Registration Dossier

Administrative data

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 MDEA and studies performed with the structurally analogous substances Monoethanolamin (MEA, CAS 141-43-5), Diethanolamin (DEA , CAS 111-42-2) and Triethanolamin (TEA, CAS 102-71-6).

A screening reproduction/developmental toxicity study with MDEA in the rat by oral gavage has been performed. The NOAEL for reproductive performance and fertility was 300 mg/kg bw/day for the F0 parental rats based upon findings such as litter loss, insufficient lactation behaviour, and increased duration of gestation. The NOAEL for parental toxicity was set at 100 mg/kg bw/day, based on body weight loss in both sexes. Reproduction toxicity was only seen in the presence of parental toxicity.

Furthermore, for the structural analogue MEA, a two-generation reproduction toxicity study is available. Under the conditions of that study (performed with MEA HCl), the NOAEL for systemic toxicity and fertility / reproductive performance in parental F0 and F1 Wistar rats was 300 mg/kg bw/day. The NOAEL for pre-and postnatal developmental toxicity in their offspring was 1000 mg/kg bw/day (highest dose tested).

The NOAEL for fertility and reproductive performance for the F0 and F1 rats in a study according OECD TG 443 performed with the structural analogue DEA is 300 ppm (ca. 37.68 mg/kg bw/day), based on a lower number of implants, prolonged/irregular estrous cycles as well as pathological changes in sexual organs, pituitary and mammary glands of both genders at the LOAEL (Lowest Observed Adverse Effect Level) of 1000 ppm (ca. 128.35 mg/kg bw/day). Most of the reported effects on reproduction and reproductive organs occurred in the range of general and systemic toxicity and have been assessed to be secondary in nature.

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.

Link to relevant study records

Referenceopen allclose all

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)
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.

Endpoint:
screening for reproductive / 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
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:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11 - 13 weeks
- Weight at study initiation: male animals: 279.9 g - 315.9 g, female animals: 173.2 g - 202.8 g
- Housing:
- During the study period, the rats were housed individually
- During overnight matings, male and female mating partners were housed together
- Pregnant animals and their litters were housed together until PND 4.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5-6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
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. The volume administered each day was 10 mL/kg bw. The calculation of the administration volume was based on the most recent individual body weight.
Details on mating procedure:
Males and females from the same dose group were mated 13 days after the beginning of treatment, overnight in a ratio of 1:1.
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration control analyses of the test substance preparations:
The method of analysis was Capilary electrophoresis (CE). All measured values for N-Methyldiethanolamin were in the expected range of the target concentrations (90-110%).
Duration of treatment / exposure:
The duration of treatment covered premating period of 2 weeks and a mating period (max. of 2 weeks) in both sexes and the entire gestation period as well as 4 days of lactation in females.
Frequency of treatment:
daily
Details on study schedule:
N-Methyldiethanolamin was given daily as an aqueous solution to groups of 10 male and 10 female Wistar rats (F0 animals) by stomach tube at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/day). Control animals were dosed daily with the vehicle only (drinking water). The duration of treatment covered premating period of 2 weeks and a mating period (max. of 2 weeks) in both sexes and the entire gestation period as well as 4 days of lactation in females. The females were allowed to deliver and rear their pups until day 4 after parturition. Four days after PND 4 of the female, which delivered last, all parental females were sacrificed and examined. Pups were sacrificed on PND 4 and gross necropsied. The male animals were sacrificed 28 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
Details on study design:
- Dose selection rationale:
In a test study (Project No. 06R0087/01248, BASF SE) N-Methyldiethanolamin was given daily as an aqueous solution to groups of 3 male and 3 female Wistar rats (F0 animals) by stomach tube at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) for 2 weeks. Control animals were dosed daily with the vehicle only (drinking water). Clinical signs, food consumption and body weights were determined. The only clearly substance-related effect was “salivation after treatment” in all males and females at the high dose level. Food consumption and body weights did not show significant differences.
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
A check for moribund or dead animals was made twice daily on working days or once daily (weekends or public holidays).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: once a week in males throughout the study and in females during premating and mating. During gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the parturition day and postnatal day (PND) 4.

FOOD CONSUMPTION:
Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4.
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 were detected, and gestational status were recorded for F0 females.
For the females, mating, fertility and gestation indices were calculated.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight and epididymis weight, and mating and fertility indices (male mating index and male fertility index) were calculated.
Litter observations:
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.

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.

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; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy.

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.

The pups were weighed on the day after birth (PND 1) and on PND 4.

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.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals 28 days after the beginning of the administration
- Maternal animals: All surviving animals. Four days after PND 4 of the female, which delivered last, all parental females were sacrificed and examined.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:

1. Anesthetized animals
2. Epididymides
3. Testes
4. Ovaries
5. Liver

The following organs or tissues of parental animals were fixed in 4% buffered formaldehyde or in modified Davidson’s solution:

1. Adrenal glands
2. All gross lesions
3. Testes (fixed in modified Davidson’s solution)
4. Epididymides (fixed in modified Davidson’s solution)
5. Pituitary gland
6. Prostate gland, seminal vesicles, coagulation glands
7. Ovaries (fixed in modified Davidson’s solution)
8. Uterus, oviducts, vagina
9. Parts of the liver (lobus dexter lateralis, processus caudatus, processus papillaris) of all male and female animals were deeply frozen and stored at -80°C for possible analysis of choline. The remaining parts of the liver were fixed in 4% buffered formaldehyde solution.

Histopathology was performed on the following organs: All gross lesions, Testes, Epididymides, Ovaries.
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: 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 (%)=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%

Female mating index (%)= number of females mated/ number of females placed with males x 100%

Female fertility index (%)= number of females pregnant/number of females mated x 100%

Gestation index (%)= number of females with live pups on the day of birth/number of females pregnant x 100%
Offspring viability indices:
Postimplantation loss (%)=number of implantations – number of pups delivered/number of implantations x 100%

Live birth index (%)= number of liveborn pups at birth/total number of pups born x 100%

Viability index (%) = number of live pups on day 4 after birth/number of live pups on the day of birth x 100%
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day:
• Decreased body weight gain in males in treatment weeks 2-3 (57% below control) and 0-3 (29% below control)
• Decreased body weights in females on gestation days 14 and 20 (up to 14% below control) and lactation day 4 (about 5% below control)
• Decreased body weight gain in females between gestation days 7-20 (up to about 46% below control)
300 mg/kg bw/day:
• Decreased terminal body weights in males and females (5% below controls in both sexes)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day:
• Decreased food consumption in females (-37%) during lactation days 1-4
Description (incidence and severity):
1000 mg/kg bw/day:
• Total litter loss in 4 females
• Undelivered pups palpable in 2 females
• Insufficient lactation behavior in 2 females (pups had no or less milk in stomach)
• Increased duration of gestation (22.8 days vs. 21.9 days in control)
• Decreased number of implantation sites (6.7 vs. 12.9 in control)
• Increased postimplantation loss (31.0% vs. 6.0% in control) and mean postimplantation loss
• Decreased number of delivered pups (4.6 vs. 12.1 in control)
• Decrease of terminal body weights in males and females (7 and 5 % below controls, respectively)

Details on fertility:
MALE FERTILITY
The male fertility index ranged between 90% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.
Beside for one male of test group 2 (300 mg/kg bw/day, animal No. 25), for all F0 parental males copulation was confirmed. Thus, the male mating index varied in the different test groups between 90% (test group 2) and 100% (test groups 0, 1 and 3). The animal No 25 showed histopathologically seminiferous tubule atrophy in the testes and aspermia in the epididymides which explained the observed infertility. However, due to the lack of a dose response relationship, this was assessed as being incidental.

