in any test group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights and average body weight gain of the F0 parental males of all test substance treated groups (100, 300 and 1000 mg/kg bw/day) were comparable to the controls throughout the entire study period. Observed differences between controls and test groups were regarded as spontaneous in nature. During premating, gestation and lactation periods, the mean body weights and body weight gains of the F0 parental females in the low- and mid-dose groups were generally comparable to the concurrent control group. During premating, gestation and lactation periods, the mean body weights and body weight gains of the F0 parental females in the low- and mid-dose groups were generally comparable to the concurrent control group. Mean body weights and body weight gains of the F0 females in test group 03 (1000 mg/kg bw/day) were similar to the controls throughout the entire premating period. During gestation, these animals gained less weight from gestation day 7 onwards (up to 38%). As a consequence, body weights on gestation day 20 were 8% lower than the control. This effect may have been caused by the statistically significantly increased postimplantation loss and the statistically significantly decreased mean number of delivered pups in test group 03. This is also indicated by the unaffected body weight of the high dose dams on post-delivery day 1. Mean body weights of the high-dose females remained comparable to the controls during entire lactation, whereas the weight gain wavered up and down in the individual lactation sections.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of the F0 parental males of all test substance-treated groups was generally comparable to the controls throughout the entire study. The statistically significant increase of food consumption in test group 02 (300 mg/kg bw/d) during study week 12-13 was regarded as spontaneous in nature. Food consumption of the F0 parental females of the low- and mid-dose groups (100 and 300 mg/kg bw/day) was comparable to the control animals during the periods of premating, gestation and lactation. Food consumption of the high-dose F0 females (1000 mg/kg bw/day) was also comparable during premating and gestation periods. However, in these animals, food consumption was statistically significantly below controls (up to 18%) in the individual lactation sections: days 1-4, 4-7, and 7-14 p.p.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- Extramedullar hematopoiesis was seen in the spleen of 1000 mg/kg male animals in a slightly higher number of animals compared to control animals, but only in a minimal (grade 1) to slight (grade 2) severity. A treatment-related increase seems unlikely, because no weight deviations were observed, the severity was very low and comparable to the control animals. All other findings noted were single observations either, or were similarly in distribution pattern and severity in control rats compared to
treatment groups. All of them are considered to be incidental and/or spontaneous in origin and without any relation to treatment.
- Fertility: The non-pregnant female and the male mating partner did not show histopathological findings explaining the infertility. Only a minimal focal atrophy of the prostate was present, which is considered an incidental finding.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data, generated during the last 3 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups including the control. For the different test groups, the mean estrous cycle duration varied between 3.8 (test group 01) and 3.9 days (test groups 00, 02 and 03).

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

- For most sperm parameters examined in the F0 parental males no treatment-related effects were noted. The number of homogenization resistant testicular spermatids, the percentages of abnormal and normal sperm and sperm motility data were comparable between the test substance-treated groups and the concurrent control and did not show any statistically or biologically significant differences. However, the number of homogenization resistant caudal epididymal sperm was statistically si
gnificantly reduced in test group 03 (1000 mg/kg bw/d) compared to the control (-13% [p≤0.05]). Hence, the number of caudal epididymal sperm in test group 02 (300 mg/kg bw/d) was additionally evaluated revealing values comparable to the control.
- For all groups, including the control, it has to be noted that the mean amount of sperm in the cauda epididymidis was below the test facility’s historical control data.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

MALE REPRODUCTION DATA:
- Cohabitation was confirmed for all F0 parental males, which were paired with females to generate F1 pups. Thus, the male mating index was 100% in all test groups.
- Fertility was proved for most of the F0 parental males with confirmed cohabitation. One high-dose male (1000 mg/kg bw/day) did not generate F1 pups although its individual sperm parameters did not show any difference to control animals. Thus, the male fertility index was 100% for the test groups 00-02 and 96% for test group 03. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. All respective values were within the range of the historical control data of the test facility.
FEMALE REPRODUCTION AND DELIVERY DATA:
- The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (day 0 p.c.) varied between 2.4 and 2.6 days without any relation to dosing. Nearly all sperm positive females delivered pups or had implants in utero. One high-dose F0 female (No. 185) did not become pregnant. Consequently, the fertility index varied between 96% (test group 03) and 100% (test groups 00-02).
- The mean duration of gestation was comparable between all test groups. The respective values were 21.8, 21.8, 21.7 and 22.2** (p≤0.01) days. Although the duration of gestation appeared to be statistically significantly increased in the high-dose group, this value was only slightly above the concurrent control and was still within the historical control range of the test facility (21.5–22.3 days). Therefore, this finding was not considered to be treatment-related.
- The gestation index was 100% in test groups 00, 01 and 03 (0, 100, 1000 mg/kg bw/d), indicating that all pregnant F0 females in these test groups had live F1 pups in their litters. The gestation index
was 96% in test group 02 (300 mg/kg bw/d), caused by one female, which delivered no pups, but had 2 implants in utero.
- With regard to the number of implantation sites, no statistically significant differences were seen between the control (11.8 implants/dam) and test groups 01 and 02 (12.4 and 11.8 implants/dam, respectively). In contrast, the number of implantation sites was statistically significantly reduced in test group 03 (8.6** [p≤0.01]). Furthermore, there were indications for test substance-induced intrauterine embryo-/fetolethality, since the postimplantation loss was statistically significantly increased in the high-dose group (23.0%** [p≤0.01] at 1000 mg/kg bw/d). There were no statistically significantly differences concerning the postimplantation loss between the remaining test groups and the control
(5.7%, 6.8% and 9.9% at 0, 100 and 300 mg/kg bw/d, respectively).
- The average litter size (F1 pups per dam) was very similar between test groups 00, 01 and 02 (11.1, 11.4, and 11.4 pups/dam, respectively). A statistically significantly lower number of F1 pups per dam were delivered in test group 03 (6.8** pups/dam [p≤0.01]). Since the number of stillborn pups was comparably low in all groups, the live birth index was 99% for test groups 00, 02 and 03, and 100% for test group 01.

PLASMA CONCENTRATIONS
The analysis of the plasma concentrations of Ethanolamine (calculated as Ethanolamine hydrochlorid) showed concentrations below 3 [mg/kg] for all control animals of both sexes for the F0 and the F1 generation. The low-dose groups resulted in values of <3 - 4 [mg/kg] for the male animals of the F0 generation and was below 3 [mg/kg] for the female animals of the F0 and for the male and female animals of the F1 generation. The mean plasma concentrations of Ethanolamine (calculated as
Ethanolamine hydrochlorid) of the animals of the mid dose were 8, 9, 11 and 10 [mg/kg] for the male animals of the F0 and the F1 generation and for the female animals of the F0 and the F1 generation, respectively. The mean plasma concentrations of Ethanolamine (calculated as Ethanolamine hydrochlorid) of the animals of the high dose were 65, 60, 66 and 81 [mg/kg] for the male animals of the F0 and the F1 generation and for the female animals of the F0 and the F1 generation, respectively. These data show a dose dependency of the plasma levels of Ethanolamine in the experimental animals and therewith prove the bioavailability of Ethanolamine hydrochloride in principle.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

Remarks on result:

not determinable

Remarks:

no clear evidence of treatment-related effects

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Remarks on result:

other: occurring together with systemic toxicity

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs or changes in general behavior, which may be attributed to the test substance, were detected in F1 male or F1 female parental animals of the test groups 11 and 12 (100 and 300 mg/kg bw/day).Intensively yellow discolored urine was recorded in all F1 males and F1 females of test group 13 (1000 mg/kg bw/day) from study week 0 onwards until the end of the treatment period. This urine discoloration mirrored the systemic availability of the test substance rather than being an adverse effect and was most likely caused by excreted test compound and/or its metabolites. Furthermore, one F1 male animal of test group 11 (100 mg/kg bw/day) had a skin lesion at its throat during study weeks 3-6.
Clinical observations for females during gestation of F2 litters: All F1 females of test group 13 showed intensively yellow discolored urine during the entire gestation period (F2 litter). No other clinical findings were observed in the test groups 10-13 (0, 100, 300 and 1000 mg/kg bw/day).One sperm-positive female of test group 10 (control), one of test group 11 (100 mg/kg bw/day) and two of test group 13 (1000 mg/kg bw/day) did not deliver F2 pups. These observations were not considered
to be associated to the test compound due to a missing dose-response relationship.
Clinical observations for females during lactation of F2 litters: All F1 females of test group 13 showed intensive yellow discolored urine during the entire lactation period for F2 litters. No other clinical findings were observed in the test groups 10-13 (0, 100, 300 and 1000 mg/kg bw/day).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

None of the male and female F1 parental animals of any test group died ahead of schedule.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Mean body weights and body weight gain of the F1 parental males in test groups 11-13 (100, 300 and 1000 mg/kg bw/day) were comparable to the control throughout the entire treatment period. The statistically significantly decreased values of body weight gain in the high-dose males during study weeks 6-7 and 9-10 were in the normal range of fluctuation of this group and the control during the course of the study and, therefore, regarded as incidental.
- Mean body weights and body weight gain of the F1 parental females in test groups 11-12 (100 and 300 1000 mg/kg bw/day) were comparable to the control throughout premating, gestation and lactation periods. Mean body weights and body weight gains of the F1 females in test group 03 (1000 mg/kg bw/day) were similar to the controls throughout the entire premating period, the statistically significantly increased body weight gain of the high-dose F1 females (1000 mg/kg bw/day) during premating week 1-2 was regarded as incidental variance.
- The average weight gain of these animals was significantly below control (26%) during gestation days 14-20, which led to an averaged decrease of weight gain for the entire gestation of 17%. This effect may have been caused by the statistically significantly increased postimplantation loss and the statisti cally significantly decreased mean number of delivered pups in test group 03. Mean body weights of the high-dose females remained comparable to the controls during entire lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of F1 male and female animals in test groups 11-12 (100, and 300 mg/kg bw/day) was generally comparable to the control group throughout the entire treatment period, covering premating, gestation and lactation periods. Food consumption of the high-dose F0 females (1000 mg/kg bw/day) was also comparable during premating and gestation periods. However, in these animals, food consumption was statistically significantly below controls (-11%) on lactation days 1-4, and remained below control on lactation days 4-7 and 7-14 (-7%), although not statistically significant. For all test groups the intake of Ethanolamine hydrochloride correlated well with the desired target doses. For the actual test substance intake see 'Any other information on results incl. tables'.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: Statistically significantly decreased absolute and relative weight of epididymides and cauda epididymidis in males.
The observed increase of absolute kidney weights of male and female animals in mid (300 mg/kg bw/day) and top dose (1000 mg/kg bw/day) groups, respectively, was statistically significant. Because no histomorphological correlate was detected, a treatment-related weight increase is less likely. The observed decrease of spleen weights in top dose males as well as the increase of thyroid glands in top dose males and mid and top dose females, respectively, is considered incidental and not treatment related due to a missing dose response relationship and no histopathological correlates.

Gross pathological findings:

no effects observed

Description (incidence and severity):

- All gross lesions observed in test animals occurred singularly. They are considered to be spontaneous lesions in origin and are not related to treatment.
- Fertility: One non-pregnant female animal did show a bilaterally severe reduced size of the ovaries as well as a moderate thickening of the uterus wall. The other three non-pregnant females did not show any gross lesions. The four male mating partners did not show any gross lesions either.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- As compared to control animals, the kidneys of low, mid, and top dose male and female animals revealed a low incidence of basophilic tubules in a slightly higher number of animals. The severity (minimal to slight) was comparable between controls and treated animals and a clear dose-response relationship was missing. The cauda epididymis and epididymides of top dose males showed no histomorphological correlates to the decreased organ weights. All other findings noted were single
observations either, or were similarly in distribution pattern and severity in control rats compared to treatment groups. All of them are considered to be incidental and/or spontaneous in origin and without any relation to treatment.
- Fertility: One non-pregnant female showed a bilateral moderate diffuse stromal hyperplasia and a unilateral severe focally extensive chronic inflammation of the ovaries as well as an ovarian cyst.
There were still corpora lutea present and the histopathological findings did not correlate with the gross lesion. The gross lesion “thickening of uterine wall” had no corresponding histological finding.
The findings on the ovaries might explain the infertility. The male mating partner did not reveal lesions affecting the fertility. One female Animal showed within the uterus a severe multifocal endometrial and glandular degeneration and within the oviducts a severe diffuse epithelial degeneration, which explains the infertility of this animal. The male mating partner did not reveal lesions affecting the fertility. The other two mating pairs did not show any lesions affecting the fertility.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

The results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant deviations between controls and animals of the top dose group.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Evaluation of the estrous cycle data, 3 weeks prior to mating for the F2 litter, revealed very regular cycles in the females of all test groups including the control. The mean estrous cycle duration varied between 3.8 days in test groups 11 and 12 and 3.9 days in test groups 10 and 13.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No treatment-related effects were noted for the different sperm parameters, examined at or after the sacrifice of the F1 parental males. after the sacrifice of the F1 parental males. The number of homogenization resistant testicular spermatids as well as caudal epididymal sperm, the percentages of abnormal and normal sperms and sperm motility data were comparable between the examined test substance-treated groups and the concurrent control group (0, 100, 300 and 1000 mg/kg bw/day). For all groups, including the control, it has to be noted that the mean sperm counts in the cauda epididymidis were below the test facility’s historical control data.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

MALE REPRODUCTION DATA:
- Cohabitation was confirmed for all F1 parental males, which were paired with females to generate F2 pups. Thus, the male mating index was 100% in all test groups.
- Fertility was proven for most of the F1 parental males with confirmed cohabitation. One control male, one low-dose male and two high-dose males did not generate F2 pups. Thus, the male fertility indices were 92% in test group 13 (1000 mg/kg bw/day), 96% in test groups 10 and 11 (0 and 100 mg/kg bw/day) and 100% in test group 12 (300 mg/kg bw/day). These values reflected the normal range of biological variation inherent in the strain of rats used for this study. In addition, these data were within the range of the historical control data of the test facility. None of the apparently infertile males showed corroborative histopathological findings, which could explain the observed infertility.
FEMALE REPRODUCTION AND DELIVERY DATA:
- The female mating index for F2 litter was 100% in all test groups.
- The mean duration until sperm was detected (day 0 p.c.) varied between 2.3 and 2.7 days without any relation to dosing.
- All sperm-positive rats delivered pups with the following exceptions: one control female, one female of test group 11 (100 mg/kg bw/day) and two females of test group 13 (1000 mg/kg bw/day) did not become pregnant. Consequently, the fertility index was 92% for test group 13 (1000 mg/kg bw/d), 96% for test groups 10 and 11 (0 and 100 mg/kg bw/d) and 100% for test group 12 (300 mg/kg bw/d). These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
- The mean duration of gestation was comparable in all test groups (10-13) and varied between 21.8
and 22.1.
- The gestation index was 100% for all test groups, indicating that all pregnant F1 females had live F2 pups in their litters.
- Implantation was clearly affected by the treatment, since the number of implantation sites was statistically significantly reduced in test group 13 (8.8** [p≤0.01] versus 11.3 implants/dam in control). There were no statistically significant differences in the number of implantation sites between test groups 11 and 12 (11.6 and 11.8 implants/dam, respectively) and the control. Furthermore, there were indications for test substance-induced intrauterine embryo-/fetolethality, since the postimplantation
loss was statistically significantly increased in the high-dose group (12.8* [p≤0.05] – 1000 mg/kg bw/day). In addition, the postimplantation loss in this test group was higher than documented in the historical control data. There were no statistically significantly differences concerning the postimplantation loss between the remaining test groups and the control (4.9%, 3.0%, and 4.0% for 0, 100, and 300 mg/kg bw/day, respectively).
- The average litter size (F2 pups per dam) was very similar between test groups 10, 11 and 12. A statistically significantly lowered number of F2 pups per dam were delivered in test group 13 (7.7**pups/dam [p≤0.01] versus 10.8, 11.2, and 11.4 in test groups 10, 11 and 12, respectively). The number of liveborn and stillborn pups was comparable between all groups, and the live birth index varied between 99% and 100%.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One low-dose pup showed a kinked tail on lactation day 21, which was confirmed by skeletal examination (misshapen caudal vertebra, cartilage changed). For one high-dose pup microphthalmia of the left eye was recorded on lactation day 21 and was confirmed by visceral examination. These indivisual finding were assumed to be incidental and can also be found in the historical control data.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

- The mean number of F1 pups/dam was statistically significantly reduced in test group 03 (6.8** pups/dam [p≤0.01]). However, the number of stillborn F1 pups was comparable between the test group 00, 01, 02, and 03 (2, 1, 2, and 2 pups/test group, respectively). Every stillborn pup belonged to a different litter.
- The viability index as indicator for pup mortality between days 0-4 p.p. was unaffected and varied between 99% (control group as well as test groups 01 and 03) and 100% (test group 02). However, cannibalized pups were found in test group 01 (3 pups), 02 (1 pup) and 03 (3 pups* [p≤0.05]). In this case, the calculated statistically significant difference for the highdose group was a consequence of the smaller litter sizes there. No pup was cannibalized in the control group.
- The lactation index, indicating pup mortality between days 4-21 p.p., was slightly, but statistically significantly lower in test group 01 (97%* [p≤0.05]). This value is, however, within the historical control range of the test facility. Thus, pup mortality in the low dose group during the lactation period was not considered to be associated to test substance-treatment. For the other test groups, the lactation index was 98% (test group 03) and 100% (test groups 00 and 02).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of F1 male and female pups in test group 03 (1000 mg/kg bw/day) were statistically significantly increased during the complete lactation period, being marginally outside the range of historical control data. Body weight gain of these animals was statistically significantly increased between lactation days 1-4 (about 19%). Afterwards the weight gain of the high-dose animals was similar to the concurrent control group. The higher pup body weights in test group 03 were rather re
garded as a consequence of the reduced number of pups/litter than a direct test substance-related effect.
No test compound-related influence on F1 pup body weights was noted in the low- and middose groups (100 and 300 mg/kg bw/day).