FEMALE FERTILITY
All sperm positive rats delivered pups or had implants in utero. Thus, the fertility index was 100% in all test groups.
The female mating index varied between 90% (test group 2) and 100% (test groups 0, 1 and 3). The non pregnant female rat of test group 2 (300 mg/kg bw/day, animal No. 125, mated with male No. 25) did not show any macroscopic findings to explain these infertility. However, as the male animal No. 25 showed reduced size of epididymides and testes, accompanied by seminiferous tubule atrophy and aspermia, the infertilily of this pair can clearly be attributed to the male animal.
The mean duration until sperm was detected (GD 0) varied between 1.6 and 2.6 days without any relation to dosing.
The mean duration of gestation was statistically significantly increased in test group 3 (22.8 days). The values in test groups 0 – 2 varied between 21.9 and 22.1 days.
The number of implantation sites was distinctly reduced in test group 3 (1000 mg/kg bw/day; 6.7 vs. 12.9 in control). In this group, both the number of resorptions as well as the % postimplantation loss was distinctly increased (21 and 31.0% vs. 8 and 6.0% in control).
The gestation index was 100% in all groups.
The rate of delivered pups was distinctly reduced in test group 3 (1000 mg/kg bw/day; 4.6. vs. 12.1 in control). However, the live birth index was not changed.
In test group 1 (100 mg/kg bw/day and 2 (300 mg/kg bw/d) the implantation, prenatal development and delivery were not affected by the treatment since neither the mean number of implantation sites nor the postimplantation loss or the average litter size showed any statistically significant differences between these groups and the control.
The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 97% to 100 %. Moreover, the number of stillborn pups did not differ statistically significant between the groups.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive performance and fertility
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on findings such as litter loss, insufficient lactation behavior, and increased duration of gestation.
Key result
Critical effects observed:
no
1000 mg/kg bw/day:
• Reduced viability index (62%), resulting from significantly higher numbers of died and cannibalized pups
• Decreased pup body weights on PND 4, average difference to the control 20%
• Decreased pup body weight gain during PND 1 – 4, average difference to the control 52%

300 and 100 mg/kg bw/day:
• No test substance-related adverse findings
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on findings such as reduced viability index and reduced postnatal offspring weight gain.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
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:
Purity: 99.5 area-%
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
Endpoint:
extended one-generation reproductive toxicity - with both developmental neuro- and immunotoxicity (Cohorts 1A, 1B without extension, 2A, 2B, and 3)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
09 Dec 2014 - 29 Jan 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study) with the developmental neurotoxicity and immunotoxicity (DNT/DIT) cohorts but without the extension of Cohort 1B to mate the F1 animals to produce an F2 generation
GLP compliance:
yes
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals: 2 weeks
- Basis for dose level selection: dose-range finder similar or according to OECD 421
- Inclusion/exclusion of extension of Cohort 1B: yes
- Termination time for F2: no F2 generation was required
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: inclusion of Cohorts 2A and 2B
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: inclusion of Cohort 3
- Route of administration: raol via drinking water
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals: regular strain in reproductive toxicity studies. 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.

The design of the present OECD 443 Extended-one-genereation Reproductive Toxicity Study has been agreed with ECHA and eMSCA Germany in the according CORPA process and at MSC.

The objective of this multi-site study was to obtain general information on the possible effects of 2,2’-iminodiethanol on the integrity and performance of the male and female reproductive systems, including gonadal function, estrous cyclicity, mating behavior, conception, gestation, parturition, lactation and weaning, as well as on growth and development of the offspring. The study should also provide information about the effects on neonatal morbidity, mortality, target organs of the pups and preliminary data on prenatal and postnatal developmental toxicity. As part of this assessment, offspring were investigated for effects on the embryonic, fetal and preadult development of the nervous and immune systems as well as alterations in endocrine function (including thyroid pertubations). In addition choline was determined in blood plasma and livers of F1 adolescents.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 000STD77L0 v. 14.05.14 / Test substane number (internal): 14/0295-1
- Expiration date of the lot/batch: 14 May 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature. The stability of the test substance under storage conditions over the test period was guaranteed by the Sponsor, and the Sponsor holds this responsibility.
- Stability under test conditions: given, The stability of test substance in drinking water was demonstrated for a period of 9 days at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: given (soluble in water) Due to the fact that the test substance preparations were true solutions, it was not considered necessary to prove homogeneity through analytical procedures.

OTHER SPECIFICS:
- All mean values for 2,2’-iminodiethanol were in the expected range of the target concentrations (90 - 110%), demonstrating the correctness of the drinking water preparations
- Determination of total N-Nitrosamine content: No measurable content of total N-Nitrosamine (calculated as N-Nitrosodiethanolamine, NDELA) was detected in the test item preparations
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 9 wks
- Housing: 1 animal per cage, polycarbonate cages type III with wooden gnawing blocks (Typ NGM E-022) and dust-free wooden bedding; during mating: 1 male/1 female per cage; during rearing up to weaning: 1 dam with her litter
- Diet: Ground Kliba maintenance diet mouse/rat “GLP”; ad libitum
- Water: Drinking water ad libitum
- Acclimation period: at least 5 days

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

Food analyses
With regard to the analytical findings of chemical and microbiological contaminants and the duration of application, the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The concentration of microorganisms did not exceed 1*105/g feed.

Drinking water analyses
On the basis of the analytical findings, the drinking water was found to be suitable. German Drinking Water Regulation (Trinkwasserverordnung, Bundesgesetzblatt, 05 Dec 1990) served as a guideline for maximum tolerable contaminants.

Bedding and Enrichment analyses
On the basis of the analytical findings, bedding and cage enrichment were found to be suitable. Levels given in Lab Animal (Nov-Dec 1979, pp. 24-34) served as a guideline for maximum tolerable contaminants.

Route of administration:
oral: drinking water
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the test substance preparation, the specific amount of test substance and deionized water will be weighed in a vessel, depending on the dose group, and mixed until it is completely dissolved (details will be retained with the raw data).

Adjustment of pH: The pH of the administration solution of test groups 1-3 is adjusted to 7.3-7.5 by the addition of hydrochloric acid (1N, analytical grade) under pH meter control.

The test substance preparations will be prepared at intervals which guarantee that the test substance concentrations in the diet will remain stable. Stock solution will be stored at room temperature, protected from light and air.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until there is evidence of copulation or when a maximum period of 14 days has elapsed
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: single
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical investigations of the test substance preparations was carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany, as a part of this study.

Duration of treatment / exposure:
The F0 animals will be offered drinking water preparations with test substance for approximately 2 weeks prior to breeding and continuing through breeding (up to two weeks), approximately 4 additional weeks (males) or gestation (three weeks) and lactation (three weeks) for females. Selected F1 offspring (cohorts 1A, 1B, 2A, 2B and 3) will be maintained on drinking water preparations with test substance until sacrifice.
Frequency of treatment:
daily
Details on study schedule:
F0 GENERATION PARENTAL ANIMALS AND F1 PUPS
Male and female rats, aged about 10 or 9 weeks when supplied, were used as F0 generation parental animals. After an acclimatization period of at least 5 days, these rats were kept for at least 2 weeks.Then the F0 animals were paired. The female F0 animals were allowed to deliver and rear their pups (F1 generation pups) until postnatal days (PND) 4 or 21. The F0 generation parental animals were sacrificed after weaning of the F1 generation pups. All F0 females were sacrificed at about the same day after weaning (+/- 2 days).

F1 PUPS AND SELECTION OF COHORTS
Before weaning of the F1 generation pups on PND 21, 75 male and 75 females per group were randomly selected, to be placed into cohorts. Obvious runts (those pups whose body weight is equal to or greater than 25% below the mean body weight of the control group, separate for sexes) were not included.

Cohorts:
Cohort 1A: One male and one female/litter (20/sex/group)
Cohort 1B: One male and one female/litter (25/sex/group)
Cohort 2A: One male or one female/litter (10/sex/group)
Cohort 2B: One male or one female/litter (10/sex/group)
Cohort 3: One male or one female/litter (10/sex/group)

Selected F1 offspring were maintained on drinking water preparations with test substance until sacrifice.

BREEDING OF THE F0 GENERATION PARENTAL ANIMALS
Male and female animals were paired overnight in a 1:1 ratio until there is evidence of copulation or a maximum period of 14 days has elapsed. Throughout the mating period, each female was paired with a predetermined male.

Normally, the female was placed in the cage of her male partner about 16:00 h and separated from the male between 7:00 h and 9:00 h on the following morning. Deviations from the specified times are possible on Saturdays, Sundays and public holidays and was documented in the raw data.

A vaginal smear was prepared after each pairing and examined for sperm. If sperm are detected, pairing of the animals was discontinued. The day on which sperm are detected will be referred to as gestation day (GD) 0 and the following day as GD 1.

STANDARDIZATION OF LITTERS (CULLING) OF F1 PUPS
On PND 4, all litters will be standardized to 10 pups per litter. Whenever possible, each litter contains 5 male and 5 female pups; as a rule, the first 5 surviving pups/sex in each litter were used. If it is not possible to have 5 pups/sex in each litter, unequal numbers of males and females can be retained (e.g. 6 male and 4 female pups). Surplus animals were sacrificed.
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
Remarks:
approx. 12.75 mg/kg body weight/day (mg/kg bw/d)
Dose / conc.:
300 ppm (nominal)
Remarks:
approx. 37.68 mg/kg bw/d
Dose / conc.:
1 000 ppm (nominal)
Remarks:
pprox. 128.35 mg/kg bw/d
No. of animals per sex per dose:
F0 parental animals: 30
F1 rearing animals, cohort 1A: 20
F1 rearing animals, cohort 1B: 25
F1 rearing animals, cohort 2A: 10
F1 rearing animals, cohort 2B: 10
F1 rearing animals, cohort 3: 10
Control animals:
yes, concurrent no treatment
Positive control:
The positive control substance (for immune system response) was used with the given specifications of the producer (Sigma-Aldrich, Taufkirchen, Germany). No further analyses will be conducted.