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:

no effects observed

Description (incidence and severity):

- The sex distribution and sex ratios of live F1 pups on the day of birth and on day 21 p.p. did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
- Vaginal opening: Each female F1 pup, which was selected to become a F1 parental animal, was evaluated for commencement of sexual maturity. The first day, when vaginal opening was observed, was day 27 p.p., the last was day 37 p.p. The mean number of days to reach the criterion in the test groups 00-03 amounted to 32.4, 31.8, 31.8, and 32.3 days (0, 100, 300 and 1000 mg/kg bw/day, respectively), indicating that female sexual maturation was not influenced by the test substance at any dose level. The mean body weight on the day, when vaginal opening was noted, amounted to 95.1, 94.1, 95.0, and 97.6 grams in test groups 00-03
- Preputial separation: Each male F1 pups, which was selected to become a F1 parental animal, was evaluated for commencement of sexual maturity. The first day, when preputial separation was observed, was day 40 p.p., the last was day 48 p.p. The mean number of days to reach the criterion in test groups 00-03 amounted to 42.2, 42.5, 41.9, and 42.8 (0, 100, 300 and 1000 mg/kg bw/day, respectively), indicating that the test substance did not influence male sexual maturation at any dose level.
The mean body weight on the day, when preputial separation was recorded, amounted to 172.1, 173.7, 168.9, and 176.2 grams in test groups 00-03.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- Absolute pup organ weights: Statistically significant changes in mean pup organ weights were only observed for the brain. The absolute mean pup brain weight was increased in high-dose (1000 mg/kg bw/day) F1 pups (males + females: +2.9% [p≤0.01]), especially in the males (+3.5% [p≤0.01]). All other mean absolute pup organ weights of the F1 pups did not show statistically significant differences to the organ weights of the control animals. The statistically significantly increased absolute brain weights of the high-dose F1 pups (1000 mg/kg bw/d) were assessed as secondary to the higher pup body weights in this group as proven by calculated the mean relative pup organ weights. The finding was neither adverse nor toxicologically relevant.
- Relative pup organ weights: Mean relative pup organ weights of the F1 pups did not show statistically significant differences to the control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At gross necropsy, a number of common findings were seen in F1 pups, such as partly cannibalized pups, post mortem autolysis, incisors sloped, microphthalmia, hemorrhagic thymus, abnormal liver lobation, empty stomach and kinked tail.
If all findings were considered together, the litter incidence and number of affected pups/litter were statistically significantly increased in test group 01 (100 mg/kg bw/day) and test group 03 (1000 mg/kg bw/day). However, each individual finding was observed only in single animals and/or can be found in the historical control data at comparable or even higher incidences. There was no evidence of a particular pattern, which might be related to a possible mode of action of the test compound. The low- (100 mg/kg bw/day) and high-dose (1000 mg/kg bw/day) litter incidences (12% and 17%, respectively) and rates of affected pups/litter with necropsy findings (1.2% and 2.7%, respectively) were clearly within the historical control data of the test facility (litter incidence: 4–52%; affected pups/litter: 0.5– 15.2%).
Thus, these findings were considered as incidental and not related to treatment.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

The F2 generation pups did not show any clinical signs up to weaning.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The viability index as indicator for pup mortality between days 0-4 p.p. was 100% in all test groups. The lactation index as indicator for pup - The mean number of F2 pups/dam was statistically significantly reduced in test group 13 (1000 mg/kg bw/day). However, a comparison of the total amount of liveborn and stillborn F2 pups per test group did not reveal statistically significant differences between test groups 10-13.
- The viability index as indicator for pup mortality between days 0-4 p.p. was 100% in all test groups.
- The lactation index as indicator for pup mortality between days 4-21 p.p. was not affected by test substance-treatment as it reached 100%, 100%, 99%, and 100% at dose levels of 0, 100, 300, and 1000 mg/kg bw/day. Any isolated pup deaths were assessed as incidental.mortality between days 4-21 p.p. was not affected by test substance-treatment as it reached 100%, 100%, 99%, and 100% at dose levels of 0, 100, 300, and 1000 mg/kg bw/day. Any isolated pup deaths were assessed as incidental.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights of F2 male and female pups in test groups 11-13 (100, 300 and 1000 mg/kg bw/day) were generally comparable to the concurrent control group throughout the entire lactation period. The statistically significantly increased body weights of female pups and the statistically significantly increased body weight value for both sexes in test group 13 on lactation day 1 were related to the decreased litter sizes and, given that, regarded to be secondary effects.

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:

no effects observed

Description (incidence and severity):

The sex distribution and sex ratios of live F2 pups on the day of birth and on day 21 p.p. did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment-related pup organ weight changes concerning absolute and relative brain, thymus and spleen weights were seen in the F2 pups. All differences observed reflected the normal biological variation in this strain of rats.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

For a number of F2 pups, findings were detected at gross necropsy such as hemorrhagic thymus, small liver, empty stomach, dilated renal pelvis, small kidney and cystic dilatation of ovary. All pup necropsy findings occurred without relation to dosing. Furthermore, equal or similar findings can be found in the historical control data at comparable or even higher incidences. The number of affected pups per litter showing hemorrhagic thymus was statistically significantly increased in test group
13 (1000 mg/kg bw/day). However, this calculated incidence of 5.7% was clearly within the range of historical control data.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

no

Test substance stability:

The stability of test substance in rat diet was demonstrated for a period of 32 days at room temperature in a different batch of comparable quality, which was not used for the study. The homogeneity of the mixtures was verified. The concentration control analyses of the samples taken revealed that the values were within a range of 90-110% of the nominal concentration in all analyses at all time points, with the exception of one concentration in the feed of the high-dose group (88%).

Plasma concentrations of 2 -aminoethanol were below 3 mg/kg for all control animals, <3 - 4 mg/kg for the low dose animals, 8 - 11 mg/kg for the mid dose animals and 60 – 81 mg/kg for the high dose animals.

Toxicokinetic data of 2 -aminoethanol (calculated as 2 -aminoethanol hydrochloride) from this two-generation reproduction toxicity study show a dose dependency of the plasma levels of 2 -aminoethanol in the experimental animals and there with prove the bioavailability of 2 -aminoethanol hydrochloride in principle.

 

Under these conditions, no test substance-related findings from clinical examinations or gross and histopathology were observed, which indicate that the administration of the test compound via the diet adversely affected the fertility or reproductive performance of the F0 or F1 parental animals up to and including a nominal dose of 300 mg/kg bw/day. Estrous cycle data, mating behavior, conception, gestation, parturition, lactation and weaning as well as sperm parameters, sexual organ weights and gross and histopathological findings of these organs (including differential ovarian follicle counts in the F1 females) were comparable between the rats of all test groups.

At the high-dose level (1000 mg/kg bw/day), absolute and relative weights of epididymides and cauda epididymidis were decreased and, in the F0 generation only, the number of homogenization resistant caudal epididymal sperm was slightly, but significantly reduced. However, histomorphological correlates for these findings were missing.

 

In the high-dose F0 and F1 generation females (1000 mg/kg bw/day), decreased numbers of implants and increased resorption rates resulted in significantly smaller litters, giving evidence for an adverse effect of the test compound on fertility and/or reproductive performance at high doses. It has to be noted that a dose of 1000 mg/kg bw/day also caused beginning systemic toxicity in these females, as was indicated by reduced food consumption and/or body weight gain during gestation/lactation.

 

All data recorded during gestation and lactation in terms of embryo-/fetal and pup development gave no indications for any developmental toxicity in the F1 and F2 offspring up to a dose level of 1000 mg/kg bw/day. The test substance did not adversely influence pup viability, body weight, sex ratio and sexual maturation.

 

Thus, under the conditions of the present two-generation reproduction toxicity study, the NOAEL (no observed adverse effect level) for fertility, reproductive performance and systemic toxicity in parental F0 and F1 Wistar rats is 300 mg/kg bw/day.

 

The NOAEL for pre-and postnatal developmental toxicity in their offspring is 1000 mg/kg bw/day.

Tables

Mean test substance intake (mg/kg bw/d; minimum value / maximum value)

	Test group 01 (100 mg/kg bw/day)	Test group 02 (300 mg/kg bw/day)	Test group 03 (1000 mg/kg bw/day)
F0 males	94.3 (72.4 / 102.5)	283.2 (218.4 / 309.4)	943.3 (716.7 / 1032.6)
F0 females (premating)	96.7 (80.5 / 100.7)	289.6 (241.2 / 304.9)	964.4 (792.4 / 1017.8)
F0 females (F1 litter) - gestation period - lactation period*	103.5 (92.6 / 111.6) 99.2 (81.6 / 120.2)	315.2 (284.8 / 337.9) 306.7 (249.7 / 370.3)	1043.2 (989.4 / 1084.7) 866.0 (668.6 / 1053.9)

* = Days 1–14 p.p. only

Absolute organ weights (P-generation)

Compared to the controls (= 100%), the following values (in %) were significantly changed (printed in bold):

	Male animals			Female animals
Group	01 100 mg/kg bw/day	02 300 mg/kg bw/day	03 1000 mg/kg bw/day	01 100 mg/kg bw/day	02 300 mg/kg bw/day	03 1000 mg/kg bw/day
Brain	99%	100%	97%*
Cauda epididymis	99%	102%	88%**
Epididymides	100%	101%	92%**
Prostate	92%	99%	86%**
Spleen				105%*	107%	97%

 *: p≤0.05; **: p≤0.01

All other mean absolute weight parameters did not show significant differences compared to the control groups.

The decrease of absolute weights of cauda epididymis, epididymides, and prostate in male top-dose animals (1000 mg/kg bw/d) were considered as treatment-related effects.

The decrease of brain weights in top-dose males (1000 mg/kg bw/day) as well as the increase of spleen weights in low-dose females (100 mg/kg bw/d) was considered as incidental and not treatment-related due to a missing dose-response relationship.

Absolute organ weights (F1 generation)

Compared to the controls (= 100%), the following values (in %)were significantly changed (printed in bold):

 

	Male animals			Female animals
Group	11 100 mg/kg bw/day	12 300 mg/kg bw/day	13 1000 mg/kg bw day	11 100 mg/kg bw/day	12 300 mg/kg bw/day	13 1000 mg/kg bw/day
Cauda epididymis	96%	99%	88%**
Epididymides	100%	101%	91%**
Kidneys	99%	106%*	111%**	103%	106%**	115%**
Spleen	99%	103%	92%*
Thyroid glands	106%	99%	109%*	110%	118%**	111%*

 *: p≤0.05; **: p≤0.01

All other mean absolute weight parameters did not show significant differences compared to the control groups.

The decrease of absolute weights of cauda epididymis and epididymides in male top-dose animals (1000 mg/kg bw/d) were considered to be treatment-related.

The increase of absolute kidney weights of male and female animals in mid- (300 mg/kg bw/d) and top-dose (1000 mg/kg bw/d) groups, respectively, was statistically significant. Because no histomorphological correlate was detected, a treatment-related weight increase was less likely. The decrease of spleen weights in top-dose males as well as the increase of thyroid glands in top-dose males and mid- and top-dose females, respectively, is considered incidental and not treatment-related due to a missingdose-response relationship.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD TG 421

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a reproduction/developmental toxicity screening study with TEA, performed according to OECD guideline 421, Wistar rats (10/sex/dose) were exposed by gavage to 0, 100, 300 or 1000 mg/kg bw/day during a premating period of 2 weeks and a mating period (max. 2 weeks) for both sexes, during approximately 1 week post-mating for males, and during the entire gestation period as well as 4 days of lactation for females.Food consumption, body weight, clinical signs, mating and reproductive performance (including determinations of the number of implantations and the calculation of the postimplantation loss in females) were examined in parental animals. At necropsy, animals were assessed for gross pathology and selected organs were weighed and examined histopathologically. In pups, bodyweight, viability and macroscopic changes were recorded. At necropsy on PND 4, all pups were examined macroscopically for external and visceral findings.At the high dose of 1000 mg/kg bw/day, a decreased number of implantation sites, increased postimplantation loss and a lower average litter size were observed. No adverse effects were observed regarding reproductive performance, fertility or systemic toxicity at any dose level. Thus, the NOAEL for systemic toxicity as well as for reproductive performance and fertility in parental animals was established at 1000 mg/kg bw/day, the NOAEL for postnatal toxicity in the offspring was 1000 mg/kg bw/day, and the NOAEL for prenatal developmental toxicity was determined to be 300 mg/kg bw/day (BASF SE, 2010).

For the structural analogue MEA-HCl (CAS 2002-24-6) a two generation reproduction toxicity study in Wistar rats with dietary administration demonstrated clear NOAELs for systemic and reproductive toxicity including fertility at 300 mg MEA-HCl/kg bw/day. Only at the highest dose, 1000 mg/kg bw/day, were minor effects noted. Males at this high dose levels showed minor effects on fertility in the form of decreased absolute and relative weights of epididymides and cauda. However, there was no histomorphological correlate of these findings in the organs, no effect upon testes or testicular sperm count, and no effect upon mating performance. Females at this dose level revealed decreased numbers of implants and increased resorption rates resulting in smaller litters associated with indications of systemic toxicity. There was virtually no effect on the pre- and postnatal development of the progeny in both generations up to the limit dose level of 1000 mg/kg bw/ day representing a clear NOAEL for developmental toxicity (ACC and CEFIC, 2010).

Effects on developmental toxicity

Description of key information

In an oral screening reproduction/developmental toxicity study (OECD 421) with TEA in rats, the NOAEL for systemic toxicity and postnatal toxicity in the offspring was 1000 mg/kg bw/day, whereas the NOAEL for prenatal developmental toxicity was determined to be 300 mg/kg bw/day based on decreased numbers of implants and delivered pups, and an increased postimplantation loss.

In an oral Chernoff-Kavlok teratogenicity screening test, TEA did not produce any evidence of developmental or maternal toxicity in CD-1 mice. Therefore, the NOAEL for maternal toxicity and developmental toxicity was established at 1125 mg/kg bw/day.

As no developmental toxicity study (OECD guideline 414) is available for TEA, read across with the structural analogue MEA, for which developmental toxicity studies are available, is applied. Based on the results of the screening studies with TEA (oral route, rats and mice) and the available developmental toxicity studies with rats and rabbits (oral and dermal route of exposure) with MEA, TEA is not considered to be a developmental toxicant.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: 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:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 000STD77L0

Species:

rat

Strain:

Wistar

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 gThe 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).

Route of administration:

oral: gavage

Vehicle:

water

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 preparationsThe 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:

yes

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:

Daily

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Maternal 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. 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 deliveryThe 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/mortalityIn 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 ratioOn 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 observationsThe 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 weightThe 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 examinationsAll 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.