Name of positive control substance: Cyclophosphamide monohydrate
CAS No.: 6055-19-2
Test-substance No.: 05/0012-6
Batch identification: SLBC0666V
Purity: 102.3% (according to supplier)
Homogeneity: Given (visually)
Stability: Expire date: March 2015
The stability of the positive control substance under storage conditions over the test period was guaranteed by the manufacturer and the manufacturer hold this responsibility.
Physical state/color: Solid/white
Storage conditions: Refrigerator (2-8°C)
The stability of Cyclophosphamide monohydrate (positive control substance) in the vehicle for 7 days at room temperature and 32 days frozen was verified in a comparable batch (Project No. 01Y0012/058064).

TEST SUBSTANCE PREPARATIONS AND ADMINISTRATION
Route of administration: Oral administration by gavage using 3 or 5 mL syringes.
Frequency of administration: Once daily
Volume to be administered: 10 mL/kg body weight; the body weight determined most recently will be used to calculate the administration volume.
Dose to be administered: 4.5 mg/kg body weight; the body weight determined most recently will be used to calculate the administration dose.
Preparation: Details on the technical procedure will be laid down in a description of the method and reported correspondingly.
Preparation frequency: At least once before the administration period.
Storage conditions of the test-substance preparations: The positive control substance preparations will be split in daily aliquots and stored closed in a freezer at -18°C.
Duration of the study: 4 weeks

Immunization and examinations (Anti SRBC-IgM ELISA) will be conducted at the same time and in exactly the same manner as with cohort 3 animals.
Parental animals: Observations and examinations:
MORTALITY
A check for moribund and dead animals will be made twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays.

CLINICAL SIGNS
A cageside examination will be conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. If such signs occur, the animals will be examined several times daily. Abnormalities and changes will be documented for each animal. The parturition and lactation behavior of the dams will generally be evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver or umbilical cord not cut) will be documented on an individual dam basis. On weekdays (except Saturdays, Sundays and public holidays) the parturition behavior of the dams will be inspected in the afternoons in addition to the evaluations in the mornings.
The day of parturition is considered to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day. Departures from this procedure may occur on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS (DCO)
All F0 parental animals will be subjected to detailed clinical observations (DCO) outside their cages at the administration period (day 0) and subsequently once per week (in the morning) by the same trained technicians, whenever possible. For observation, the animals will therefore be removed from their cages and placed in a standard arena (50 × 37.5 × 25 cm). The scope of examinations and the scoring of the findings observed will be based on the current index of findings in GROSSE-Reprotox software and includes but is not limited to the following parameters listed:

1.Abnormal behavior in handling
2.Fur
3.Skin
4.Posture
5.Salivation
6.Respiration
7.Activity/arousal level
8.Tremors
9.Convulsions
10.Abnormal movements
11.Gait abnormalities
12.Lacrimation
13.Palpebral closure
14.Exophthalmos (Protruding eyeball)
15.Assessment of the feces excreted during the examination (appearance/consistency)
16.Assessment of the urine excreted during the examination
17.Pupil size

FOOD CONSUMPTION
Generally, food consumption will be determined once a week for the male and female F0 parental animals, with the following exceptions:
• Food consumption will not be determined after the 2nd premating week (male F0 animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm will be determined for GD 0-7, 7-14 and 14-20.
• Food consumption of the F0 females, which gave birth to a litter, will be determined for PND 1-4, 4-7, 7-14 and 14-21.
Food consumption will not be determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

WATER CONSUMPTION
Generally, water consumption will be determined twice a week for male and female F0 parental animals.
• Additionally, after the 2nd premating week water consumption of the females during gestation (animals with evidence of sperm plugs) will be determined for GDs 0-1, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21.
• Water consumption of the F0 females, which gave birth to a litter, will be determined for PNDs 1-2, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21.
Water consumption will not be determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

BODY WEIGHTS
In general, the body weight of the male and female F0 parental animals will be determined once a week at the same time of the day (in the morning). The following exceptions are notable for the female parental animals:
• During the mating period of the F0 parental animals, the females will be weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter will be weighed on the day after parturition (PND 1) and on PND 4, 7, 14 and 21.
Body weight will not be determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

CLINICAL PATHOLOGY IN F0 PARENTAL ANIMALS
Samples will be withdrawn from 10 F0 parental males and females per group at termination.

Blood samples will be taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood examinations will be carried out in a randomized sequence. The list of randomization instructions will be compiled with a computer.

In the afternoon preceding the day of urinalysis, the animals will be individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens will be examined in a randomized sequence (the list of randomization instructions will be compiled with a computer).

The following parameters will be examined in all animals:
Hematology
1.Leukocytes
2.Erythrocytes
3.Hemoglobin
4.Hematocrit
5.Mean corpuscular volume (MCV)
6.Mean corpuscular hemoglobin (MCH)
7.Mean corpuscular hemoglobin concentration (MCHC)
8.Platelets
9.Differential blood count
10.Reticulocytes
11.Blood smear (only evaluated preparations will be archived)
12.Prothrombin time

Clinical chemistry
1.Alanine aminotransferase
2.Aspartate aminotransferase
3.Alkaline phosphatase
4.Serum glutamyl transferase
5.Sodium
6.Potassium
7.Chloride
8.Inorg. phosphate
9.Calcium
10.Urea
11.Creatinine
12.Glucose
13.Total bilirubin
14.Total protein
15.Albumin
16.Globulins
17.Triglycerides
18.Cholesterol

Hormone evaluations
The following hormones will be determined in the serum samples:
1.T4 (thyroxine)
2.TSH

Urinalysis
1.Volume
2.Color
3.Turbidity
4.pH value
5.Protein
6.Glucose
7.Ketones
8.Urobilinogn
9.Bilirubin
10.Blood
11.Specific gravity
12.Microscopy of sediment
Oestrous cyclicity (parental animals):
For all F0 females, estrous cycle length and normality will be evaluated by preparing vaginal smears during a minimum of 2 weeks prior to mating and throughout cohabitation until there is evidence of sperm in the vaginal smear.

In all cohort 1A and 1B females, vaginal smears will be collected after vaginal opening until the first cornified smear (estrous) is recorded. The estrous cycle also will be evaluated in cohort 1A and 1B females for 2 weeks around PND 75.

Additionally, on the day of scheduled sacrifice, the estrous status will be determined in all female F0 animals and all females of cohorts 1A and 1B.
Sperm parameters (parental animals):
After the organ weight determination, the following parameters will be determined in the right testis or right epididymis of all male F0 parental animals and cohort 1A males sacrificed on schedule:

• Cauda epididymis sperm motility
• Sperm morphology
• Spermatid head count in the testis
• Sperm head count in the cauda epididymis

Initially, sperm morphology and sperm head count (cauda epididymis and testis) will be evaluated for the control and highest dose group, only. The intermediate doses will only be evaluated if deemed necessary based on the obtained results or other findings.

On completion of the final report, the testis and epididymis samples that have not been processed will be discarded. Sperm morphology slides will be archived.
Litter observations:
MORTALITY
A check for moribund and dead animals will be made twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS (DCO)
All F1 animals in cohorts 1A, 1B, 2A and 3 will be subjected to detailed clinical observations (DCO) outside their cages at the administration period (day 0) and subsequently once per week (in the morning) by the same trained technicians, whenever possible. For observation, the animals will therefore be removed from their cages and placed in a standard arena (50 × 37.5 × 25 cm). The scope of examinations and the scoring of the findings observed will be based on the current index of findings in GROSSE-Reprotox software and includes but is not limited to the following parameters listed:
1.Abnormal behavior in handling
2.Fur
3.Skin
4.Posture
5.Salivation
6.Respiration
7.Activity/arousal level
8.Tremors
9.Convulsions
10.Abnormal movements
11.Gait abnormalities
12.Lacrimation
13.Palpebral closure
14.Exophthalmos (Protruding eyeball)
15.Assessment of the feces excreted during the examination (appearance/consistency)
16.Assessment of the urine excreted during the examination
17.Pupil size

FOOD CONSUMPTION
Generally, food consumption will be determined once a week for the F1 rearing animals.

WATER CONSUMPTION
Generally, water consumption will be determined twice a week for F1 rearing animals.