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

Details on maternal toxic effects:

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 effect
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.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

> 1 000 mg/kg bw/day

Basis for effect level:

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

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance and fertility

Effect level:

> 1 000 mg/kg bw/day

Basis for effect level:

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

Key result

Abnormalities:

no effects observed

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

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: decreased numbers of implants and delivered pups as well as an increased postimplantation loss.

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

no

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 91/351

Species:

rat

Strain:

not specified

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

VEHICLE
- Concentration in vehicle: 0, 500, 1500, 3000, 5000 mg/100 mL
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Recharacterization was carried out after the end of all studies

Duration of treatment / exposure:

day 6-15 of gestation

Frequency of treatment:

daily

Duration of test:

Until gestation day 16

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 females

Control animals:

yes

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes:
- Soft tissue examinations: Yes:
- Skeletal examinations: Yes:
- Head examinations: Yes:

Statistics:

ANOVA + Dunnett's test (two-sided; * for p ≤ 0.05, ** for p ≤ 0.01)

Clinical signs:

no effects observed

Description (incidence and severity):

During the whole study period the animals showed no abnormal clinical signs or findings .

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no mortalities.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of the high dose animals were statistically significantly lower on days 10, 13 and 15 p .c . when compared with the control group . Furthermore body weight gain of the 500 mg/kg rats was clearly reduced on days 6 - 15 p .c . During this period the relevant animals gained about 25 % less weight than the controls .

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption of the high dose females (500 mg/kg body weight/day) was statistically significantly reduced during days 8-15 p.c. The reduced food intake of the 300 mg/kg females on days 6-8 p.c. might be a spontaneous effect due to the missing dose-response relationship.

The results of the corrected body weight gain (terminal body weight on day 15 p .c . minus weight of the unopened uterus minus body weight on day 6 p.c.) was clearly reduced (to about 65% of the actual control value) in the 500 mg/kg group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Total protein and albumin values were statistically significantly reduced in the highest dose group (500 mg/kg body weight/day), which has to be related to the substance-induced impairment of the body weight gain of the dams

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The weights of the gravid uteri were not clearly influenced by the administration of the test substance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

According to the pathology report besides the reduced mean body weights of the high dose dams, the only necropsy finding which was assessed as substance -related was a thickening of the wall of the forestomach in 3 high dose animals.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no substance-related and/or statistically significant differences between the groups in the values calculated for the pre- and post-implantation losses.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no substance-related and/or statistically significant differences between the groups in the number of resorptions and viable fetuses.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate varied between 90 % (high dose group) and 100 % (all other groups). There were no substance-related and/or statistically significant differences between the groups in conception rate.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

not specified

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No substance-related effects were observed.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The external examination of the fetuses, which was limited due to the stage of development of the fetuses at this part of the gestation period, revealed no malformation and variation in any test group. Only one so-called unclassified observation (placentae fused) was recorded for one fetus of test group 3 (300 mg/kg body weight/day).

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Weight of placentae
No sub stance-related effects were observed. The marginal, however statistically significant decrease in the mean placental weights in test group 3 (300 mg/kg body weight/day) is without any biological relevance.

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sample 25 (91/351)

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sexually mature, virgin Wistar rats (Chbb :THOM (SPF)) supplied by Karl THOMAE, Biberach an der Riss, Germany,
- Age at study initiation: 60 days
- Weight at study initiation: mean weight approx. 223.7 g
- Fasting period before study: none
- Housing: singly in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm²)
- Diet: ground Kliba 343 feed rat/mouse/hamster supplied by Klingentalermuehle AG, Kaiseraugst, Switzerland ad libitum
- Water: tap water ad libitum
- Acclimation period: 5 days

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

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Each day the test substance solutions were freshly prepared shortly before the test substance was administered. For the preparation of the solutions, an appropriate amount of the test substance was weighed in a volumetric flask and subsequently topped up with doubly distilled water and intensively shaken.

VEHICLE
- Amount of vehicle (if gavage): 10 mL kg bw.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity of the test substance was proven by visual inspection. The content of active ingredient was 99 .4% before the beginning of the study. The reanalysis of the test substance proved its stability (content: 99 .5%)

Details on mating procedure:

- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: [yes]
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy

Duration of treatment / exposure:

day 6 - 15 of gestation

Frequency of treatment:

daily, once per day

Duration of test:

up to day 21 of gestation

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

40 dams per dosing group

Control animals:

yes, concurrent vehicle

Details on study design:

On day 0, the animals were assigned to the different test groups according to a randomization plan. The test substance was administered to the animals orally (by gavage) once a day during the period of major organogenesis (day 6 to day 15 p .c .) always at approx. the same time of day (in the morning). The animals of the control group were treated in the same way with the vehicle (doubly distilled water). The volume administered each day was 10 mL/kg body weight. The calculation of the volume administered was based on the individual body weight determined at the beginning of the administration period (day 6 p.c.). On day 20 p.c., the first 25 animals/group were sacrificed in a randomized order and examined macroscopically. The fetuses were dissected from the uterus and further investigated with different methods. The other animals (15/group) were allowed to litter and rear their pups up to day 21 p.p. (post partum). On day 21 post partum (p.p.) or one of the following days the relevant dams and pups were sacrificed and examined macroscopically.

Maternal examinations:

Clinical examinations
Food consumption
With the exception of day 0 p.c. (all animals) and days 0 p.p. and 21 p.p. (for animals with terminal sacrifice on day 21 p.p. only), the consumption of food was determined on the same days as was body weight. Food consumption was not determined for the females without litter during the lactation period of the dams used in parallel.

Body weight data
All animals were weighed on days 0, 1, 3, 6, 8, 10, 13, 15, 17 and 20 p.c. Body weights of the animals with terminal sacrifice on day 21 p.p. were additionally determined on the day of birth and on days 4, 7, 14 and 21 p.p. The body weight change of the animals was calculated from these results. Body weights of the animals without litter were not determined during the lactation period of the dams used in parallel.

Corrected body weight gain (net maternal body weight change)
Furthermore, the corrected body weight gain was calculated for all animals with terminal sacrifice on day 20 p.c. (terminal body weight on day 20 p.c. minus weight of the uterus before it was opened minus body weight on day 6 p.c.).

Clinical symptoms
All animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited. The nesting, littering, and lactation behavior of the dams with terminal sacrifice day 21 p.p. was generally evaluated in the mornings in connection with the daily clinical inspection of the dams. Only if there were any special findings (e.g., animal could not litter, umbilical cord not cut), these specific findings were documented with the dam concerned.

The littering behavior of the relevant dams was also inspected on weekdays (except holidays) in the afternoons in addition to the evaluations in the mornings. These reevaluation were documented separately, but, as before, findings were only recorded with the dams concerned. Moreover, the duration of gestation, the number of live and dead pups at birth and litter size were recorded for the animals with terminal sacrifice on day 21 p.p. For these animals the fertility and the gestation indices were calculated according to the following formulae :

fertility index = (n pregnant animals/ n mated animals) x 100

gestation index = (n animals with litters/ n pregnant animals) x 100

The values listed in the Summary Tables are group means determined from the fertility/gestation indices of the individual animals.

Mortality
A check was made twice a day on working days or once a day (Saturday, Sunday or on public holidays).

Ovaries and uterine content:

Examinations of the dams at termination
Dams with terminal sacrifice on day 20 p.c.
On day 20 p.c., the dams were sacrificed in randomized order by cervical dislocation and the fetuses dissected from the uterus. After the dams had been sacrificed , they were necropsied and assessed by gross pathology. The uterus and the ovaries were removed and the following data were recorded :

Weight of uterus before it was opened
- Number of corpora lutea
- Number and distribution of implantation sites classified as :
• live fetuses
• dead implantations:
a) early resorptions (only decidual or placental tissues visible or according to Salewski from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy)
b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

Furthermore, calculations of conception rate and pre- and postimplantation losses were carried out:
- The conception rate (in %) was calculated according to the following formula :

conception rate = (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 post implantation loss (in % ) was calculated from the following formula:

((number of implantations - number of live fetuses)/number of implantations) x 100

Dams with terminal sacrifice on day 21 p.p.
On day 21 post partum (p. p. ) the relevant dams were sacrificed by cervical dislocation. After the dams had been sacrificed, the following examinations were carried out:
- gross - pathological examination
- staining of uterus according to Salewski for determination of the number of implantations

Furthermore, calculations of conception rate and postimplantation loss were carried out:
- The conception rate (in %) was calculated according to the following formula :

conception rate = (number of pregnant animals/ number of fertilized animals) x 100

- The post implantation loss (in % ) was calculated from the following formula:

((number of implantations - number of live fetuses)/number of implantations) x 100

Fetal examinations:

Examination of the fetuses:
Examination of the fetuses after dissection from the uterus
At necropsy each fetus was weighed, sexed 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. Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes and fluids were examined. Individual placental weights were recorded. After these examinations, approximately one half of the fetuses per dam was placed in ethyl alcohol and the other half was placed in Bouin's solution for fixation and further evaluation.

Soft tissue examination of the fetuses
After fixation in Bouin's solution, approximately one half of the fetuses of the dams of all groups was examined for any findings in the organs according to the method of Barrow and Taylor with special attention being paid to the kidneys and the ureters. After the examination, these fetuses were discarded with the exception of the kidneys, which were placed into cassettes separately for each fetus and kept in 4% formaldehyde solution for possible further examination by light microscopy. Moreover, after fixation of the fetuses placed in ethyl alcohol for further evaluation of the fetal skeletons the organs of these fetuses
were examined macroscopically. Thereafter, the kidneys of each fetus were placed into cassettes and kept in 4% formaldehyde solution for a possible further examination by light microscopy, while the other organs were discarded. Afterwards the carcasses of these fetuses were stained according to a modified method (Dawson) for the presentation of the skeletons.

Skeletal examination of the fetuses
After fixation in ethyl alcohol and examination of the organs, the skeletons of the fetuses were stained according to a modified method of Dawson. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After these examinations the relevant fetuses were retained by litter.

Evaluation criteria for assessing skeletons and organs of the fetuses
In the present investigations the following terms (definitions) were used for describing a change:
- Malformations (concerning external, soft tissue and skeletal observations)
Rare and/or probably lethal changes were classified as malformations (e.g. exencephaly, atresia ani, hernia umbilicalis).

- Variations (concerning external, soft tissue and skeletal observations)
Changes which occur regularly also in control groups and have generally no adverse effect on survival were regarded as variations (e.g. dilated renal pelvis).

- Retardations (concerning skeletal observations only)
Delays in skeletal development compared with the norm at the time of the examination were considered to be retardations (e.g. sternebra(e) not ossified)

- Unclassified observations (concerning external and soft tissue observations, only)
External or soft tissue observations, which could not be classified as malformations or variations (e.g. blood coagulum around placenta).

Examination of the pubs:
Pup number and status at delivery
All pups derived from the females 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.

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. Dead pups were evaluated by the methods which will be described in detail before.
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 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. Furthermore, viability and lactation indices were calculated according to the following formulas :

Viability index (%) = (number of live pups on day 4 after birth/ number o f liveborn pups on the day of birth)
x 100

Lactation index (%) = (number of live pups on day 21 after birth/ number of live pup s on day 4 after birth)
x 100

Sex ratio
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. During the following time 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 sex ratio was calculated for day 0 and day 21 after birth according to the following formula:

sex ratio = (number of live male or female pups on day 0/21 / number of live male and female pups on day 0/21) x 100

Pup body weight data
The pups were weighed on the day after birth (day 1 p.p.) and on days 4, 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). In the relevant summary tables pup body weights and pup body weight gains are listed for males, females and males + females.

Pup clinical observations
The pups were examined each day for clinical symptoms (including gross-morphological findings).

Pup necropsy observations
After sacrifice on day 21 p.p. or one of the following days (by means of CO2) or intercurrent death, the pups were examined externally, eviscerated and their organs were assessed macroscopically with special attention being paid to the urinary tract. After the macroscopic examination of the pups, the kidneys of each pup were placed into cassettes and fixed in 4% formaldehyde solution for a possible further examination by light microscopy. If there were notable findings or if abnormalities were found in the daily clinical observation of the animals after their delivery, the affected animals were, if it was deemed necessary, examined additionally using appropriate methods (e.g., skeletal staining according to modified Dawson's method and/or further processing of head according to Wilson's method. The stained skeletons were evaluated under a stereomicroscope or a magnifying glass. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation (with the exception of the kidneys (see above)).

Statistics:

Dunnett-Test was used for a simultaneous comparison of several dose groups with the control. The hypothesis of equal means was tested. This test was performed two-sided and was used for the statistical evaluation of food consumption, body weights and body weight change (females and pups), corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, number of corpora lutea, number of implantations, number of resorptions and number of live fetuses; proportion of preimplantation loss, postimplantation loss, resorptions and live fetuses in each litter; litter mean fetal body weight and litter mean placental weight, duration of gestation and number of pups delivered per litter. For the body weight and the body weight change of the pups the mean weight of each litter was used for the statistical analysis (statistical unit = litter).
Fisher' s Exact Test was used for a pairwise comparison of each dose group with the control for the hypothesis of equal proportions. This test was performed one - sided was used for statistical evaluation of the following parameters: female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings, female fertility index, gestation index, females with liveborn, stillborn and with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy.
The Wilcoxon Test was used for a comparison of each dose group with the control for the hypothesis of equal medians. This test was performed one-sided and was used for the proportion of fetuses with malformations, variations, retardations and/or unclassified observations in each litter and for the proportion of affected pups per litter with necropsy observations. If the results of these tests were significant, labels (*for ≤ 5 0.05, ** for p ≤ 0.01) were printed in the summary tables.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No signs that might be attributed to the test substance administered were detected during gestation and lactation periods. During gestation, piloerection was recorded for one high dose animal on day 13. Without any dose-response relationship insufficient nesting activity was observed for several dams of all groups. During lactation one dam of the 40 mg/kg/day group was found dead on day 0 p.p. after an incomplete delivery. Moreover one dam of the 120 mg/kg/day group had a total litter loss on day 1 after birth. All of these findings are spontaneous in nature and cannot be attributed to the test substance administration.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One dam of the 40 mg/kg/day group died intercurrently during delivery. Undelivered pups were found in the uterus.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean maternal body weight of the 450 mg/kg/day group was statistically significantly lower than that of the control group on days 15, 17, and 20 of gestation. The high dose females gained statistically significantly less weight than the controls during the treatment-free interval of the gestation period (days 15-20) and on days 0, 4, 7, and 21 p.p. The results of the corrected body weight gain on gestation day 20 of all groups did not show any differences of biological significance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption of the high dose animals (450 mg/kg/day) was statistically decreased within the first days of the treatment period (days 6-8 of gestation) and also after termination of the treatment on the last days of the gestation period (17-20 of gestation). During the beginning of the lactation period (days 0-4 p.p.) there was also a slight, but statistically significant reduction in the food consumption of the high dose animals.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The uterus weights, which were determined for the animals with termination on day 20 of gestation only, were not influenced by the administration of the test substance. The differences between the groups is without biological relevance and do not show any dose-response relationship.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no substance-related observations at necropsy in any of the dams. Hydrometra (a spontaneous finding) was recorded for one female of the control group, for 2 females of the 40 mg/kg/day group, and 3 females of the 120 mg/kg/day group. These animals did not become pregnant. Edema of the lungs which has to be related to the termination of the rats was recorded for several dams of the control, low and intermediate groups without any relation to dosing. For the one low dose female that died intercurrently during parturition, undelivered pups were found in the uterus.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

No fetuses were aborted or delivered early in any of the groups.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean % pre-implantation loss was 3.4, 6.8, 9.9 and 11.7 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. The mean % post-implantation loss for the 20 gestational females was 10.3, 7.3, 7.0 and 6.3 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. The mean % post-implantation loss for the 21 p.p. females was 5.7, 3.9, 9.7 and 7.3 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. In the animals euthanized on day 20 of gestation, there were no-substance related and/or statistically significant differences in the values calculated for the pre- and postimplantation losses. The females euthanized on day 21 p.p. showed no substance-associated effects on the postimplantation loss.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

Mean early resorptions were 1.4, 0.9, 1.0 and 0.9 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. Mean late resorptions were 0.2, 0.3, 0.1 and 0.0 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. In the animals euthanized on day 20 of gestation, there were no-substance related and/or statistically significant differences in the number of resorptions.