BODY WEIGHTS
In general, the body weight of the F1 rearing animals will be determined once a week at the same time of the day (in the morning).

AUDITORY STARTLE RESPONSE HABITUATION IN COHORT 2A ANIMALS
On PND 24±1, the auditory startle response test will be carried out in all animals of cohort 2A using the SR-LAB; STARTLE RESPONSE SYSTEM (San Diego Instruments, San Diego, CA, U.S.A.). The examinations are started in the morning. Age-appropriate sized enclosures are used. The animals are given a 5 minute acclimation period in the response chamber with a 70 dBA background noise. Then the startle response is recorded in 50 trials at a startle stimulus sound level of 120 dBA with a 5 - 10 second variable interval between the trials. Response is recorded for 50 milliseconds. Measurement is carried out with the light and ventilator switched on in the measurement chambers; no feed or water is provided during the test. Data (maximum amplitude, latency to the peak of the response) are analyzed in 5 blocks of 10 trials each.

On completion of measurement, the data will be printed. The printouts will be signed with the project No., date and name, and will be part of the raw data.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB) IN COHORT 2A ANIMALS
The FOB will be carried out once, between PND 63-75, in all animals of cohort 2A. The examinations will generally start in the morning at about 10:00 h. The FOB will be carried out in a randomized sequence. The animals will not be transferred to new cages before the test, nor will food or drinking water be withdrawn. The FOB will start with passive observations without disturbing the rats, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings will be ranked according to their degree or severity, if applicable.

Home cage observation
The animals will be observed for a short period (about 10-30 seconds) in their cages with the lids closed in the rack, while disturbing influences (touching of the cage and loud noises) are avoided. While other abnormalities will be recorded, particular attention will be paid to the following parameters:
1.Posture
2.Tremors
3.Convulsions
4.Abnormal movements
5.Gait
6.Other findings

Open field observation
For observation, the animals will be removed from their cages by the investigator and placed in a standard arena (50 × 50 × 25 cm). Besides noting other abnormalities, the following parameters will be assessed:
1.Behavior on removal from the cage
2.Fur
3.Skin
4.Salivation
5.Nasal discharge
6.Lacrimation
7.Eyes/ pupil size
8.Posture
9.Palpebral closure
10.Respiration
11.Tremors
12.Convulsions
13.Abnormal movements/ stereotypes
14.Gait
15.Activity/ arousal level
16.Feces (consistency/color) excreted during the examination (2 minutes)
17.Urine (amount/color) excreted during the examination (2 minutes)
18.Rearing within 2 minutes
19.Other findings

Sensory-motoric test/Reflexes
The animals will be removed from the open field and will be subjected to the sensory motor and reflex tests listed below:
1.Reaction to an object being moved towards the face (Approach response)
2.Touch sensitivity (Touch response)
3.Vision (Visual placing response)
4.Pupillary reflex
5.Pinna reflex
6.Audition (Startle response)
7.Coordination of movements (Righting response)
8.Behavior during handling
9.Vocalization
10.Pain perception (Tail pinch)
11.Other findings
12.Grip strength of forelimbs
13.Grip strength of hindlimbs
14.Landing foot-splay test

Motor activity measurement
The Measurement of motor activity (MA) will be carried out between PND 63-75, in all animals of cohort 2A. The MA will be measured from 14:00 h onwards on the same day as the FOB will be performed. The examinations will be performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals will be placed in clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams are allocated per cage. The number of beam interrupts will be counted over 12 intervals for 5 minutes per interval. The sequence at which the animals are placed in the cages will be selected at random. Since the starting time of each animal will be vary by the time needed to place the animals in the cages, the measurement periods will be staggered accordingly, beginning at about 14:00 h for the first animal. The individual measurement period begins when the 1st beam is interrupted and finishes exactly 1 hour later. No food or water will be offered to the animals during these measurements. After the transfer of the last animal in each case, the measurement room will be darkened. The program requires a file name for the measured data to be stored. This name consists of the reference number and a serial number.

CLINICAL EXAMINATIONS OF F1 GENERATION PUPS
Pup status and litter size after birth
The status (sex, live-born or stillborn) and number of all pups delivered from the F0 parents will be determined as soon as possible after birth. At the same time, the pups will also be examined for gross-morphological changes.

Pup viability/mortality
In general, a check will be 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.

Pups, which died before the first determination of their status on the day of birth, will be defined as stillborn pups.

Clinical signs
All live pups will be examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups show particular findings, these will be documented with the dam concerned.

Nipple/areola presence
All surviving F1 male pups will be examined for the presence of nipples/areolas on PND 12 and again towards the end (either PND 20 or 21) of the lactation phase.

Anogenital distance
Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements will be conducted in a blind randomized fashion, using a measuring ocular, on all live F1 pups on postnatal day 1.

Anogenital index
The anogenital index will be calculated according to the following formula:

anogenital index = anogenital distance [mm] / cubic root of pup weight [g]

Pup body weights
The pups will be weighed on the day after birth (PND 1) and on PND 4, 7, 14 and 21. Whenever possible, the individual weights will always be determined at the same time of the day (in the morning) and on PND 4 before standardization of the litters.

Additionally, the body weight of all F1 rearing animals will be determined on the day of vaginal opening and preputial separation.

Puberty onset
Developmental landmarks of puberty onset will be recorded on the following days in all cohort 1A, 1B, 2A, and 3 animals:
- From PND 27 onward: vaginal patency
- From PND 38 onward: balanopreputial separation

Recording will be completed when all animals are sexually mature or latest on PND 66 in males and in females.

CLINICAL PATHOLOGY IN COHORT 1A ANIMALS
Samples will be withdrawn from 10 cohort 1A males and females per group at termination.

Blood samples will be taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood examinations will be carried out in a randomized sequence. The list of randomization instructions will be compiled with a computer.

In the afternoon preceding the day of urinalysis, the animals will be individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens will be examined in a randomized sequence (the list of randomization instructions will be compiled with a computer).

The following parameters will be examined in all animals:
Hematology
1.Leukocytes
2.Erythrocytes
3.Hemoglobin
4.Hematocrit
5.Mean corpuscular volume (MCV)
6.Mean corpuscular hemoglobin (MCH)
7.Mean corpuscular hemoglobin concentration (MCHC)
8.Platelets
9.Differential blood count
10.Reticulocytes
11.Blood smear (only evaluated preparations will be archived)
12.Prothrombin time

Clinical chemistry
1.Alanine aminotransferase
2.Aspartate aminotransferase
3.Alkaline phosphatase
4.Serum glutamyl transferase
5.Sodium
6.Potassium
7.Chloride
8.Inorg. phosphate
9.Calcium
10.Urea
11.Creatinine
12.Glucose
13.Total bilirubin
14.Total protein
15.Albumin
16.Globulins
17.Triglycerides
18.Cholesterol

Hormone evaluations
The following hormones will be determined in the serum samples:
1.T4 (thyroxine)
2.TSH

Urinalysis
1.Volume
2.Color
3.Turbidity
4.pH value
5.Protein
6.Glucose
7.Ketones
8.Urobilinogen
9.Bilirubin
10.Blood
11.Specific gravity
12.Microscopy of sediment

HORMONES IN PND 4 AND 22 F1-OFFSPRING
Blood sampling
Blood samples will be withdrawn from 10 surplus (culled) PND 4 offspring (as far as possible of different litters) per sex and group. PND 4 samples may be pooled per sex and litter if the available amount is not sufficient for a hormone analysis.
Blood samples will be withdrawn from 10 surplus PND 22 offspring (as far as possible of different litters) per sex and group.
The blood samples will be collected after decapitation (following isoflurane anesthesia) from the Vena cava cranialis.

Hormone evaluations
The following hormones will be determined in the serum samples:
1.T4 (thyroxine)
2.TSH

BLOOD SAMPLING FOR CHOLINE DETERMINATION IN COHORT 1B ANIMALS
Blood will be sampled from all cohort 1B males and females shortly before sacrifice. Blood samples will be taken from non-fasted animals by puncturing the retrobulbar venous plexus under Isoflurane anesthesia. On the specific sampling days, samples will be withdrawn about 4 hours after lights turned on.

Blood samples (1 mL) will be collected in prefilled commercial EDTA tubes (e.g. Fa. Sarstedt). The samples will be centrifuged. The plasma will be separated. The preparation of the samples will be done under cooling. All samples will be stored at -80°C until analysis.