Dead fetuses:

no effects observed

Description (incidence and severity):

No cases of dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened) were reported in all groups.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

The mean duration of gestation for the 21 postpartum (p.p.) females was 21.8 days, 21.6 days, 21.6 days, and 21.4 days for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively. In the females euthanized on day 21 p.p., the duration of gestation and the gestation index were substantially similar in all groups.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate varied between 85% (450 mg/kg/day group) and 75% (40 mg/kg/day group). The conception per dose level was 33 in the control group, 30 in the 40 mg/kg/day group, 33 in the 120 mg/kg/day group and 34 in the 450 mg/kg/day group. The number pregnant at caesarian-section was 21 in the control, 20 in the 40 and 120 mg/kg/day groups and 24 in the 450 mg/kg/day group. The females euthanized on day 21 p.p. showed no substance-associated effects on the fertility index which was 80%, 67%, 87% and 67% for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively.

Key result

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

not specified

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal body weights were not influenced by the test substance administration. The mean body weight of viable fetuses was 3.9 grams for all groups.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Of the females euthanized on day 21 p.p. the number of females with liveborn was 12, 10, 13, and 10 and the number of pups delivered was 165, 132, 170, and 125 for the control, the 40 mg/kg/day, 120 mg/kg/day and the 450 mg/kg/day groups, respectively.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in the test groups was comparable with the control fetuses. Sex ratios (M/F in %) on day 0 were:

control: 54.6/45.4
40 mg/kg/day: 55.7/44.3
120 mg/kg/day: 46.1/53.9
450 mg/kg/day: 53.6/46.4

Sex ratios (M/F in %) on day 21 were:
control: 54.4/45.6
40 mg/kg/day: 54.0/46.0
120 mg/kg/day: 45.2/54.8
450 mg/kg/day: 54.2/45.8

The sex distribution and sex ratios of live pups on the day of birth and on day 21 p.p. did not show any substantial difference between controls and treated test groups.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The litter size was not influenced by the test substance administration.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

The mean number of delivered pups/dam was not influenced by the administration of test substance. There were no substantial biological relevant differences concerning pup viability/ mortality in any of the groups. Viability and lactation indices were unaffected.

External malformations:

no effects observed

Description (incidence and severity):

The only external malformation which was found was an anasarca in one high-dose fetus. This malformation is also present at a low incidence in the historical control data. The external examination of the fetuses revealed no variations in any group. One unclassified observation, fused placentae, was recorded in one fetus of the 40 mg/kg/day group and one fetus of the 450 mg/kg/day group. In all groups, including the control, some soft tissue malformations were found. These malformations were related to the eyes (microphthalmia), the heart (dilatation of the right or both ventricles; dextrocardia), the lung (uni-lobular) or the kthidneys (hyper-/hypoplasia) and did not show any relation to dose. Two soft tissue variations, which were related to the urinary tract (dilated renal pelvis; hydroureter) occurred in all groups without any dose-response relationship and were fully within the historical control range. One unclassified observation (bloody inhibition of the kidneys) was recorded for 3 control and one high dose fetus.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Only a few pups showed findings at necropsy. Post mortem autolysis, incisor sloped and dilated renal pelvis (1 high-dose pup) occurred in single pups of the control, the 40 and 450 mg/kg/day groups. The only skeletal malformations which occurred were related to the thoracic part of the vertebral column (thoracic vertebral body/bodies dumbbell-shaped (asymmetrical) or bipartite (asymmetrical)). One or both of these malformations were found in a few fetuses of each test group including the controls without any biological relevant differences. The variations elicited were related to the ribs (shortened 13th rib(s), accessory 14th rib(s), rudimentary cervical rib(s), and the sternum (sternebra(e) of irregular shape or bipartite). These variations had no clear dose-response relationship, can be found in a similar frequency in historical control data, and/or the differences between groups are without biological significance. In all groups signs of retardations (incomplete or missing ossification of vertebral bodies/arches and the sternebra(e)) were found. The differences between the groups, however, are not associated with the test substance administration. All of the skeletal retardations are to be found at a comparable frequency in the historical control data and most often a clear dose-response relationship is not present. The only statistically significant difference, an increased rate of total variations in the 120 mg/kg/day group, is without biological relevance because it shows no dose-dependence.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 450 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Pregnant New Zealand White rabbits were exposed dermally to 0, 10, 25, and 75 mg/kg bw/day of monoethanolamine (MEA) for approximately 6 hr/day on Days 6 through 18 of gestation.

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazelton Research Products, Inc. (Denver, PA), USA
- Weight at study initiation: 3 .0-4 .0 kg)
- Fasting period before study: none
- Housing: in wire-bottom cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

dermal

Vehicle:

water

Details on exposure:

TEST SITE
- Area of exposure: shaved skin of the back
- Type of wrap if used: sterile gauze held in place by Lycra-Spandex jacket

REMOVAL OF TEST SUBSTANCE
- Washing (if done): water-dampened towel was used to wipe remaining test material off
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2 mL/kg
- Constant volume or concentration used: no

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 2/1
- Length of cohabitation: over night
- Verification of same strain and source of both sexes: [yes]
- Proof of pregnancy: [copulation] referred to as [day 0] of pregnancy

Duration of treatment / exposure:

day 6 - 18 of gestation

Frequency of treatment:

6 hours/day, daily

Duration of test:

up to day 29 of gestation

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

75 mg/kg bw/day (nominal)

No. of animals per sex per dose:

15 dams/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels selected for these studies were chosen based upon the results of dermal range-finding and teratology probe studies conducted in rabbits

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 3, 6, 9, 12, 15, 18, 24 and 21

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: weights of liver, kidneys

OTHER: Blood analysis prior to caesarian section; skin irritation was evaluated once daily during the postdosing period

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Uteri with no visible implantations were stained with a 10% sulfide solution.

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter ]
All fetuses were weighed and examined for evidence of external alterations and palate closure. All of the rabbit fetuses in each litter were examined for visceral alterations (Staples,1974). The sex of all live fetuses was determined. The heads of one half of the rabbit fetuses not selected for skeletal examination were removed, placed in Bouin's solution, and subsequently sectioned and examined for craniofacial defects (Wilson, 1 965 ; Van Julsingha and B ennet, 1977). All fetuses were eviscerated and stained with alizarin red-S ( Dawson, 1926; Crary, 1962). Skeletal examinations were conducted only on the rat fetuses not selected for Bouin's examination.

Statistics:

Continuous data were evaluated for homogeneity of variance using Levene's test (Levene, 1960). Based upon the outcome of this test, a parametric or nonparametric analysis of variance (ANOVA) was performed. If the ANOVA was significant, analysis by Dunnett's test (Steel and Torrie, 1960), the Wilcoxen Rank-Sum test with Bonferroni's correction (Miller, 1966), or a pooled t test was performed as appropriate. The level of statistical significance was set a priori at a = 0.05. Nonparametric data were compared using Fischer's exact probability test (Siegel, 1956).

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

Significant findings in the areas of intact treated skin included exfoliation, crust, necrosis, and ecchymosis in the 75 mg/kg/day group. Exfoliation, crusting, and areas of-necrosis persisted subsequent to the dosing period. However, after the treatment period, the skin of the majority of does in the 75 mg/kg/day group began to heal (as evidenced by scabbing. In the 25 mg/kg/day group, crust was noted on the treated skin of 3 does during the treatment period: There were no significant findings
in does from the 10 mg/kg/day group. Barely perceptible to well defined erythema was observed in 3 dams after 1 day of dosing in the 75 mg/kg/day group. Erythema was consistently observed in most does from the 75 mg/kg/day group after 4-5 dosing days. In the 25 mg/kg/day group, transient barely perceptible to well-defined erythema was observed during the treatment period. No significant erythema was observed for treated females in the 10 mg/kg/day groups. (Consistent but transient barely perceptible to well-defined erythema noted in 1 doe each from control and 10 mg/kg/day groups were considered to be due to the occlusion procedure.) Barely perceptible to moderate edema was observed in half of the does from the 75 mg/kg/day group after 6 dosing days. Transient, barely perceptible edema was also noted in 2 does from the 25 mg/kg/day group and 1 doe each from the control and 10 mg/kg/day groups. Subsequent to the dosing period, barely perceptible edema was observed in only one high dose female.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically identified changes were observed in body weight and body weight gain.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no consistent treatment-related effects on gestational food consumption. Statistically significant reductions in food consumption for Days 25-26 and 18-29 were not considered to be a direct effect of treatment due to the magnitude of the reductions and the fact that the reductions occurred after completion of the treatment period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on the numbers of leukocytes, erythrocytes, or platelets. Mean values for hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were equivalent across groups. Differential leukocyte evaluations also indicated no treatment-related effects.

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

Description (incidence and severity):

There were no effects of treatment on gravid uterine weight, or liver and kidney weights.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Findings listed for treated skin were consistent with clinical observations skin irritation scores for the 75 mg/kg/day group. There were no other pertinent necropsy findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed on reproductive parameters including the number of implantations and resorptions. The reduced percent preimplantation loss at 75 mg/kg/day was considered to be spurious due to the lack of a dose-response relationship.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed on reproductive parameters including the number of implantations and resorptions.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed on reproductive parameters including the number of dead fetuses.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed on reproductive parameters including pregnancy rate.

Other effects:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed on the number of corpora lutea.

Details on maternal toxic effects:

Rabbits administered 75 mg/kg/day of MEA exhibited severe skin irritation (erythema, edema, ecchymosis, necrosis, exfoliation, and crusting) at the site of exposure. Subsequent to the dosing period, exfoliation, crusting, and areas of necrosis persisted. The skin of the majority of these rabbits began to heal as evidenced by scab formation late in the gestation period. Crusting, transient erythema, and edema were noted in a few rabbits administered 25 mg MEA/kg/day. No significant dermal irritation or lesions were observed among rabbits administered 10 mg MEA/kg/day.

No females died, aborted, delivered early, or were removed from the test during the study. There were also no significant treatment-related effects observed on feed consumption, hematologic parameters, or kidney and liver weights of MEA-exposed rabbits at any dose level tested.

No statistically identified changes were observed in body weight and body weight gain, the average body weight gain of high-dose rabbits over the course of gestation was decreased when compared to that of the control and other dose groups, mainly due to weight loss or very little weight gain during the treatment period.

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

dermal irritation

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not examined

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Skeletal malformations:

effects observed, non-treatment-related

Visceral malformations:

effects observed, treatment-related

Details on embryotoxic / teratogenic effects:

No treatment-related effects were observed on reproductive parameters including pregnancy rate, number of corpora lutea, number of implantations, resorptions, litter size, number of dead fetuses, fetal sex ratio, fetal body weight, or gravid uterine weight among MEA exposed rabbits at any dose level when compared to controls.
There were no statistically or biologically significant treatment-related differences in the incidence of any fetal variation or malformation, or in the number of malformed fetuses in any dose group. Among control litters the following types of malformations were noted: ventricular septal defect, common opening at entry of the vessels of the heart, missing lung lobe, missing gallbladder, and extra lumbar centrum and arches.

Malformations observed in litters from rabbits given 10 mg/kg/day included dilated lateral cerebral ventricle with tissue depression, missing lung lobe, missing gallbladder, misaligned or fused thoracic centra, extra lumbar centrum and arches, and fused ribs. Limited numbers of malformations noted at 25 mg/kg/day included dilated lateral cerebral ventricle with tissue depression and missing lung lobe. Malformations observed in fetuses from the 75 mg/kg/day dose group litters included the following: dilated lateral cerebral ventricle with tissue depression, missing lung lobe, diagonally displaced thoracic centra, missing thoracic arch, and a single missing rib.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

> 75 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: No treatment related effects at highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Reference 5

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Pregnant Sprague-Dawley rats were exposed dermally to 0, 10, 25, 75 and 225 mg/kg/day of the test substance for approximately 6 hr/day on Days 6 through 15 of gestation.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. (Kingston, NY), USA
- Weight at study initiation: 250-300 g
- Housing: in wire-bottom cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 40-60

Route of administration:

dermal

Vehicle:

water

Details on exposure:

TEST SITE
- Area of exposure: shaved skin of the back
- Type of wrap if used: absorbant gauze pad followed by nonabsorbant cotton; an elastic bandage was wrapped securely around the animal to hold the patch in place and to prevent accidental ingestion of the test material via grooming during the exposure.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): water-dampened towel was used to wipe remaining test material off
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1 mL/kg
- Constant volume or concentration used: no

Details on mating procedure:

- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: over night
- Verification of same strain and source of both sexes: [yes]
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy

Duration of treatment / exposure:

day 6 - 15 of gestation

Frequency of treatment:

6 hours/day, daily

Duration of test:

up to day 21 of gestation

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

25 mg/kg bw/day

Dose / conc.:

75 mg/kg bw/day

Dose / conc.:

225 mg/kg bw/day

No. of animals per sex per dose:

30-45 rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels selected for these studies were chosen based upon the results of dermal range-finding and teratology probe studies conducted in rats.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 3, 6-16, and 21

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: weights of liver, kidneys

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Uteri with no visible implantations were stained with a 10% sulfide solution.

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations:Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter ]

All fetuses were weighed and examined for evidence of external alterations and palate closure. At least one-half of the rat fetuses in each litter were examined for visceral alterations (Staples,1974). The sex of all live fetuses was determined. The heads of rat fetuses not selected for skeletal examination were removed, placed in Bouin's solution, and subsequently sectioned and examined for craniofacial defects (Wilson, 1965; Van Julsingha and Bennet, 1977). All fetuses were eviscerated and stained with alizarin red-S ( Dawson, 1926; Crary, 1962). Skeletal examinations were conducted only on the rat fetuses not selected for Bouin's examination.

Statistics:

Continuous data were evaluated for homogeneity of variance using Bartlett's test (Winer, 1971). Based upon the outcome of these tests, a parametric or nonparametric analysis of variance (ANOVA) was performed. If the ANOVA was significant, analysis by Dunnett's test (Steel and Torrie, 1960), the Wilcoxen Rank-Sum test with Bonferroni's correction (Miller, 1966), or a pooled t test was performed as appropriate. The level of statistical significance was set a priori at a = 0.05. Nonparaznetric data were statistically treated using the Kruskal-Wallis test followed by the Mann-Whitney U test (Sokal and Rohlf, 1969), when appropriate. Incidence data for rats were analyzed using the Wilcoxon test as modified by Haseman and Hoel (1974). Fetal sex ratios were analyzed using a binomial distribution test (Steel and Torrie, 1960).

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

Rats administered 225 mg/kg/day exhibited a treatment-related increased incidence of skin irritation at the site of exposure. In general, the dermal irritation followed a progression, beginning with erythema and leading to necrosis, scabs, and scar formation. No significant dermal irritation or lesions were observed among rats administered lower doses of the test substance.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain of rats given 225 mg/kg/day was significantly decreased during the exposure period. No effect on weight gain was observed in dams treated with lower levels of the substance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No significant differences were observed in the feed consumption of exposed rats relative to controls.

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

no effects observed

Description (incidence and severity):

No significant differences were observed in water consumption of exposed rats relative to controls.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No effects on liver or kidney weights were observed at any dose level.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no postmortem treatment-related effects observed in any dose group.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no differences in the number of implantations

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no differences in resorptions among any of the dose groups when compared to controls.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no differences in resorptions among any of the dose groups when compared to controls.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no differences in number of dead fetuses among any of the dose groups when compared to controls.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were no differences in pregnancy rate among any of the dose groups when compared to controls.

Other effects:

no effects observed

Description (incidence and severity):

There were no differences in number of corpora lutea mong any of the dose groups when compared to controls.

Key result

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day

Basis for effect level:

body weight and weight gain

dermal irritation

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no differences in fetal body weight, or gravid uterine weight among any of the dose groups when compared to controls.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no differences in fetal sex ratio among any of the dose groups when compared to controls.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no differences in litter size among any of the dose groups when compared to controls.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see details on embryonic / teratogenic effects

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see details on embryonic / teratogenic effects

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see details on embryonic / teratogenic effects

Details on embryotoxic / teratogenic effects:

There were no treatment-related increases in the incidence of variations or malformations observed externally, viscerally, or at skeletal examination by individual category, or in total variations or malformations when compared to controls. Among controls, the following types of malformations were observed : microphthalmia, retroesophageal right subclavian artery, and an extra cervical rib . No malformed fetuses were observed in the 10 mg/kg/day dose group.
Malformations observed in the 25 mg MEA/kg/day dose group included retroesophageal right subclavian artery and an extra cervical rib. A single fetus was malformed in the 75 mg MEA/ kg/day dose group . This fetus had multiple craniofacial malformations consisting of micrognathia, cleft lip and soft palate, and aglossia. No malformations were observed in fetuses from dams administered 225 mg MEA/kg/day.