DEVELOPMENTAL IMMUNOTOXICITY EXAMINATIONS IN COHORT 3 ANIMALS
T-cell dependent antibody response
All males and females of cohort 3 will be used to assess the functional responsiveness of major components of the immune system to a T-cell dependent antigen, sheep red blood cells (SRBC). For this purpose, the Anti SRBC-IgM ELISA of Life Diagnostics Inc, West Chester, USA (cat. no. 4200-2), will be performed. Each sample will be diluted 1:500. SRBC-IgM concentrations outside the standard curve range will be measured in a second test run with an appropriate dilution. Generally, two in-house controls will be measured with each test run. The ELISA will be measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Immunization:
Route of administration: Intraperitoneal, using 1 mL tuberculin-syringes
Preparation: Details of preparation of the sheep red blood cells will be described in the raw data (4×108 SRBC/mL)
Frequency of administration: twice (within one action)
Administration volume: 0.5 mL per animal, split into two portions of 0.25 mL
Five days after immunization blood samples will be taken by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood examinations will be carried out in a randomized sequence. The list of randomization instructions will be compiled with a computer.

Splenic lymphocyte subpopulation analysis
Ten males and females per group of cohort 1A will be used to perform a splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells) using one half of the spleen, the other half of the spleen being preserved for histopathological evaluation.

Cyclophosphamide dependent immune system response
Ten male and ten female offspring derived from test group 10 will be selected at weaning to become a positive control group in this study. These animals will be treated with Cyclophosphamide monohydrate to prove the functional responsiveness of major components of the immune system of the rats against an immunosuppressant.

The following dose level of Cyclophosphamide monohydrate was selected to be sufficient to cause immunosuppressive activity as positive control substance:
4.5 mg/kg body weight/day: as dose level with expected immunosuppressive effects

CHOLINE DETERMINATION
Choline content will be determined in the liver of the blood-sampled PND 4 and 22 offspring.
Choline content will be determined in blood plasma and liver of cohort 1B animals. Details of the sampling are specified above, and in the section on pathology.

The choline analysis will be carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany, as a part of this study.

Methods of analysis
The analytical investigations will be performed according to the most recently authorized version of the control procedure. The control procedure will be described in the raw data and the report.
Postmortem examinations (parental animals):
NECROPSY
All F0 parental animals will be sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals will be necropsied and assessed by gross pathology, special attention being given to the reproductive organs. Animals which die intercurrently or are sacrificed in a moribund state will be necropsied as soon as possible after their death and assessed by gross pathology.

Organ weights
The following weights will be determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Lymph nodes, axillary (10 animals per sex per group, Cohort 1A animals only)
10. Lymph nodes, mesenteric (10 animals per sex per group, Cohort 1A animals only)
11. Ovaries
12. Pituitary gland
13. Prostate
14. Testes
15. Seminal vesicles including coagulating gland
16. Spleen
17. Thymus
18. Thyroid glands (with parathyroid glands)
19. Uterus (with cervix)

Organ/Tissue fixation
The following organs or tissues will be fixed in 4% formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix uteri
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left (fixed in modified Davidson´s solution)
11. Esophagus
12. Eyes with optic nerve (fixed in modified Davidson’s solution)
13. Heart
14. Ileum
15. Jejunum (with Peyer’s patches)
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries (fixed in modified Davidson´s solution)
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Rectum
28. Sciatic nerve
29. Seminal vesicles
30. Skeletal muscle
31. Spinal cord (cervical, thoracic and lumbar cord)
32. Spleen
33. Stomach (forestomach and glandular stomach)
34. Target organs
35. Testis, left (fixed in modified Davidson ´s solution)
36. Thymus
37. Thyroid glands (with parathyroid glands)
38. Trachea
39. Urinary bladder
40. Uterus
41. Vagina
42. Vas deferens

The testes, epididymides, ovaries and eyes with optic nerve of animals that die or will have to be sacrificed intercurrently will be fixed in 4% buffered formaldehyde solution.

The left testis and left epididymis of all male F0 parental animals sacrificed at scheduled dates will be fixed in modified Davidson’s solution, whereas the right testis and epididymis will be used for sperm parameters.

In case of macroscopic findings in the right testis or right epididymis, this testis as well as the corresponding epididymis will be fixed for histopathological examination and the left testis and epididymis will be used for sperm analysis.

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 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method). Then the uteri will be rinsed carefully in physiologic salt solution (0.9 % NaCl). When the examinations are completed, the uteri will be transferred to the Pathology Laboratory for further processing.

Histopathology
Fixation will be followed by histotechnical processing, examination by light microscopy and assessment of findings.

Special attention will be given to stages of spermatogenesis in the male gonads.

Special attention will be given to the synchrony of the morphology in ovaries, uterus, cervix, and vagina to the estrous cycle status. Any morphological patterns of asynchrony will be reported.

Animals that die or are sacrificed in a moribund state will be processed histotechnically and assessed like control animals. Special stains of individual organs of individual animals will be prepared if required.

Reproductive organs of all low- and mid-dose F0 parental animals suspected of reduced fertility, or for which estrous cyclicity or sperm quality were affected, will be subjected to histopathological investigation. Organs demonstrating potential treatment–related changes will also be examined in the lower dose groups.

Further examinations or procedures will depend on the results obtained in the study.
Postmortem examinations (offspring):
POSTMORTEM EXAMINATION OF F1 GENERATION PUPS
Pups sacrificed on schedule
On PND 4, all surplus F1 pups as a result of standardization will be sacrificed by decapitation under isoflurane anesthesia and blood will be sampled for determination of serum thyroid hormone concentrations. After sacrifice, these pups will be examined externally, eviscerated and their organs will be assessed macroscopically.

On PND 22, the surplus F1 generation pups that will not be used for the formation of the cohorts will be sacrificed by decapitation under isoflurane anesthesia with CO2 and blood will be sampled for thyroid hormone analyses.

Pups showing clinical symptoms or gross-morphological findings may be further examined using appropriate methods. Organs/tissues with gross-morphological findings may be preserved in a suitable manner for potential histopathological examination.

All F1 pups not used for other purposes without any notable findings will be discarded after their macroscopic evaluation.

Prematurely dead or sacrificed pups
Pups that die or are sacrificed in a moribund state will be eviscerated and examined for possible defects and/or the cause of death using appropriate methods. These animals will be preserved for this purpose, if necessary.

Liver sampling for choline determination in PND 4 pups
Livers of 10 male and 10 female culled PND 4 offspring per group (as far as possible of different litters) will be sampled.

As soon as possible after dissection of the animals, the livers will be weighed in toto. The weight of the livers will be recorded but will not be reported. Immediately after weighing, the liver samples will be frozen in liquid nitrogen. Until analysis, the samples will be stored at -80°C.

NECROPSY
COHORT 1A
All cohort 1A animals will be sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals will be necropsied and assessed by gross pathology, special attention being given to the reproductive organs. Animals which die intercurrently or are sacrificed in a moribund state will be necropsied as soon as possible after their death and assessed by gross pathology.

Organ weights
The following weights will be determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Lymph nodes, axillary
10. Lymph nodes, mesenteric
11. Ovaries
12. Pituitary gland
13. Prostate
14. Testes
15. Seminal vesicles including coagulating gland
16. Spleen
17. Thymus
18. Thyroid glands (with parathyroid glands)
19. Uterus (with cervix)

Organ/Tissue fixation
The following organs or tissues will be fixed in 4% formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix uteri
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left (fixed in modified Davidson´s solution)
11. Esophagus
12. Eyes with optic nerve (fixed in modified Davidson’s solution)
13. Heart
14. Ileum
15. Jejunum (with Peyer’s patches)
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries (fixed in modified Davidson´s solution)
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Rectum
28. Sciatic nerve
29. Seminal vesicles
30. Skeletal muscle
31. Spinal cord (cervical, thoracic and lumbar cord)
32. Spleen
33. Stomach (forestomach and glandular stomach)
34. Target organs
35. Testis, left (fixed in modified Davidson ´s solution)
36. Thymus
37. Thyroid glands (with parathyroid glands)
38. Trachea
39. Urinary bladder
40. Uterus
41. Vagina
42. Vas deferens

The testes, epididymides, ovaries and eyes with optic nerve of animals that die or will have to be sacrificed intercurrently will be fixed in 4% buffered formaldehyde solution.
The left testis and left epididymis of all male animals sacrificed at scheduled dates will be fixed in modified Davidson’s solution, whereas the right testis and epididymis will be used for sperm parameters.
In case of macroscopic findings in the right testis or right epididymis, this testis as well as the corresponding epididymis will be fixed for histopathological examination and the left testis and epididymis will be used for sperm analysis.
Spleens of 10 animals per sex per group of cohort 1A will be split in two comparable parts (transversally). One part of the spleen will be fixed in 4% buffered formaldehyde and afterwards be embedded in paraplast. The other part of the spleen will be frozen at -80°C, being used to perform a splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells).