Key result

Dose descriptor:

NOAEL

Effect level:

225 mg/kg bw/day

Basis for effect level:

other: developmental toxicity- No treatment related effects observed.

Key result

Abnormalities:

effects observed, non-treatment-related

Key result

Developmental effects observed:

no

Reference 6

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: basic data given

Qualifier:

no guideline followed

Principles of method if other than guideline:

Method: Chernoff-Kavlok teratogenicity screening test

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, New York
- Age at study initiation: 6-8 weeks
- Fasting period before study: none
- Housing: individually house in polycarbonate shoebox cages
- Diet: Purina Certified Rodent Chow 115002 ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1 (72 +/-3°F)
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The daily dosing solutions were prepared and color coded by the chemistry staff at study initiation and were stored refrigerated in amber glass vials to prevent photodegradation. All dosing concentrations were verified as accurate by the chemistry staff. The animal technicians dosing the animals were not aware of the test article name and were blind to doses. Each test article was identified by the number assigned by the Radian Corp. and a corresponding color. This technique allowed for unbiased clinical observations.

VEHICLE
- Amount of vehicle (if gavage): 10 mg/kg bw

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

no data

Duration of treatment / exposure:

on days 6-15 of gestation

Frequency of treatment:

daily

Duration of test:

until post partum day 3

Dose / conc.:

10 mg/kg bw/day

Remarks:

Phase I

Dose / conc.:

100 mg/kg bw/day

Remarks:

Phase I

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Phase I

Dose / conc.:

600 mg/kg bw/day

Remarks:

Phase II

Dose / conc.:

1 200 mg/kg bw/day

Remarks:

Phase II

Dose / conc.:

2 400 mg/kg bw/day

Remarks:

Phase II

Dose / conc.:

4 800 mg/kg bw/day

Remarks:

Phase II

Dose / conc.:

9 600 mg/kg bw/day

Remarks:

Phase II

Dose / conc.:

1 125 mg/kg bw/day

Remarks:

Phase III

No. of animals per sex per dose:

3 virgin females (Phase I)
2-4 mated females (Phase II)
50 mated females (Phase III)

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: female
Duration of test: up to 3 days post partum

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:


BODY WEIGHT: Yes
- Time schedule for examinations:
Phase 1: at the time of randomization, on day 1, 5 of treatment and day 3 after treatment
Phase 2 : at the time of randomization, on days 6 through 15 and day 17 of gestation,
Phase 3 : at the time of randomization, on days 6 through 15 and day 17 of gestation, post partum day 0 and 3


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 17 (phase 2); Sacrifice on day 3 post partum
- Organs examined: uteri

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: No data
- Soft tissue examinations: No data
- Skeletal examinations: No data
- Head examinations: No data

Statistics:

An overall test for homogeneity of variance (Bartlett's test) and F-test were performed on the weight data of each group following randomization. Average body weight per group and average body weight change per group were calculated for treatment and control groups. Probit analysis of mortality and morbidity data generated in Phase II of the range finding study was used to determine the predicted LD10 for the Phase III.
The following statistical procedures were used for analyzing maternal data:
- Random weights: analysis of variance (ANOVA) (2-tail)
- Survival: Fisher's exact test (one-tail)
- Weight gains: Mann-Whitney U-Test (2-tail)
- Proportion of viable litters Fisher's exact test (one-tail)
- Survival of pups Chi-Square test (one-tail)

The Mann-Whitney U-test (2-tail) was used to compare each group to the concurrent control.
- Number live pups/litter (day 0, day 3)
- Length of gestation
- Average wt. pup (day 0, day 3)
- Average wt. gain/litter (day 3-day 0)

The p-value listed is not corrected for multiple comparisons.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
See Remarks on results section.

Key result

Dose descriptor:

NOAEL

Effect level:

1 125 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

1 125 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Abnormalities:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
See Remarks on results section.

Remarks on result:

not measured/tested

Abnormalities:

not specified

Developmental effects observed:

not specified

Phase I: (range finding)

Mortality:

No deaths occurred.

 

Body weight development:

Dose (mg/kg bw)	10	100	1000
TD 5 – TD 1	-0.5	+1.2	+1.3
PTD 7 – TD 5	+1.9	+2.3	+0.3
PTD 7 – TD 1	+1.4	+3.5	+1.6

 

Clinical signs

No signs of systemic toxicity were observed at any dose level.

 

 

Phase II: (range finding)

 

Body weight development

Dose mg/kg bw/day	600	1200	2400	4800	9600
TD 10 - TD 1	+13.33	+12.66	+6.55	+15.01	-
PTD 2 - TD 10	-4.06	+7.27	+2.97	+6.70	-
PTD 2 - TD 1	+17.39	+19.93	+9.52	+21.71	-

 

Mortality and Pregnancy Status

Dose mg/kg bw/day	600	1200	2400	4800	9600
No of animals treated	4	4	4	4	4
Animals found dead	1	0	1	2	4
Ammonium sulfide positive	1	-	-	2	2
Ammonium sulfide negative	-	-	1	-	2
Animals sacrificed	3	4	3	2	0
Live pups	3	2	1	2	-
Ammonium Sulfide positive	-	-	1	-	-
Ammonium sulfide negative	-	2	1	-	-

  

Clinical signs

600 mg/kg

A rough haircoat was seen in all animals. On treatment day 4, one animal was noted as having hunched posture, pale eyes and extremities. This animal was found dead during the morning observation on treatment day 5. This did not appear to be caused by dosing trauma. Two of the surviving animals appeared normal by treatment day 9. One surviving animal exhibited a rough haircoat until post-treatment day 2.

 

1200 mg/kg

All animals at this dose level exhibited a rough haircoat by treatment day 2. This was the only deviation from normal noted for these animals. All animals appeared normal by treatment day 9.

 

2400 mg/kg

All animals at this dose level exhibited a rough haircoat by treatment day 2. On post-treatment day 1, one animal appeared languid, exhibited hunched posture, rough haircoat and squinted eyes. This animal was found dead during the morning observation of post-treatment day 2. This death did not appear to be caused by dosing trauma.

 

4800 mg/kg

Animals at this dose level exhibited a rough haircoat by treatment day 2. On treatment day 4, one animal was noted as not using its left rear leg. This animal’s leg appeared normal by the afternoon observation on treatment day 5. Two animals were found dead, onde during the morning observation of treatment day 7 and one during the morning observation of treatment day 9. These deaths did not appear to be caused by dosing trauma.

 

9600 mg/kg

All animals in this dose level died. One animal was found dead during the afternoon observation period of treatment day 1. The remainder of the animals were found dead during the morning observation of treatment day 2. These deaths did not appear to be caused by dosing trauma.

 

Conclusion

Based on the data obtained from phases I and II, the recommended dose for phae III was 1125 mg/kg/day (LD₁₀).

 

 

Phase III

 

Mortality

There were no deaths in the treatment and control group.

 

Clinical signs

One animal was noted as having a subcutaneous lump (treatment day 2). This did not appear to be related to treatment. One other animal exhibited hunched posture, languid behavior, rough haircoat,, wheezing, labored respiration, head tilt, squinted eyes, and pale extremities throughout the treatment and post-treatment period. All other animals appeared normal throughout the treatment and post-treatment period.

 

Maternal body weights

No statistically significant group differences were found for the randomization weights. Statistical analysis was performed to compare the maternal body weights on postpartum day 0 and 3. The treatment group was significantly heavier from the control group at postpartum day 0 but not at postpartum day 3. The average weight gain from pretreatment to postpartum day 0 was significantly greater for the treatment group.

 

Reproductive index and litter data

No statistically significant effect was observed upon the percent viable litters and upon pup survival.

 

Duration of gestation

No statistically significant group differences were noted in the length of gestation.

 

Pup weight data

No statistically significant differences were noted.

 

Conclusion

Triethanolamine(1125mg/kg/day) did not affect maternal mortality, the number of viable litters, bitter size, percent survival of the pups, birth weight of the pups and weight gainedbythe pups. Using the ranking system of Hardin with a maximum score of 22 and for low maternal toxicity the following scores should be subtracted: viable litters (4); litter size (2); percent survival of pups(3);birth weight of pups(1);and weight gain of pups (2).

Therefore, the score for triethanolamine is 12 which represents intermediate priority for further testing. Since the dose used of triethanolamine did not result in maternal mortality and since the other two ethanolamines (mono- and di-) are positive in the Chernoff/Kavlock test,it is recommended that triethanolamine be retested in the Chernoff/Kavlock test at a higher dose which results in approximately10%maternal mortality. Until it is retested, triethanolamine is judged to be inaccurately tested in the Chernoff/Kavlock preliminary developmental toxicity test.

Reference 7

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

03 Aug 2006 - 15 Jan 2009

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:

other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

Jan 2001

Deviations:

yes

Remarks:

Food consumption was not determined between days 14 and 21 after parturition

Qualifier:

according to guideline

Guideline:

other: Corrigendum to EC Commission Directive 2004/73/EC, Part B: Methods for the determination of toxicity: Two-Generation Reproduction Toxicity Study; Official Journal of the European Communities; No. L216, pp. 236–246

Version / remarks:

29 Apr 2004

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

Aug 1998

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- No.of test material: 1) 05/0372-2; 2) 05/03723; 3) 05/0372-4
- Lot/batch No.: ad 1) JB116/2+3 (from 09 Aug – 04 Oct 2006); ad 2) JB116/4 (from 04 Oct – 29 Nov 2006); ad 3) JB116/9-17 (from 29 Nov 2006 until the scheduled termination of the in life part of the study)
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, under N2

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 44 (+/- 1) days
- Weight at study initiation: (P) Males: 162.1 (142.5 – 186.5) g; Females: 126.2 (110.6 – 145.1) g;
- Fasting period before study: none
- Housing: 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:
• overnight mating: male and female mating partners were housed together in type DK III cages
• gestation day 18 – lactation day 21: pregnant animals and their litters were housed in Makrolon type M III cages (floor area of about 800 cm²). The M III cages were also supplied by Becker & Co. 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 in racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum
- Acclimation period: 16 days
- Other: 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
The test substance was weighed and thoroughly mixed with a small amount of food. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer. Test diets were prepared at intervals, which guaranteed that the test substance in the diet remained stable throughout the feeding period.

During the first week of the premating period, F0 parental animals received dietary Ethanolamine hydrochloride (EAH) concentrations based on the body weight of randomization and historical food consumption data given below:
Food consumption males: 19 g
Food consumption females: 15 g
The dietary concentration of EAH was calculated using the following formula: BWx . D / FCx = ppm where
BWx = mean body weight on day x [g]
D = desired dose [mg/kg body weight/day]
FCx = mean daily food consumption on day x [g]
ppm = dietary EAH concentration for the week/period following day x

- During the remaining premating period, the dietary concentrations of EAH were adjusted weekly for each group and sex based on body weight and food consumption measurements from the preceding week.
- During the mating period of the F0 parental animals, each group and sex received the concentrations of EAH used during the last week of the premating period. This concentration was maintained throughout the mating period with the following exception: During cohabitation, both sexes received the test substance preparation for females as soon as the male was placed in the cage of its female partner. Both sexes returned to their normal test diet when they were separated the following morning. This test diet cycle remained in effect until there was evidence of successful mating. At that time, the mated animals received the test substance preparations described below at the first opportunity in the specific week.
- During the gestation period, dietary concentrations of EAH for the F0 males were again adjusted weekly on the basis of body weight and food consumption data from the preceding week. The EAH concentrations in the diet of the F0 females were the same as those used during the last week of the premating period.
- During the lactation period, dietary concentrations of EAH for the F0 males continued to be adjusted weekly on the basis of body weight and food consumption data from the preceding week. The EAH concentrations in the diet of the F0 females were 50% of those used during the last week of the premating period. This dietary adjustment, derived from historical body weight and food consumption data, maintained the dams at the desired doses of EAH during this period of increased food intake.
- Post weaning, dietary EAH levels for parental male animals awaiting necropsy were adjusted weekly based on body weight and food consumption data from the preceding week. The EAH concentration of parental female diets was the same as those used during the last week of the preceding premating period.
- Until all litters were weaned (when the last selected F1 pup reached age of day 21 p.p.), the food for the weaned F1 pups selected as F1 parental animals was prepared with EAH concentrations on the basis of historical body weight and food consumption data for rats of similar age.
- During the first week of the premating period of F1 parental animals, dietary EAH concentrations were formulated on the basis of actual body weight on day 0 and historical food consumption data. Subsequently, dietary EAH levels for each F1 dose group and sex were adjusted as described for F0 parental animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of EAH in the diet over 32 days at room temperature was investigated analytically before the beginning of the study. Homogeneity and concentration control analyses were carried out at the beginning and toward the end of the premating periods. At least one analysis of test substance preparations for female animals was carried out during the gestation and lactation periods.

The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: over night
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged (how): individual

In general, male and female animals were mated overnight at a 1 : 1 ratio for a maximum of 2 weeks. Each female animal was paired with a predetermined male animal from the same dose group throughout the entire mating period. Mating was accomplished by placing the male in the cage of the female mating partner from about 4.00 p.m. until 7.00 - 9.00 a.m. of the following morning. Deviations from these 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 was examined for the presence of sperm. If sperm were detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "day 0" and the following day "day 1" post coitum (p.c.).

Duration of treatment / exposure:

semichronic duration (> 75 days)

Frequency of treatment:

continuously

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

in diet

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

in diet

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

in diet

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Details on study design:

F0 PARENTAL ANIMALS
After the acclimatization period, the F0 generation parental animals continuously received the test substance at the appropriate concentrations in the diet up to about 16 hours before they were sacrificed. At least 75 days after the beginning of treatment, males and females from the same dose group were mated. The females were allowed to litter and rear their pups (F1 generation pups) until day 4 (standardization) or 21 after parturition. After weaning of F1 pups the F0 generation parental animals were sacrificed.

F1 PARENTAL ANIMALS
After weaning, 25 males and 25 females of the F1 pups of test groups 00, 01, 02 and 03 (0, 100, 300 and 1000 mg/kg bw/d) were taken per group as the basis of the F1 generation parental animals. These animals were chosen by lot and it was attempted to take each litter into account. If fewer than 25 litters were available in a group or if one sex was missing in a litter, more animals were taken from the other litters of the respective test group to obtain the full number. All selected animals were treated with the test substance at the same dose level as their parents from their growth into adulthood up to about one day before they were sacrificed. At least 75 days after assignment of the F1 generation parental animals, the males and females were generally mated at a ratio of 1:1. Partners were randomly assigned, mating of siblings was excluded. The females were allowed to litter and rear their pups (F2 generation pups) until day 4 (standardization) or 21 after parturition. Shortly after the F2 generation pups had been weaned, the F1 generation parental animals
were sacrificed.

STANDARDIZATION OF LITTERS (F1 AND F2 GENERATION PUPS)
On day 4 p.p., individual litters were standardized in such a way that, where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If it was not possible for individual litters to have 4 pups/sex, it was proceeded in such a way that 8 pups per litter were present for further rearing (e.g. 5 male and 3 female pups). Standardization of litters was not performed in litters with ≤ 8 pups.

PUPS AFTER STANDARDIZATION AND AFTER WEANING
After standardization or weaning, all pups were sacrificed by means of CO2 with the exception of those F1 generation pups, which were chosen as F1 generation parental animals. All sacrificed pups, including stillborn pups and those that died during their rearing period, were subject of a macroscopic (external and visceral) examination. All pups without any notable findings or abnormalities were discarded after their macroscopic
evaluation.

Maternal examinations:

MORTALITY:
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on 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:
All parental animals were checked daily for clinically evident signs of toxicity. For technical reasons, however, the clinical observations recorded during the premating periods were printed out on a weekly basis (the daily observations can be found in the raw data). 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 special findings, e.g. disability to deliver, were documented on an individual dam basis. In addition to the evaluations in the mornings, parturition behavior of the dams was also inspected on weekdays (except public holidays) in the afternoons. The day of parturition 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 Saturday, Sunday and on public holidays.

FOOD CONSUMPTION:
In general, food consumption was determined once a week (each time for a period of at least 6 days) for the male and female F0 and F1 parental animals. For the females during pregnancy (animals with evidence of sperm), food consumption was determined weekly 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 will begin to consume considerable amounts of solid food offered, and therefore, there was no point in such measurement. Furthermore, food consumption was not determined for females without positive evidence of sperm and for females without litter.