Histopathology
Fixation will be followed by histotechnical processing, examination by light microscopy and assessment of findings.
Special attention will be given to stages of spermatogenesis in the male gonads.
Special attention will be given to the synchrony of the morphology in ovaries, uterus, cervix, and vagina to the estrous cycle status. Any morphological patterns of asynchrony will be reported.
Animals that die or are sacrificed in a moribund state will be processed histotechnically and assessed like control animals. Special stains of individual organs of individual animals will be prepared if required.
A differential ovarian follicle count (DOFC) will be conducted in test groups 10 and 13 (Cohort 1A females) according to Plowchalk et.al..
Further examinations or procedures will depend on the results obtained in the study.

COHORT 1B
All cohort 1B animals will be sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals will be necropsied and assessed by gross pathology, special attention being given to the reproductive organs. Animals which die intercurrently or are sacrificed in a moribund state will be necropsied as soon as possible after their death and assessed by gross pathology.
Organ weights
The following weights will be determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Cauda epididymis
3. Epididymides
4. Liver
5. Ovaries
6. Pituitary gland
7. Prostate
8. Target organs
9. Testes
10. Seminal vesicles including coagulating gland
11. Uterus (with cervix)
Organ/Tissue fixation
The following organs or tissues will be fixed in 4% formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Cervix uteri
3. Coagulating glands
4. Epididymis, left (fixed in modified Davidson ´s solution)
5. Liver
6. Ovaries (fixed in modified Davidson´s solution)
7. Pituitary gland
8. Prostate
9. Seminal vesicles
10. Target organs
11. Testis, left (fixed in modified Davidson ´s solution)
12. Uterus
13. Vagina

The testes, epididymides and ovaries of animals that die or will have to be sacrificed intercurrently will be fixed in 4% buffered formaldehyde solution.

Tissue sampling for choline determination
Tissue portions of the liver of all cohort 1 B males and females per group will be sampled.
As soon as possible after dissection of the animals, the livers will be weighed in toto. Immediately thereafter, the lobus sinister medialis of the liver will be separated from the remaining parts of the liver and weighed. The weight of the lobus sinister medialis will be recorded online in the pathology data system but will not be reported within the pathology report. Immediately after weighing, the liver lobe samples will be frozen in liquid nitrogen. Until analysis, the samples will be stored at -80°C.
For the remaining parts of the liver the standard procedures as described above will be followed.

Histopathology
All organs listed above will be preserved in adequate fixative. Histopathological processing and examination by light microscopy will only be conducted if results from cohort 1A are equivocal or in case the test compound is, from the results obtained so far, suspected to be a reproductive or endocrine toxicant.

Specifically, fixation of reproductive organs will be followed by histotechnical processing and examination by light microscopy and assessment of findings.

COHORT 2A
On postnatal day 77, cohort 2A animals will be weighed, counterbalanced, subjected to deep anesthesia (pentobarbital) and sacrificed by perfusion fixation.
SOERENSEN phosphate buffer will be used as the rinsing solution, and a fixation solution according to KARNOVSKY will be used as a fixative.
The perfusion fixed animals will be necropsied with regard to the question of neuropathology, and the visible organs will be assessed by gross pathology as accurately as is possible after a perfusion fixation. The cranial vault and the spinal cord will be opened and the skin will be removed from both hind extremities. In this state, the perfused animals will be stored in a fixation solution according to KARNOVSKY for at least 48 hours.
Animals which die intercurrently or are sacrificed in a moribund state will be necropsied as soon as possible after their death and assessed by gross pathology. These animals will be sacrificed under isoflurane anesthesia with C02.

Organ weights
The following weights will be determined (the brain will be weighed after its removal but before further preparation):
1. Brain (including olfactory bulb)

The terminal body weights will be recorded to calculate the relative organ weights.

Length and width of brain
The length and maximum width of the brain will be measured in all animals (length: on a line extending from the rostral end of the frontal lobe to the caudal medulla oblongata of the cerebellum; width: pituitary region).

Organ/Tissue fixation
The following organs/tissue specimens will be carefully removed, processed histotechnically in accordance with the data given in the sections of this part of the study plan and examined:
1. All gross lesions
2. Brain with olfactory bulb
3. Eyes with retina and optic nerve
4. M. gastrocnemius
5. Nose (nasal cavity)
6. Pituitary gland
7. Sciatic nerve, proximal section
8. Spinal cord, cervical part (C1-C5)
9. Spinal cord, thoracic part (Th5-8)
10. Spinal cord, lumbar part (L1-L4)
11. Spinal ganglia (C1-C5 [3x])
12. Spinal ganglia (L1-L4 [3x])
13. Tibial nerve (on the knee), proximal section
14. Tibial nerve (nerve branch in the lower leg muscles), distal section
15. Trigeminal ganglia (s. Gasserian)
16. Root fibers, dorsal (C1-C6 and L1-L4)
17. Root fibers, ventral (C1-C6 and L1-L4)

The remaining animal body after trimming will be stored in neutrally buffered, 4% formaldehyde solution.

Neurohistopathology
The histotechnical processing, examination by light microscopy and assessment of findings is performed on organs/tissues mentioned above.
Tissues demonstrating potential treatment–related changes will also be examined in the lower dose groups.
Further examination procedures (e.g. special stains) will depend on the results of the study, especially on the presence of neurofunctional or neuropathological changes.

Morphometry
Thickness measurements of major brain layers (neocortex: frontal and parietal cortices, caudate nucleus/putamen, hippocampus, corpus callosum, cerebellum) will be performed. Measurements will be carried out bilaterally in the left and right halves of the brain with the exception of the corpus callosum and the cerebellum.

Selection of the planes:
• Measurements for the thickness of the neocortex, corpus callosum and caudate nucleus/putamen will be carried out in a cross section which approximates the plane of section on page 88 in Sherwood and Timiras (1970).
• Measurements for the thickness of the hippocampus will be carried out in a cross section which approximates the plane of section on page 110 in Sherwood and Timiras (1970).
• Measurements for the thickness of select folia of the cerebellum will be carried out in a midsagittal section through the vermis of the cerebellum which approximates the plane of section on page 134 in Sherwood and Timiras (1970).
Conduct of the measurements:
• Neocortex (frontal and parietal cortices): The width of the total cortical mantle (layers I-VI – from the surface of the pia mater to the white substance) will be measured vertically to a tangent over a region of the frontal and parietal cortices determined beforehand.
• Caudate nucleus/putamen: The largest lateral extension will be measured.
• Corpus callosum: The width will be measured at the middle line of the cross section.
• Hippocampus: The largest dorsoventral extension will be measured.
• Cerebellum: The width of a select folium (e.g. folium pyramis) will be measured in the middle of a line which runs vertically to a tangent from the tip to the base of the folium.

COHORT 2B
On postnatal day 22, cohort 2B animals will be weighed, counterbalanced, subjected to deep anesthesia (isoflurane) and sacrificed by perfusion fixation.
SOERENSEN phosphate buffer will be used as the rinsing solution, and neutrally buffered, 4% formaldehyde solution will be used as a fixative.
The perfusion fixed animals will be necropsied with regard to the question of neuropathology, and the visible organs will be assessed by gross pathology as accurately as is possible after a perfusion fixation. The cranial vault and the spinal cord will be opened and the skin will be removed from both hind extremities. In this state, the perfused animals will be stored in neutrally buffered, 4% formaldehyde solution for at least 48 hours.
Animals which die intercurrently or are sacrificed in a moribund state will be necropsied as soon as possible after their death and assessed by gross pathology. These animals will be sacrificed under isoflurane anesthesia with C02.

Organ weights
The following weights will be determined (the brain will be weighed after its removal but before further preparation):
1. Brain (including olfactory bulb)

The terminal body weights will be recorded to calculate the relative organ weights.

Length and width of brain
The length and maximum width of the brain will be measured in all animals (length: on a line extending from the rostral end of the frontal lobe to the caudal medulla oblongata of the cerebellum; width: pituitary region).

Organ/Tissue fixation
The following organs/tissue specimens will be carefully removed, processed histotechnically in accordance with the data given in the sections of this part of the study plan and examined:

The following organs or tissues will be fixed in 4% formaldehyde solution:
1. All gross lesions
2. Brain with olfactory bulb
3. Eyes with retina and optic nerve
4. Nose (nasal cavity)
5. Pituitary gland
6. Trigeminal ganglia (s. Gasserian)

The animals and the tissue or organ material remaining after trimming will be stored in neutrally buffered, 4% formaldehyde solution.

Neurohistopathology
The histotechnical processing, examination by light microscopy and assessment of findings is performed on organs/tissues mentioned above.

COHORT 3
All Cohort 3 animals will be sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals will be necropsied and assessed by gross pathology. Animals which die intercurrently or are sacrificed in a moribund state will be necropsied as soon as possible after their death and assessed by gross pathology.

Organ weights
The following weights will be determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Spleen
3. Thymus

Organ/ tissue fixation
The following organs or tissues will be fixed in 4% buffered formaldehyde solution:
1. All gross lesions
2. Spleen
3. Thymus

Histopathology
Histotechnical processing and examination will be performed at the request of the sponsor only.

SURPLUS F1 GENERATION PUPS
All surplus F1 generation pups will be sacrificed by decapitation under isoflurane anesthesia with CO2. The exsanguinated animals will be necropsied and assessed by gross pathology with special emphasis on the reproductive organs.

Organ weights
The following weights will be determined in up to 10 animals per sex per group sacrificed on schedule:
1. Anesthetized animals
2. Brain
3. Spleen
4. Thymus

Organ/ Tissue fixation
The following organs or tissues of up to 10 animals per sex per group will be fixed in 4% buffered formaldehyde solution:
1. All gross lesions
2. Target organs
3. Brain
4. Mammary gland (male and female)
5. Spleen
6. Thymus
7. Thyroid glands

Histopathology
Histotechnical processing and examination will be performed at the request of the sponsor only.

Liver sampling for choline determination
Livers of the blood-sampled surplus PND 22 offspring (10 male and 10 female pups per group) will be sampled.
As soon as possible after dissection of the animals, the livers will be weighed in toto. The weight of the livers will be recorded but will not be reported. Immediately after weighing, the liver samples will be frozen in liquid nitrogen. Until analysis, the samples will be stored at -80°C.
Statistics:
Means and standard deviations will be calculated. In addition, the following statistical analyses will be carried out:
- Water consumption, food consumption, body weight and body weight change (parental animals, rearing animals and pups); estrous cycle length; mating days; duration of gestation; number of delivered pups per litter; developmental landmarks (days up to preputial separation or opening of the vagina), anogenital distance and index; implantation sites; postimplantation loss: DUNNETT test (two-sided)
- Number of live and dead pups and different indices (e.g. mating index, fertility index and gestation index) and number of litters with necropsy findings in pups; developmental landmarks (preputial separation or opening of the vagina): FISHER's exact test
- Absolute and relative pup organ weights; feces, rearing, grip strength forelimbs, grip strength hind-limbs, landing foot-splay test, motor activity, startle response: KRUSKAL-WALLIS and WILCOXON test (two-sided)
- Proportion of pups with necropsy findings per litter, presence of areolas/nipples: WILCOXON test (one-sided)
- Clinical pathology and sperm parameters: KRUSKAL-WALLIS and WILCOXON test
- Weight of the anesthetized animals and absolute and relative organ weights: KRUSKAL-WALLIS and WILCOXON test
- DOFC: WILCOXON test (one-sided)
- Brain weights: KRUSKAL-WALLIS and WILCOXON test
- Brain morphometry: linear measurements of selected brain regions: WILCOXON test
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical observations for males and females (except gestation and lactation period). No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the groups.

Detailed clinical observations (DCO)
Male and female animals of all dose groups (100, 300 and 1000 ppm) did not show any abnormality.

Clinical observations for females during gestation of F1 litters:
There were no test substance-related clinical findings in any F0 parental females of all dose groups during the gestation period for F1 litter.
One sperm positive and one sperm negative female of test group 02 (Nos. 262 and 275 - 300 ppm) and one sperm positive female of test group 03 (No. 291 - 1000 ppm) did not deliver F1 pups and had no implants in the uterus. One sperm negative female of test group 02 (No. 266) did not deliver F1 pups, but had implants in the uterus. These observations were not considered to be associated with the test compound.

Clinical observations for females and offspring during lactation of F1 litters:
There were no test substance-related clinical observations observed in all F0 parental females of all dosed groups during the lactation period.
Two high-dose females (Nos. 292 and 311 - 1000 ppm) had lost their complete litters by PND 4. One female pup in control (dam No. 219, pup No. 7 - 0 ppm) showed chromodacryorrhea (both eyes) during PND 20 - 21 and one female pup in test group 03 (dam No. 306, pup No. 9 - 1000 ppm) showed microphthalmia (both eyes) during PND 18 - 21. These finding were not considered to be treatment-related.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights of the high-dose F0 parental males (1000 ppm) were statistically significantly below the concurrent control from study day 7 onwards (up to 9%), as were the body weights of the mid-dose F0 parental males (300 ppm) from study day 28 onwards (up to 7%), until the end of the study.
The body weights of the low-dose F0 parental male rats (100 ppm) were comparable to the concurrent control values throughout the entire study. The body weights of the high-dose F0 parental females were statistically significantly below the concurrent control from premating day 7 onwards, throughout gestation and lactation (up to 5%, 15% and 13%, respectively).
The body weights of the mid-dose F0 parental females were statistically significantly below the concurrent control values during PND 4 - 7 (up to 5%). There were no effects on body weights during premating and gestation in this dose group.
The body weights of the low-dose females were comparable to the concurrent control during the entire study. A consistently lower body weight gain was noted in the high-dose F0 parental males (1000 ppm) which became statistically significant during several study sections. The overall average decrease of weight gain was about 25%. The mid-dose F0 parental males (300 ppm) still gained less weight than the controls (overall about 23% less), however the difference to the control was statistically significant in fewer sections than in the high-dose group.
A lower body weight gain was also noted for the low-dose F0 parental males (100 ppm) during the first week of treatment (14% below control). However, this was the only study section where this was seen and there was no effect on body weight during this study section and the remaining study. Thus, this is considered of no toxicological relevance.
The body weight gain of the high-dose F0 parental females (1000 ppm) was consistently below the concurrent control during gestation (overall about 33%) through early lactation (about 65%). In the other study sections periods of lower weight gain alternated with periods of comparable or higher weight gains, displaying an inconsistent course. The body weight gain of the low- and mid-dose F0 parental females (100 and 300 ppm) was widely comparable to the concurrent control, with some brief episodes of statistically significantly lower (GD 0-7) or higher (premating days 7-14) weight gains. Those minor changes were considered as spurious findings.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose F0 parental males (1000 ppm) was statistically significantly below the concurrent control values during premating (up to 9%), while food consumption of all low- (100 ppm) and mid-dose (300 ppm) males was comparable to the concurrent control values.
Food consumption of the high-dose F0 females was statistically significantly below the concurrent control values throughout premating, gestation and lactation periods (up to 17%, 13% and 29%, respectively). For the mid-dose females (300 ppm) food consumption was statistically significantly below the concurrent control values during PND 4 - 7 (about 7%). There were no effects on food consumption during premating and gestation in this dose group. Food consumption of the low-dose females was comparable to the concurrent control during the entire study.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption of all F0 parental male rats of all test groups was comparable to the concurrent control throughout the entire study. The statistically significantly increased water consumption in the low-dose males during study days 18 - 21 and in the mid- and high-dose males during study days 42 - 46 was considered as spontaneous in nature.