COMPOUND INTAKE:
The intake of test substance was calculated from the amount of food consumed and is expressed as mg/kg body weight/day (mg/kg bw/d). The calculation of the group values/day was carried out according to the following formula: intake of test substance on day x in mg/kg bw/d = (daily food consumption on day x in grams) x (concentration in ppm) / (body weight on day y in grams (last weighing before day x))

BODY WEIGHT DATA:
In general, the body weight of parental animals was determined on the first day of the premating period and then once a week at the same time of day (in the morning). Based on these results, the body weight change of the animals was calculated. The following exceptions are notable for the female parental animals:
a) During each gestation period the F0 and the F1 generation parental females were weighed on the day of positive evidence of sperm (day 0 p.c.) and on days 7, 14 and 20 p.c.
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 p.p.
d) Females without litter were not weighed during the lactation phase.

FEMALE REPRODUCTION AND DELIVERY DATA:
For F0 and F1 females, following parameters were noted: mating partners, number of mating days until positive evidence of sperm, and gestational status.

BLOOD SAMPLINGS:
Blood samples were taken from all F0 and F1 parental animals of each sex and test group during week 10 of premating treatment and the plasma was analyzed for the concentration of the test substance.

POST-MORTEM EXAMINATIONTS:
All F0 and F1 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.
As soon as possible after termination, one portion of the liver (lobus medialis) of each 10 dams per group was sampled to be analyzed for choline concentration.

ORGAN WEIGHTS:
Weight assessment was carried out on all animals sacrificed at scheduled dates. The following weights were determined:
1. Anesthetized animals, 2. Liver, 3. Kidneys, 4. Adrenal glands, 5. Testes, 6. Epididymides, 7. Cauda epididymis, 8. Prostate, 9. Seminal vesicles including coagulation glands, 10. Ovaries, 11. Uterus, 12. Spleen, 3. Brain, 14. Pituitary gland, 15. Thyroid glands (with parathyroid glands).

ORGAN/TISSUE FIXATION:
The following organs or tissues of the F0 and F1 generation parental animals were fixed in 4% neutral buffered formaldehyde solution or in BOUIN’s solution, respectively:
1. Vagina, 2. Cervix uteri, 3. Uterus, 4. Ovaries (fixed in BOUIN´s solution), 5. Oviducts, 6. Left testis (fixed in BOUIN´s solution), 7. Left epididymis (fixed in BOUIN´s solution), 8. Seminal vesicles, 9. Coagulation glands, 10. Prostate, 11. Pituitary gland, 12. Adrenal glands, , 3. Liver, 14. Kidneys, 15. Spleen, 16. Brain, 17. Thyroids (with parathyroids), 18. All gross lesions. After fixation, the organs fixed in BOUIN´s solution were embedded in Paraplast. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings. All gross lesions were examined. Of the fixated organs the organs of all animals in the control group and the high dose group were evaluated. Additionally, the organs for mating pair suspected of reduced fertility were evaluated.

BLOOD SAMPLINGS:
Blood samples were taken from all F0 and F1 parental animals of each sex and test group during week 10 of premating treatment and the plasma was analyzed for the concentration of the test substance.

Ovaries and uterine content:

From both ovaries (”ovary 1” and “ovary 2”) of F1 female animals (control and top dose), five sections were taken from the proximal and the distal part of the ovaries, respectively, at least 100 μm apart from the inner third of the ovary. All ovarian sections were prepared and evaluated. Primordial follicles and growing follicles were counted by light microscope (magnification: 100x) on each of these slides, – according to the definitions given by Plowchalk et al. (PLOWCHALK, D. R., B. J. SMITH, and D. R. MATTISON: Assessment of Toxicity to the Ovary Using Follicle Quantitation and Morphometrics. In: Methods in Toxicology, Vol. 3, Part B: Female Reproductive Toxicology (J. J. HEINDEL and R. E. CHAPIN, Editors), p. 57-68, 1993, Academic Press). To prevent multiple counting on serial slides – especially of the growing follicles – only follicles with an oocyte with visible chromatin on the slide were counted. The number of each type of follicle was recorded individually for ovary 1 and ovary 2 of every animal on any of the slide levels (level 1-10), giving in summary the incidence of each type of the follicles by using EXCEL sheets for the reporting of the results. Finally, the results of all types of follicles were summarized for all animals per group in dose groups 10 and 13. As primordial follicles continuously develop into growing follicles, the assessment of the follicles was extended to the combined incidence of primordial plus growing follicles. In general, the fifth slide of the left and right ovary was evaluated for histological findings. A correlation between gross lesions and histopathological findings was performed.

Fetal examinations:

PUP NUMBER AND STATUS AT DELIVERY:
On the day of birth, all pups derived from the F0 parents (F1 litter) and the F1 parents (F2 litter) were examined as soon as possible to determine the total number of pups, and the number of liveborn as well as stillborn pups of each litter. Pups, which died before examination, were designated as stillborn pups.

PUP VIABILITY/MORTALITY:
- In general, a check was made for dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) and once in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated as dexribed in the section 'Postmortem examinations (offspring)'
- 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.

SEX RATIO:
- On the day of birth (day 0 p.p.), 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 and was finally confirmed at necropsy.
- The sex ratio was calculated at day 0 and day 21 p.p. according to the following formula:
Sex ratio = (number of live male or female pups on day 0/day 21 p.p. / number of live male and female pups on day 0/day 21 p.p.) X 100

PUP 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 any special findings, these were documented with the dam concerned.

PUP BODY WEIGHT DATA:
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 based on 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.

SEXUAL MATURATION:
- Vaginal opening: All female F1 pups selected to become the F1 parental generation females (25/group) were examined daily for vaginal opening beginning on day 27 p.p. On the day of vaginal opening, the body weights of the respective animals were additionally determined.
- Preputial separation: All male F1 pups selected to become the F1 parental generation males (25/group) were examined daily for preputial separation beginning on day 40 p.p. On the day of preputial separation, the body weights of the respective animals were additionally determined.

PUP ORGAN WEIGHTS:
After scheduled sacrifice brain, spleen and thymus of 1 pup/sex and litter from the F1 and F2 pups were weighed. Normally, the first male and the first female pups/litter were taken for these examinations. For the calculation of the respective relative organ weights, pup body weights were taken, which were determined routinely during the in-life phase on day 21 p.p.

PUP NECROPSY OBSERVATIONS:
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. or subsequent days) were killed by means of CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically. All stillborn pups and all pups that died up to weaning were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.

Statistics:

see in 'Any other information on materials and methods incl. tables'.

Indices:

For females, mating, fertility and gestation indices were calculated for F1 and F2 litters according to the following formulas:
- Female mating index (%) = (number of females mated / total number of pups born) x 100
- Female fertility index (%) = (number of females pregnant / number of females mated) x 100
- Gestation inex (%) = (number of females with live pups on the day of birth / number of females pregnant) x 100

The number of females mated was defined as the number of females with vaginal sperm or with implants in utero.
The number of females pregnant was defined as the number of females with implants in utero.

The total amount of delivered pups/dam was recorded and the number of liveborn and stillborn pups noted. The live birth index was calcualted for F1 and F2 litters according to the following formula:
- Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100

The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:
Postimplantation loss (%) = ((number of implantations – number of pups delivered) / number of implantations) x100Viability index (%) = (number of live pups on day 4 (before standardization of litters) after birth / number of live pups on the day of birth) X 100
Lactation index (%) = (number of live pups on day 21 after birth / number of live pups on day 4(after standardization of litters) after birth) X 100

Viability and lactation indices were calculated according to the following formulas:
- Viability index (%) = (number of live pups on day 4 (before standardization of litters) after birth / number of live pups on the day of birth) x 100
- Lactation index (%) = (number of live pups on day 21 after birth / number of live pups on day 4(after standardization of litters) after birth) x 100

Historical control data:

yes

Clinical signs:

no effects observed

Description (incidence and severity):

F0: No clinical signs or changes of the general behavior, which may be attributed to the test substance, were detected in F0 female parental animals of the test groups 01 and 02 (100 and 300 mg/kg bw/d). Intensively yellow discolored urine was recorded in all F0 females of test group 03 (1000 mg/kg bw/d) from study week 3 onwards until the end of the treatment period. This urine discoloration mirrored the systemic availability of the test substance rather than being an adverse effect and was most likely caused by the excreted test compound and/or its metabolites.

Clinical observations for females during gestation of F1 litters:
All F0 females of test group 03 showed intensively yellow discolored urine during the entire gestation period for F1 litter. No other clinical findings were observed in the test groups 00-03 (0, 100, 300 and 1000 mg/kg bw/d). One sperm positive female of test group 02 (300 mg/kg bw/d) and one of test group 03 (1000 mg/kg bw/d) did not deliver F1 pups. This observation was not considered to be associated to the test compound.

Clinical observations for females during lactation of F1 litters:
All F0 females of test group 03 showed intensively yellow discolored urine during the entire lactation period for F1 litter. One high-dose female (1000 mg/kg bw/d) had just one pup (female), which was cannibalized by its mother on lactation day 8. No other clinical findings were observed in the test groups 00-03 (0, 100, 300 and 1000 mg/kg bw/d).

F1: No clinical signs or changes in general behavior, which may be attributed to the test substance, were detected in F1 female parental animals of the test groups 11 and 12 (100 and 300 mg/kg bw/d).
Intensively yellow discolored urine was recorded in all F1 females of test group 13 (1000 mg/kg bw/d) from study week 0 onwards until the end of the treatment period. This urine discoloration mirrored the systemic availability of the test substance rather than being an adverse effect and was most likely caused by excreted test compound and/or its metabolites.

Clinical observations for females during gestation of F2 litters:
All F1 females of test group 13 showed intensively yellow discolored urine during the entire gestation period (F2 litter). No other clinical findings were observed in the test groups 10-13 (0, 100, 300 and 1000 mg/kg bw/d).

One sperm-positive female of test group 10 (control), one of test group 11 (100 mg/kg bw/d) and two of test group 13 (1000 mg/kg bw/d) did not deliver F2 pups. These observations were not considered to be associated to the test compound due to a missing dose-response relationship.

Clinical observations for females during lactation of F2 litters:
All F1 females of test group 13 showed intensive yellow discolored urine during the entire lactation period for F2 litters. No other clinical findings were observed in the test groups 10-13 (0, 100, 300 and 1000 mg/kg bw/d).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled mortalities of maternal animals in any test group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F0: During premating, gestation and lactation periods, the mean body weights and body weight gains of the F0 parental females in the low- and mid-dose groups were generally comparable to the concurrent control group. During premating, gestation and lactation periods, the mean body weights and body weight gains of the F0 parental females in the low- and mid-dose groups were generally comparable to the concurrent control group. Mean body weights and body weight gains of the F0 females in test group 03 (1000 mg/kg bw/d) were similar to the controls throughout the entire premating period. During gestation, these animals gained less weight from gestation day 7 onwards (up to 38%). As a consequence, body weights on gestation day 20 were 8% lower than the control. This effect may have been caused by the statistically significantly increased postimplantation loss and the statistically significantly decreased mean number of delivered pups in test group 03. This is also indicated by the unaffected body weight of the high dose dams on post-delivery day 1. Mean body weights of the high-dose females remained comparable to the controls during entire lactation, whereas the weight gain wavered up and down in the individual lactation sections.

F1: Mean body weights and body weight gain of the F1 parental females in test groups 11-12 (100 and 300 1000 mg/kg bw/d) were comparable to the control throughout premating, gestation and lactation periods. Mean body weights and body weight gains of the F1 females in test group 03 (1000 mg/kg bw/d) were similar to the controls throughout the entire premating period, the statistically significantly increased body weight gain of the high-dose F1 females (1000 mg/kg bw/d) during premating week 1-2 was regarded as incidental variance. The average weight gain of these animals was significantly below control (26%) during gestation days 14-20, which led to an averaged decrease of weight gain for the entire gestation of 17%. This effect may have been caused by the statistically significantly increased postimplantation loss and the statistically significantly decreased mean number of delivered pups in test group 03. Mean body weights of the high-dose females remained comparable to the controls during entire lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

F0: Food consumption of the F0 parental females of the low- and mid-dose groups (100 and 300 mg/kg bw/d) was comparable to the control animals during the periods of premating, gestation and lactation. Food consumption of the high-dose F0 females (1000 mg/kg bw/d) was also comparable during premating and gestation periods. However, in these animals, food consumption was statistically significantly below controls (up to 18%) in the individual lactation sections: days 1-4, 4-7, and 7-14 p.p.

F1: Food consumption of F1 female animals in test groups 11-12 (100, and 300 mg/kg bw/d) was generally comparable to the control group throughout the entire treatment period, covering premating, gestation and lactation periods. Food consumption of the high-dose F0 females (1000 mg/kg bw/d) was also comparable during premating and gestation periods. However, in these animals, food consumption was statistically significantly below controls (-11%) on lactation days 1-4, and remained below control on lactation days 4-7 and 7-14 (-7%), although not statistically significant.

For all test groups the intake of Ethanolamine hydrochloride correlated well with the desired target doses. For the actual test substance intake see 'Any other information on results incl. tables'.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

F0: The observed increase of absolute kidney weights of female animals in mid (300 mg/kg bw/day) and top dose (1000 mg/kg bw/day) groups, respectively, was statistically significant. Because no histomorphological correlate was detected, a treatment-related weight increase is less likely. The observed increase of thyroid glands in mid and top dose females, is considered incidental and not treatment-related due to a missing dose response relationship and no histopathological correlates.

F1: The observed increase of absolute kidney weights of female animals in mid (300 mg/kg bw/day) and top dose (1000 mg/kg bw/day) groups, respectively, was statistically significant. Because no histomorphological correlate was detected, a treatment-related weight increase is less likely. The observed increase of thyroid glands IN mid and top dose females, is considered incidental and not treatment-related due to a missing dose response relationship and no histopathological correlates.

Gross pathological findings:

no effects observed

Description (incidence and severity):

F0: All gross lesions observed in test animals occurred singularly. They are considered to be spontaneous lesions in origin and are not related to treatment. The female animal which was not pregnant as well as the male mating partner did not show relevant gross lesions.
F1: All gross lesions observed in test animals occurred singularly. They are considered to be spontaneous lesions in origin and are not related to treatment.
One non-pregnant female animal did show a bilaterally severe reduced size of the ovaries as well as a moderate thickening of the uterus wall. The other three non-pregnant females did not show any gross lesions. The four male mating partners did not show any gross lesions either.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

F0:
- All findings noted were single observations either, or were similarly in distribution pattern and severity in control rats compared to treatment groups. All of them are consideredto be incidental and/or spontaneous in origin and without any relation to treatment.
- Fertility: The non-pregnant female and the male mating partner did not show histopathological findings explaining the infertility. Only a minimal focal atrophy of the prostate was present, which is considered an incidental finding.

F1:
- As compared to control animals, the kidneys of low, mid, and top dose female animals revealed a low incidence of basophilic tubules in a slightly higher number of animals. The severity (minimal to slight) was comparable between controls and treated animals and a clear dose-response relationship was missing. The cauda epididymis and epididymides of top dose males showed no histomorphological correlates to the decreased organ weights. All other findings noted were single observations either, or were similarly in distribution pattern and severity in control rats compared to treatment groups. All of them are considered to be incidental and/or spontaneous in origin and without any relation to treatment.
- Fertility: One non-pregnant female showed a bilateral moderate diffuse stromal hyperplasia and a unilateral severe focally extensive chronic inflammation of the ovaries as well as an ovarian cyst. There were still corpora lutea present and the histopathological findings did not correlate with the gross lesion. The gross lesion “thickening of uterine wall” had no corresponding histological finding. The findings on the ovaries might explain the infertility. The male mating partner did not reveal lesions affecting the fertility. One female Animal showed within the uterus a severe multifocal endometrial and glandular degeneration and within the oviducts a severe diffuse epithelial degeneration, which explains the infertility of this animal. The male mating partner did not reveal lesions affecting the fertility. The other two mating pairs did not show any lesions affecting the fertility.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

The results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant deviations between controls and animals of the top dose group.

Number of abortions:

no effects observed

Description (incidence and severity):

F1 pups: The mean number of F1 pups/dam was statistically significantly reduced in test group 03 (6.8** pups/dam [p≤0.01]). However, the number of stillborn F1 pups was comparable between the test groups 00, 01, 02, and 03 (2, 1, 2, and 2 pups/test group, respectively). Every stillborn pup belonged to a different litter.

F2 pups: The mean number of F2 pups/dam was statistically significantly reduced in test group 13 (1000 mg/kg bw/d). However, a comparison of the total amount of liveborn and stillborn F2 pups per test group did not reveal statistically significant differences between test groups 10-13.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

F0: No statistically significant differences were seen between the control (11.8 implants/dam) and test groups 01 and 02 (12.4 and 11.8 implants/dam, respectively). In contrast, the number of implantation sites was statistically significantly reduced in test group 03 (8.6** [p≤0.01]). Furthermore, there were indications for test substance-induced intrauterine embryo-/fetolethality, since the postimplantation loss was statistically significantly increased in the high-dose group (23.0%** [p≤0.01] at 1000 mg/kg bw/d). There were no statistically significantly differences concerning the postimplantation loss between the remaining test groups and the control (5.7%, 6.8% and 9.9% at 0, 100 and 300 mg/kg bw/d, respectively).

F1: There were no statistically significant differences in the number of implantation sites between test groups 11 and 12 (11.6 and 11.8 implants/dam, respectively) and the control. Furthermore, there were indications for test substance-induced intrauterine embryo-/fetolethality, since the postimplantation loss was statistically significantly increased in the high-dose group (12.8* [p≤0.05] – 1000 mg/kg bw/d). In addition, the postimplantation loss in this test group was higher than documented in the historical control data. There were no statistically significantly differences concerning the postimplantation loss between the remaining test groups and the control (4.9%, 3.0%, and 4.0% for 0, 100, and 300 mg/kg bw/d, respectively).

Total litter losses by resorption:

not specified

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

In the high-dose F0 and F1 generation females (1000 mg/kg bw/d), decreased numbers of implants and increased resorption rates resulted in significantly smaller litters, giving evidence for an adverse effect of the test compound on fertility and/or reproductive performance at this dose. It has to be noted that a dose of 1000 mg/kg bw/d also caused systemic toxicity in these females, as was indicated by reduced food consumption and/or body weight gain during gestation/lactation.

Dead fetuses:

no effects observed

Description (incidence and severity):

The number of liveborn and stillborn pups was comparable between all groups, and the live birth index varied between 99% and 100%.

Changes in pregnancy duration:

effects observed, non-treatment-related

Description (incidence and severity):

F0: The mean duration of gestation was comparable in all test groups. The respective values were 21.8, 21.8, 21.7 and 22.2** (p≤0.01) days. Although the duration of gestation appeared to be statistically significantly increased in the high-dose group, this value was only slightly above the concurrent control and was still within the historical control range of the test facility (21.5–22.3 days). Therefore, this finding was not considered to be treatment-related.
F1: The mean duration of gestation was comparable in all test groups (10-13) and varied between 21.8 and 22.1.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

F0: The female mating index calculated after the mating period for F1 litter was 100% in all test groups. Nearly all sperm positive females delivered pups or had implants in utero. One high-dose F0 female (No. 185) did not become pregnant. Consequently, the fertility index varied between 96% (test group 03) and 100% (test groups 00-02).
F1: The female mating index for F2 litter was 100% in all test groups. All sperm-positive rats delivered pups with the following exceptions: one control female, one female of test group 11 (100 mg/kg bw/d) and two females of test group 13 (1000 mg/kg bw/d) did not become pregnant. Consequently, the fertility index was 92% for test group 13 (1000 mg/kg bw/d), 96% for test groups 10 and 11 (0 and 100 mg/kg bw/d) and 100% for test group 12 (300 mg/kg bw/d). These values reflect the normal range of biological variation inherent in the strain of rats used for this study.

Other effects:

effects observed, treatment-related

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

pre and post implantation loss

other: less implantation sites, smaller litters

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

F1: Mean body weights of F1 male and female pups in test group 03 (1000 mg/kg bw/d) were statistically significantly increased during the complete lactation period, being marginally outside the range of historical control data. Body weight gain of these animals was statistically significantly increased between lactation days 1-4 (about 19%). Afterwards the weight gain of the high-dose animals was similar to the concurrent control group. The higher pup body weights in test group 03 were rather regarded as a consequence of the reduced number of pups/litter than a direct test substance-related effect. No test compound-related influence on F1 pup body weights was noted in the low- and middose groups (100 and 300 mg/kg bw/d).

F2: Mean body weights of F2 male and female pups in test groups 11-13 (100, 300 and 1000 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire lactation period. The statistically significantly increased body weights of female pups and the statistically significantly increased body weight value for both sexes in test group 13 on lactation day 1 were related to the decreased litter sizes and, given that, regarded to be secondary effects.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

F1: The mean number of F1 pups/dam was statistically significantly reduced in test group 03 (6.8** pups/dam [p≤0.01]). However, the number of stillborn F1 pups was comparable between the test groups 00, 01, 02, and 03 (2, 1, 2, and 2 pups/test group, respectively). Every stillborn pup belonged to a different litter.

F2: The mean number of F2 pups/dam was statistically significantly reduced in test group 13 (1000 mg/kg bw/d). However, a comparison of the total amount of liveborn and stillborn F2 pups per test group did not reveal statistically significant differences between test groups 10-13.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

F1: The sex distribution and sex ratios of live F1 pups on the day of birth and on day 21 p.p. did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

F2: The sex distribution and sex ratios of live F2 pups on the day of birth and on day 21 p.p. did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

F1: The average litter size (F1 pups per dam) was very similar between test groups 00, 01 and 02 (11.1, 11.4, and 11.4 pups/dam, respectively). A statistically significantly lower number of F1 pups per dam were delivered in test group 03 (6.8** pups/dam [p≤0.01]). Since the number of stillborn pups was comparably low in all groups, the live birth index was 99% for test groups 00, 02 and 03, and 100% for test group 01.

F2: The average litter size (F2 pups per dam) was very similar between test groups 10, 11 and 12. A statistically significantly lowered number of F2 pups per dam were delivered in test group 13 (7.7** pups/dam [p≤0.01] versus 10.8, 11.2, and 11.4 in test groups 10, 11 and 12, respectively). The number of liveborn and stillborn pups was comparable between all groups, and the live birth index varied between 99% and 100%.

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

F1:
- The viability index as indicator for pup mortality between days 0-4 p.p. was unaffected and varied between 99% (control group as well as test groups 01 and 03) and 100% (test group 02). However, cannibalized pups were found in test group 01 (3 pups), 02 (1 pup) and 03 (3 pups* [p≤0.05]). In this case, the calculated statistically significant difference for the highdose group was a consequence of the smaller litter sizes there. No pup was cannibalized in the control group.
- The lactation index, indicating pup mortality between days 4-21 p.p., was slightly, but statistically significantly lower in test group 01 (97%* [p≤0.05]). This value is, however, within the historical control range of the test facility. Thus, pup mortality in the low dose group during the lactation period was not considered to be associated to test substance-treatment. For the other test groups, the lactation index was 98% (test group 03) and 100% (test groups 00 and 02).

F2:
- The viability index as indicator for pup mortality between days 0-4 p.p. was 100% in all test groups.
- The lactation index as indicator for pup mortality between days 4-21 p.p. was not affected by test substance-treatment as it reached 100%, 100%, 99%, and 100% at dose levels of 0, 100, 300, and 1000 mg/kg bw/d. Any isolated pup deaths were assessed as incidental.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

F1: One low-dose pup showed a kinked tail on lactation day 21, which was confirmed by skeletal examination (misshapen caudal vertebra, cartilage changed). These indivisual finding were assumed to be incidental and can also be found in the historical control data

F2: no effects reported

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

F1: One low-dose pup showed a kinked tail on lactation day 21, which was confirmed by skeletal examination (misshapen caudal vertebra, cartilage changed). These indivisual finding were assumed to be incidental and can also be found in the historical control data

F2: no effects reported

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

F1: For one high-dose pup microphthalmia of the left eye was recorded on lactation day 21 and was confirmed by visceral examination. These indivisual finding were assumed to be incidental and can also be found in the historical control data

F2: no effects reported

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at highest dose tested

Key result

Abnormalities:

no effects observed

Developmental effects observed:

no

Test substance stability:

The stability of test substance in rat diet was demonstrated for a period of 32 days at room temperature in a different batch of comparable quality, which was not used for the study. The homogeneity of the mixtures was verified. The concentration control analyses of the samples taken revealed that the values were within a range of 90-110% of the nominal concentration in all analyses at all time points, with the exception of one concentration in the feed of the high-dose group (88%).

Plasma concentrations of 2 -aminoethanol were below 3 mg/kg for all control animals, <3 - 4 mg/kg for the low dose animals, 8 - 11 mg/kg for the mid dose animals and 60 – 81 mg/kg for the high dose animals.

Toxicokinetic data of 2 -aminoethanol (calculated as 2 -aminoethanol hydrochloride) from this two-generation reproduction toxicity studyshow a dose dependency of the plasma levels of 2 -aminoethanol in the experimental animals and there with prove the bioavailability of 2 -aminoethanol hydrochloride in principle.

 

Under these conditions, no test substance-related findings from clinical examinations or gross and histopathology were observed, which indicate that the administration of the test compound via the diet adversely affected the fertility or reproductive performance of the F0 or F1 parental animals up to and including a nominal dose of 300 mg/kg bw/d. Estrous cycle data, mating behavior, conception, gestation, parturition, lactation and weaning as well as sperm parameters, sexual organ weights and gross and histopathological findings of these organs (including differential ovarian follicle counts in the F1 females) were comparable between the rats of all test groups.

At the high-dose level (1000 mg/kg bw/d), absolute and relative weights of epididymides and cauda epididymidis were decreased and, in the F0 generation only, the number of homogenization resistant caudal epididymal sperm was slightly, but significantly reduced. However, histomorphological correlates for these findings were missing.

 

In the high-dose F0 and F1 generation females (1000 mg/kg bw/d), decreased numbers of implants and increased resorption rates resulted in significantly smaller litters, giving evidence for an adverse effect of the test compound on fertility and/or reproductive performance at high doses. It has to be noted that a dose of 1000 mg/kg bw/d also caused beginning systemic toxicity in these females, as was indicated by reduced food consumption and/or body weight gain during gestation/lactation.

 

All data recorded during gestation and lactation in terms of embryo-/fetal and pup development gave no indications for any developmental toxicity in the F1 and F2 offspring up to a dose level of 1000 mg/kg bw/d. The test substance did not adversely influence pup viability, body weight, sex ratio and sexual maturation.

 

Thus, under the conditions of the present two-generation reproduction toxicity study, the NOAEL(no observed adverse effect level) for fertility, reproductive performance and systemic toxicity in parental F0 and F1 Wistar rats is 300 mg/kg bw/d.

 

The NOAEL for pre-and postnatal developmental toxicityin their offspring is 1000 mg/kg bw/d.

Tables

Mean test substance intake (mg/kg bw/d; minimum value / maximum value)

	Test group 01 (100 mg/kg bw/day)	Test group 02 (300 mg/kg bw/day)	Test group 03 (1000 mg/kg bw/day)
F0 males	94.3 (72.4 / 102.5)	283.2 (218.4 / 309.4)	943.3 (716.7 / 1032.6)
F0 females (premating)	96.7 (80.5 / 100.7)	289.6 (241.2 / 304.9)	964.4 (792.4 / 1017.8)
F0 females (F1 litter) - gestation period - lactation period*	103.5 (92.6 / 111.6) 99.2 (81.6 / 120.2)	315.2 (284.8 / 337.9) 306.7 (249.7 / 370.3)	1043.2 (989.4 / 1084.7) 866.0 (668.6 / 1053.9)

* = Days 1–14 p.p. only

Absolute organ weights (P-generation)

Compared to the controls (= 100%), the following values (in %) were significantly changed (printed in bold):

	Male animals			Female animals
Group	01 100 mg/kg bw/day	02 300 mg/kg bw/day	03 1000 mg/kg bw/day	01 100 mg/kg bw/day	02 300 mg/kg bw/day	03 1000 mg/kg bw/day
Brain	99%	100%	97%*
Cauda epididymis	99%	102%	88%**
Epididymides	100%	101%	92%**
Prostate	92%	99%	86%**
Spleen				105%*	107%	97%

 

*: p≤0.05; **: p≤0.01

 

All other mean absolute weight parameters did not show significant differences compared to the control groups.

 

The decrease of absolute weights of cauda epididymis, epididymides, and prostate in male top-dose animals (1000 mg/kg bw/d) were considered as treatment-related effects.

 

The decrease of brain weights in top-dose males (1000 mg/kg bw/d) as well as the increase of spleen weights in low-dose females (100 mg/kg bw/d) was considered as incidental and not treatment-related due to a missing dose-response relationship.

Absolute organ weights (F1 generation)

Compared to the controls (= 100%), the following values (in %)were significantly changed (printed in bold):

 

	Male animals			Female animals
Group	11 100 mg/kg bw/day	12 300 mg/kg bw/day	13 1000 mg/kg bw day	11 100 mg/kg bw/day	12 300 mg/kg bw/day	13 1000 mg/kg bw/day
Cauda epididymis	96%	99%	88%**
Epididymides	100%	101%	91%**
Kidneys	99%	106%*	111%**	103%	106%**	115%**
Spleen	99%	103%	92%*
Thyroid glands	106%	99%	109%*	110%	118%**	111%*

 

*: p≤0.05; **: p≤0.01

All other mean absolute weight parameters did not show significant differences compared to the control groups.

The decrease of absolute weights of cauda epididymis and epididymides in male top-dose animals (1000 mg/kg bw/d) were considered to be treatment-related.

 

The increase of absolute kidney weights of male and female animals in mid- (300 mg/kg bw/d) and top-dose (1000 mg/kg bw/d) groups, respectively, was statistically significant. Because no histomorphological correlate was detected, a treatment-related weight increase was less likely.

 

The decrease of spleen weights in top-dose males as well as the increase of thyroid glands in top-dose males and mid- and top-dose females, respectively, is considered incidental and not treatment-related due to a missing dose-response relationship.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD TG 421

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

For the endpoint developmental toxicity/teratogenicity a WoE Approach is conducted using results from the registered substance TEA and studies performed with the structurally analogous substance MEA-HCl (CAS 2000-42-7).

In a reproduction/developmental toxicity screening study with TEA, performed according to OECD guideline 421, Wistar rats (10/sex/dose) were exposed by gavage to 0, 100, 300 or 1000 mg/kg bw/day during a premating period of 2 weeks and a mating period (max. 2 weeks) for both sexes, during approximately 1 week post-mating for males, and during the entire gestation period as well as 4 days of lactation for females. Food consumption, body weight, clinical signs, mating and reproductive performance (including determinations of the number of implantations and the calculation of the postimplantation loss in females) were examined in parental animals. At necropsy, animals were assessed for gross pathology and selected organs were weighed and examined histopathologically. In pups, bodyweight, viability and macroscopic changes were recorded. At necropsy on PND 4, all pups were examined macroscopically for external and visceral findings. At the high dose of 1000 mg/kg bw/day, a decreased number of implantation sites, increased postimplantation loss and a lower average litter size were observed. No adverse effects were observed regarding reproductive performance, fertility or systemic toxicity at any dose level. Thus, the NOAEL for systemic toxicity as well as for reproductive performance and fertility in parental animals was established at 1000 mg/kg bw/day, the NOAEL for postnatal toxicity in the offspring was 1000 mg/kg bw/day, and the NOAEL for prenatal developmental toxicity was determined to be 300 mg/kg bw/day (BASF SE, 2010).

In a Chernoff-Kavlok teratogenicity screening test, CD-1 mice were exposed to TEA by gavage in 3 phases: 1) 3 virgin females were exposed to 10, 100 of 1000 mg TEA/kg bw/day during 5 consecutive days; 2) 2 -4 mated females were exposed to 600, 1200, 2400, 4800 or 9600 mg TEA/kg bw/day on gestation days (GD) 6 -15; 3) 50 mated females were exposed to 1125 mg TEA/kg bw/day on GD 6 -15. In the main study (phase 3), exposure to TEA did not produce any evidence of developmental or maternal toxicity. Therefore, the NOAEL for maternal toxicity and developmental toxicity was established at 1125 mg/kg bw/day (NTP, 1987).

As no developmental toxicity study (OECD guideline 414) is available for TEA, read across with the structural analogue MEA, for which developmental toxicity studies are available, is applied.

In a GLP-compliant prenatal developmental toxicity study with rats, performed according to OECD guideline 414 (BASF SE, 1994) pregnant Wistar rats were exposed to the structure analogue MEA by gavage at dose levels 0, 40, 120, 450 mg/kg bw/day on days 6 - 15 of gestation. Signs of maternal toxicity were observed at the highest dose, manifested as reduced food consumption, lower mean body weights and impaired body weight gain. No reproductive and developmental toxicity parameters were affected. The NOAEL for developmental effects was thus established to correspond to 450 mg/kg bw/day; the NOAEL for maternal toxicity was 120 mg/kg bw/day.

 

In another comparable to guideline prenatal developmental toxicity study (Liberacki, 1996) rats and rabbits were exposed. Pregnant Sprague-Dawley rats were exposed dermally to 0, 10, 25, 75 and 225 mg/kg bw/day of MEA. Rats administered 225 mg MEA/kg bw/day exhibited a treatment-related increased incidence of skin irritation and the body weight gain was significantly decreased during the exposure period. Despite maternal effects observed among dams in the high dose group, reproductive and developmental toxicity parameters among exposed rats were unaffected at all dose levels. The NOAEL for maternal toxicity was set at 75 mg/kg bw/day and the NOAEL for developmental toxicity was set at the highest dose level of 225 mg/kg bw/day.

In the rabbit study exposure was via the dermal route to 0, 10, 25, and 75 mg/kg/day of MEA. The rabbits in the mid and high dose group exhibited signs of skin irritation, severe at the highest dose level. No treatment-related effects were observed on reproductive and developmental toxicity parameters. The NOAEL for maternal toxicity was set at 10 mg/kg bw/day and the NOAEL for developmental toxicity was set at the highest dose level of 75 mg/kg bw/day (Liberacki,1996).

In a preliminary study on the prenatal toxicity of MEA, female rats (10/dose) were exposed to 0, 50, 150, 300 or 500 mg/kg bw/day by gavage on gestation days 6 -15. Maternal toxicity was observed at the high dose only, and included reduced food consumption, impaired body weight gain, decreased total protein and albumin levels, and a thickened wall of the forestomach in 3 dams. No adverse effects on the fetuses occurred. Therefore, NOAELs for maternal toxicity and teratogenicity were established at 300 and 500 mg/kg bw/day (the highest dose tested), respectively (BASF SE, 1992).

For the structural analogue substance MEA a two generation reproduction toxicity study of the hydrochloric acid performed in Wistar rats with dietary administration demonstrated clear NOAELs for systemic and reproductive toxicity including fertility at 300 mg /kg bw/day. Only at the highest dose, 1000 mg/kg bw/day, were minor effects noted. Males at this high dose levels showed minor effects on fertility in the form of decreased absolute and relative weights of epididymides and cauda . However, there was no histomorphological correlate of these findings in the organs, no effect upon testes or testicular sperm count, and no effect upon mating performance. Females at this dose level revealed decreased numbers of implants and increased resorption rates resulting in smaller litters associated with indications of systemic toxicity. There was virtually no effect on the pre- and postnatal development of the progeny in both generations up to the limit dose level of 1000 mg/kg bw/day representing a clear NOAEL for developmental toxicity (ACC and Cefic, 1994).

Based on the results of the screening studies with TEA (oral route, rats and mice) and the available developmental toxicity studies with rats and rabbits (oral and dermal route of exposure) with MEA, TEA is not considered to be a developmental toxicant.

Reproductive toxicity was also not identified as an additional concern during substance evaluation (cf. Substance Evaluation Report, August 2015).

Mode of Action Analysis / Human Relevance Framework

Available data indicate the capability of Ethanolamines to impair choline homeostasis. This underlines the hypothesis of an involvement of choline and an impaired choline uptake and/or metabolism after Ethanolamine exposure as “mode-of-action”. Rodents appear to be more sensitive towards effects on choline homeostasis and effects observed have been assessed to lack human relevance. For more details please refer to "Justification for non-classification" and to the Read Across Justification in IUCLID Section 13.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available information is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available experimental information, the test substance is not classified for toxicity to reproduction or developmental toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.

The basis for this non-classification is as follows:

1. Effects on reproductive parameters were affected only in the presence of clear parental toxicity. In a standard screening study to OECD TG 421 (BASF, 2010), Triethanolamine (TEA) was administered by gavage (vehicle water) to groups of 10 male and 10 female Wistar rats at dose levels of 0, 100, 300, or 1000 mg TEA/kg bw/day. At the highest dose level there was a statistically significant decrease in litter size and increase in post-implantation loss. The number of implantation sites was decreased by 20%, but this was not statistically significant. A reduction in maternal bodyweight gain during gestation is attributed to the smaller litter sizes in the high dose group. There were no treatment-related effects on postnatal survival or pup bodyweights. Although bodyweights in the high dose group were ca. 8% higher than control, this was not statistically significant and probably reflects, if anything, the smaller litter sizes. For the structural analogue Monoethanolamine (MEA) a two generation reproduction toxicity study in Wistar rats with dietary MEA-HCl administration demonstrated clear NOAELs for systemic and reproductive toxicity including fertility at 300 mg MEA-HCl/kg bw/day. Only at the highest dose, 1000 mg/kg bw/day, were minor effects noted. Males at this high dose levels showed minor effects on fertility in the form of decreased absolute and relative weights of epididymides and cauda epididymidis and, in the F0 generation only, a significantly lower number of homogenization resistant caudal epididymal sperm compared to control. However, there was no histomorphological correlate of these findings in the organs, no effect upon testes or testicular sperm count, and no effect upon mating performance. Females at this dose level revealed decreased numbers of implants and increased resorption rates resulting in smaller litters associated with indications of systemic toxicity. There was virtually no effect on the pre- and postnatal development of the progeny in both generations up to the limit dose level of 1000 mg/kg bw/day representing a clear NOAEL for developmental toxicity.

2. Effects of Ethanolamines can be explained by perturbation of choline-homeostasis as these effects have also been reported to occur in choline-deficient states either by nutritional choline deficiency or by genetic knockout of key enzymes such as choline kinase mimicking a choline deficient state as well. Thus, Ethanolamines-induced effects are plausibly secondary and in consequence of perturbation of choline homeostasis. This underlines the hypothesis of an involvement of choline and an impaired choline uptake and/or metabolism after Ethanolamine exposure as “mode-of-action”.

3. Rodents appear to be more sensitive towards effects on choline homeostasis and effects observed have been assessed to lack human relevance. Choline is an essential nutrient; however, rodents appear to be more susceptible towards an impaired choline-homeostasis than humans. Leung et al. (2005) summarized the evidence why humans are less susceptible for choline-deficiency than rodents in the context of the carcinogenicity endpoint (further references given within the original article):“…choline is an essential nutrient in all mammals, the proposed mechanism of DEA-induced choline deficiency is qualitatively applicable to humans. However, there are marked species differences in susceptibility to choline deficiency, with rats and mice being far more susceptible than other species including humans.

These differences are attributed to quantitative differences in the enzyme kinetics controlling choline metabolism. Rats and mice rapidly metabolize choline to betaine in the liver and it is likely that choline oxidase activity determines choline requirements and controls species sensitivity to choline deficiency.

For example, choline oxidase activity is much lower in primates than rodents and primates are less sensitive to choline deficiency. Humans have the lowest choline oxidase activity of all species and are generally refractory to choline deficiency, with evidence of choline deficiency observed only after prolonged fasting, significantly depressed liver function or deficient parenteral feeding. It is noteworthy that there was no evidence of GJIC inhibition in human hepatocytes treated with DEA or cultured in choline-deficient media.”

For TEA it is reported that it decreases the hepatic levels of Phosphatidylcholine and Betaine, the primary oxidation product, up to 26-42% indicating a disturbance when TEA is given dermally to female B6C3F1 mice (Stott, 2004) at the high dose of 1000 mg/kg bw/day. In this study by Stott et al. (2004) no changes on hepatic Phosphatidylcholine and Betaine were reported in F344-derived rats. However, only a single dose of 250 mg TEA/kg bw/day was tested in female rats for 3 weeks (5days/week). Higher doses of TEA applied orally as it has been done in the available OECD 421 might cause the same effects as observed in mice. Furthermore, a strain difference in rats’ sensitivity to choline depletion cannot be excluded. TEA also inhibited the ³H-choline uptake in vitro in Chinese hamster ovary cells.

Moore and co-workers investigated the potential role of choline antagonism in the aetiology of Monoethanolamine (MEA)-induced implantation loss. When administered to pregnant rats during gestation days (GD) 1–3, 4–5, or 6–7, MEA had no effect upon implantation success. In a second experiment, MEA was administered either in the diet or by oral gavage from two weeks prior to mating through to GD 8. Parallel groups also received a diet supplemented with choline. In the absence of supplementary choline, MEA induced early resorptions, statistically significant only when administered in the diet. A slight reduction in implantation success was ameliorated by supplementary choline. It was concluded that implantation is affected by MEA only when exposure starts before mating; that dietary administration is more effective than gavage dosing; and that interference with choline homeostasis may play a role in the aetiology of this lesion. Two possible molecular targets were hypothesized for amine alcohols: the platelet activating factor (PAF) and the endocannabinoids. PAF is a choline-derived phospholipid autacoid, which is critical in pre-implantation development and implantation (Moore et al. 2018). Studies show that injection of a specific PAF antagonist into the uterine horn of pregnant rats once during GD 1-4 reduced the number of implanted embryos significantly (Acker et al., 1988). Competition between choline and ethanolamine for uptake into local tissues or within the pathways for PAF synthesis might explain the ameliorating effect of choline supplementation upon MEA-induced implantation loss (Moore et al., 2018).

In a OECD TG 443 study performed with the structural analogue DEA the platelet activating factor (PAF) concentration in serum of the F0 females was reduced dose-dependently when regarding medians with a decrease of 31% in test group 3 (1000 ppm) compared to controls. As supporting evidence, in the OECD TG 443 study which is available for DEA a clear decrease in the choline levels was seen. The analytical results demonstrated the clear presence of choline in all plasma samples from the animals dosed with the test substance DEA (100 ppm, 300 ppm and 1000 ppm dosed animals) and in those from control, non-dosed animals. In general, it can be stated that the presence of the test substance DEA led to a reduction in the content of choline in the plasma samples analysed. This effect appears to be dose-dependent, in that higher dose levels were associated with greater choline reduction. This effect is most clearly visible at lower dose levels (100 ppm and 300 ppm), at which dramatic plasma choline levels could be seen. At higher dosing levels, although further minor plasma choline content reduction was observed, this was by no means as drastic. Furthermore, also in the offspring the analytical results demonstrated the clear presence of choline in all liver samples from the animals dosed with the test substance DEA (100 ppm, 300 ppm and 1000 ppm dosed animals) and in those from control, non-dosed animals. This was true from all time points investigated (4-day old pups, 22-day old pups and ~90-day old adolescents). In general, it can be stated that the presence of the test substance DEA led to a reduction in the content of choline in the liver samples analyzed. This effect appears to be dose-dependent, in that higher dose levels were associated with greater choline reduction, but only up to moderate dosing levels (300 ppm and 100 ppm, depending on the sampling day). At higher dosing levels, no further dramatic liver choline content reduction was observed. This effect was however, not observed in 4-day old animals, in which no clearly definable dose dependent trend is evident. In 22-day old animals this effect could be clearly observed, although the choline levels of the 100 ppm dosed animals have not yet attained minimal concentrations. In ~90-day old animals the effect is dramatic in that the liver choline levels of all non-control animals have reached an approximate minimum. Only a relatively minor further dose-dependency can be observed at this time point.

For DEA various mechanistic in vitro and in vivo studies identified that choline depletion is the key event in hepatic carcinogenicity. DEA decreased gap junctional intracellular communication (GJIC) in primary cultured mouse and rat hepatocytes; induced DNA hypomethylation in mouse hepatocytes; decreased phosphatidylcholine synthesis; and increased S-phase DNA synthesis in mouse hepatocytes, but had no effect on apoptosis. All of these effects were mediated by the inhibition of choline sequestration, and were prevented with choline supplementation. No such effects were noted in human hepatocytes in vitro. Apparent differences in the susceptibility of two different mice strains (B6C3F1 > C57BL) were noted. B6C3F1 mice are extremely sensitive to non-genotoxic effects and are susceptible to spontaneous liver tumors. Moreover, chronic stimulation and compensatory adaptive changes of hepatocyte hypertrophy and proliferation are able to enhance the incidence of common spontaneous liver tumors in the mouse by mechanisms not relevant to humans (adapted from the DEA OECD SIAR, 2009).

However, there are marked species differences in susceptibility to choline deficiency, with rats and mice being far more susceptible than other species including humans. It is reported that primates are much more resistant towards adverse effects of choline‐deficiency and associated changes (Hoffbauer and Zaki, 1964). Moreover, quantitative data underline this species difference: choline oxidase, the key enzyme in converting choline into betaine is highly active in rodents whereas it plays a minor role in Humans (Sidransky & Faber, 1960). The reaction / metabolism with betaine occur mainly in the liver and is of minor importance for the overall metabolism of homocysteine in humans; it is of major significance only in rodents. This is because betaine is derived from choline, a pathway of minimal importance and hence of little relevance in primates, who have a paucity of choline oxidase in the liver (Lieber and Packer, 2002). Choline metabolism is connected to Phosphatidylcholine and Betaine. The latter is reported to be central for the synthesis of SAM (S-Adenosyl-Methionine), a principle methylating agent for biosynthetic pathways and maintenance of critical gene methylation patterns (Stott et al. 2004; Zeisel and Blusztajn, 1994).

Taken together, similar effects on pre- and/or post-implantation losses were observed for Mono-, Di- and Triethanolamine. Additionally, Ethanolamines show similar effects on choline-metabolism. It is likely that the effects of TEA and its structurally analogues substances MEA and DEA on pre- and post-implantation in laboratory animals are mediated by effects on choline homeostasis rather than through direct embryo toxicity. These effects are inhibition of choline-uptake in the liver, subsequent perturbation of choline-homeostasis, with subsequent impairment of C1-metabolism, DNA-methylation, lipid metabolism, and intercellular communication. These effects are judged to be relevant for systemic toxicity of this group of substances, but are not evaluated to be direct effects on reproductive toxicity. However, rodents appear to be more sensitive towards effects on choline homeostasis and effects observed have been assessed to lack human relevance. Furthermore, effects observed for TEA on reproduction were observed in the presence of systemic toxicity and are regarded as secondary effect.

 

Therefore, TEA is not subjected for classification on toxicity to reproduction or developmental toxicity according to Regulation (EC) No 1272/2008,as amended for the tenth time in Regulation (EU) No 2017/776. Reproductive toxicity was also not identified as an additional concern during substance evaluation (cf. Substance Evaluation Report, August 2015).

References

Acker et al, Role of platelet-activating factor (PAF) in the ovoimplantation in the rat: effect of the specific PAF-acether agonist, BN 52021, Prostagaldins 35 (1988) 233-241

Ethanolamine hydrochloride Modified Developmental Toxicity Study to detect potential effects on implantation after window dose exposure in Wistar Rats (Range-finding), Ethanolamines REACH Consortium, 2015

Hoffbauer FW and Zaki FG (1965).Choline Deficiency in Baboon and Rat Compared. Arch Path 79: 364‐369

Leung HW, Kamendulis LM, Stott WT.(2005).Review of the carcinogenic activity of diethanolamine and evidence of choline deficiency as a plausible mode of action. Reg Tox and Pharmacol, 43: 260-271

Lieber CS and Packer L (2002). S‐Adenosylmethionine: molecular, biological, and clinical aspects-an introduction. Am J Clin Nutr 76(suppl): 1148–1150

Moore et al (2018) Implantation loss induced by ethanolamine in the rat is ameliorated by a choline-supplemented diet, Reproductive Toxicology 78 (2018) 102–110

OECD SIDS (2009). Diethanolamine.

Smyth et al, (1951).Range-finding Toxicity Data: List IV.Arch.Hyg. Occup. Med.4: 119-122

Stott WT, Radtke BJ, Linscombe VA, Mar M-H, Zeisel SH (2004).Evaluation of the potential of triethanolamine to alter hepatic choline levels in female B6C3F1 mice. Toxicol Sci 79:242-247.

Sidransky & Faber (1960). Liver choline oxidase activity in man and in several species of animals. Arch Biochem Biophys. 1960 Mar; 87:129-33.

Zeisel SH and Blasztajn JK (1994). Cholin and human nutrition.Ann. Rev. Nutr.14: 269-296