In the high-dose F0 females (1000 ppm) water consumption was statistically significantly below concurrent control during GD 14 – 18 (up to 18%) and during the entire lactation period (up to 45%). The overall mean water consumption during lactation was about 35% below control. There was no effect on water consumption during premating in this dose group. For the mid-dose females (300 ppm) water consumption was statistically significantly below the concurrent control values during PND 1 - 2, 7 - 8 and 20 - 21 (about 11%, 11% and 15%, respectively). The overall mean water consumption during lactation was about 10% below control. There was no effect on water consumption during premating and gestation in this dose group. Water consumption of the low-dose females (100 ppm) was comparable to the concurrent control during the entire study.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In the F0 generation rats of both sexes in test group 3 (1000 ppm) red blood cell (RBC) counts, hemoglobin and hematocrit values as well as the calculated red blood cell indices mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were decreased. Additionally, in the mentioned test group in males mean corpuscular hemoglobin concentration (MCHC) and in females relative reticulocyte counts were lower compared to controls, but MCHC was within the historical control range (MCHC 20.36-21.48 mmol/L) and in females mean values of the reticulocyte counts were not different to the controls. Therefore, both latter parameter changes were regarded as incidental and not treatment-related. In rats of both sexes of test group 2 (300 ppm) hemoglobin values were decreased. In males of this test group MCV and MCH and in females hematocrit values were decreased. MCV was already decreased in males of test group 1 (100 ppm). In males of test group 2 the measured parameter hemoglobin was within the historical control range (males: hemoglobin 8.6-9.3 mmol/L). In these individuals also the the calculated red blood cell parameters MCV and MCH were within or marginally below (MCV in test group 2) historical control ranges (males: MCH 1.00-1.10 fmol; MCV 48.4-51.9 fl). In females of test group 2 (300 ppm) as well as in female study controls hemoglobin and hematocrit values were not below, but above historical control ranges (females hemoglobin 8.7-9.3 mmol/L; hematocrit 0.410-0.435 L/L). Measured red blood cell parameters, hemoglobin in both sexes and hematocrit in females of test group 2 were only slightly lower compared to study controls (hemoglobin males mean – 6%; females mean -3%, hematocrit females mean -4% compared to respective control means, Mueller et al., 2006). Therefore, the alterations of the red blood cell parameters in test group 2 and test group 1 were regarded as treatment-related, but not adverse.
In rats of both sexes of test groups 2 and 3 (300 and 1000 ppm) prothrombin time (Hepatoquick’s test HQT) was reduced and in males of both test groups platelet counts were increased. Regarding differential blood cell counts in test group 3 (1000 ppm), in rats of both sexes absolute basophil counts were decreased. Additionally, in males absolute neutrophil counts and in females absolute and relative monocyte and eosinophil counts and relative basophil counts were lower compared to controls. All absolute cell counts were within historical control ranges (males absolute neutrophils 0.70-1.35 Giga/L; absolute basophils 0.00-0.02 Giga/L; females absolute basophils 0.00-0.02 Giga/L; absolute monocyte 0.06-0.08 Giga/L; absolute
eosinophils 0.08-0.11 Giga/L). Relative monocyte and eosinophil counts in females of test group 3 were marginally below the historical control ranges (relative monocytes 1.7-2.8 %, relative eosinophils 2.2-3.7 %). However, because no relevant change above historical control ranges occurred in the total and absolute differential cell counts all alterations in the differential blood cell counts in males and females of test group 3 (1000 ppm) were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In male F0 generation rats of test group 3 (1000 ppm) aspartate aminotransferase (AST) and alkaline phosphatase (ALP) activities were increased. ALP activities were already higher in males of test group 2 (300 ppm), but in this test group it was the only relevantly changed clinical pathology parameter. ALP means in males of both mentioned test groups was less than 2fold higher compared to controls. Therefore, the ALP change in males of test group 2 was regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002). Alanine aminotransferase (ALT) activities were lower in males of test group 2 (300 ppm) compared to controls, but the change was not dose-dependent and therefore it was regarded as incidental and not treatment-related. In male and female rats of test group 3 (1000 ppm) urea and albumin levels were increased. Albumin levels were already higher in females of test group 2 (300 ppm), but in these individuals, this was the only relevantly changed clinical pathology parameter (total calcium levels were higher in these individuals as consequence of higher albumin levels). Therefore, the albumin increase in females of test group 2 (300 ppm) was regarded as treatment-related,
but not adverse (ECETOC Technical Report No. 85, 2002). Calcium levels were increased in rats of both sexes of test groups 2 and 3 (300 and 1000 ppm). Unbound and protein-bound calcium levels were measured. In order to maintain a constant
free calcium level, total calcium level has to increase with higher albumin levels. Therefore, calcium changes were secondary to the albumin level increases and were regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002). Calcium levels in males were within, those of females slightly above the historical control range (calcium males 2.45-2.67 mmol/L, females 2.49-2.61 mmol/L).
The following clinical chemistry values were within historical control ranges and therefore, the alterations were regarded as incidental and not treatment-related: increased sodium and chloride values in rats of both sexes of test groups 2 and 3 (300 and 1000 ppm), increased total protein levels in females of test group 2 and 3, Decreased cholesterol levels in females of test group 3, increased triglyceride levels in males of test group 2 and 3 and in females of test group 2 (males: sodium 141.1-146.7 mmol/L, chloride 98.8-105.9 mmol/L, triglycerides 0.61-1.29 mmol/L; females: sodium 140.3-145.8 mmol/L, chloride 98.7- 103.2 mmol/L, total protein 60.65-67.01 g/L, cholesterol 0.99-1.70 mmol/L). Triglyceride levels in females of test group 2 (300 ppm) were higher compared to controls, but the change was not dose-dependent and therefore, it was regarded as incidental and not treatment-related.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.
In males of test groups 2 and 3 (300 and 1000 ppm) urine specific gravity was lower and urine volume was higher (not statistically significantly) compared to controls. In conjunction with histopathological alterations in the kidneys, this change was regarded as treatment-related and adverse.
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Please see tables (Tables IC 10 – IC 14) of the attachment.
In the proximal tubules of the kidneys, there were signs of degeneration and regeneration characterized by apoptotic cell death, debris in the tubular lumen, increased numbers of large vesicular nuclei, increased basophilic staining and nuclear crowding. These findings were regarded to be treatment-related. Within the papilla, females of test group 02 and 03 (300 and 1000 ppm) showed an increase of multifocal mineral depositions. Nine males of test group 03 (1000 ppm) showed multifocal mineral depositions at the transition between the outer and inner medulla. The mineralization in males and females was regarded to be treatment-related.

The liver revealed a minimal to mild centrilobular hypertrophy in single males of test group 03 (1000 ppm) and females of test groups 02 and 03 (300 and 1000 ppm). This finding was regarded to be treatment-related.

The submucosa of the glandular stomach of females was distended by an edema with infiltration of inflammatory cells. Furthermore, some females of test group 02 and 03 (300 and 1000 ppm) revealed erosion/ulcers in the mucosa of the glandular stomach. These findings in test groups 02 and 03 (300 and 1000 ppm) were regarded to be treatment-related. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Fertility
The female animals (Nos. 262, 275), which were not pregnant as well as their male mating partners (Nos. 62, 75) did not show relevant histopathological findings consistent with impaired fertility.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
CHOLINE CONCENTRATION IN BLOOD AND LIVER SAMPLES
The analytical results demonstrated the clear presence of choline in all plasma samples from the animals dosed with the test substance 2,2’-iminodiethanol (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 2,2’-iminodiethanol led to a reduction in the content of choline in the plasma samples analyzed. 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. The analytical results demonstrated the clear presence of choline in all liver samples from the animals dosed with the test substance 2,2’-iminodiethanol (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 2,2’-iminodiethanol 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.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration in the different test groups was similar: 3.9 days in control and in the low- and middose groups and 4.0 days in the high-dose group.

Differential ovarian follicle count - F0 generation parental animals
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 differences between the control group 00 and animals of test group 03.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Concerning motility of the sperms and sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed. In males of test group 3 (1000 ppm) incidence of abnormal sperms was slightly higher compared to controls. This was mainly due to missing heads, shortened heads, an abnormal hook of the head and in some samples combined morphology changes of head and tail. However, at least the first three mentioned morphological changes occurred also in the controls and the incidences were still in the range of historical controls (abnormal sperms 6.0-7.5 %). Therefore, these changes were regarded as incidental and not treatment-related.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Male reproduction data
For nearly all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Copulation was not confirmed for test group 02 male No. 75 paired with test group 02 female No. 275. Thus, the male mating index was 100% in the control and test groups 01 and 03 and 97% in test group 02. Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. Two males of test group 02 (Nos. 62 and 75 - 300 ppm) did not generate F1 pups. One male of test group 02 (No. 66) and one male of test group 03 (No. 91) did not generate F1 pups, but implants were found in the uterus of the corresponding females (No. 266 - test group 02 and No. 291 - test group 03).
Thus, the male fertility index ranged between 93% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study. The apparently infertile male rats did not show relevant gross or microscopic lesions. Thus, the test substance 2,2’-iminodiethanol did not adversely affect fertility and reproduction of the F0 generation parental males.

Female reproduction and delivery data
The female mating index calculated after the mating period for F1 litter was 100% in the control and test groups 01 and 03 and 97% in test group 02. The mean duration until sperm was detected (GD 0) varied between 2.2 and 3.1 days without any relation to the dose level. All female rats delivered pups or had implants in utero with the following exceptions:
-Test group 02 female No. 262 (mated with male No. 62) did not become pregnant, female No. 275 (mated with male No. 75) did not become pregnant
The fertility index varied between 97% and 100% without showing any relation to the dose level. This reflects the normal range of biological variation inherent in the strain of rats used for this study. The non-pregnant females had no relevant gross lesions or microscopic findings. The mean duration of gestation was 22.0 / 22.0 / 22.1 and 22.5** (**:p<=0.01) days in the control, low-, mid- and high-dose groups (0, 100, 300 and 1000 ppm), respectively. The gestation index was 100% in the control and test groups 01, 96% in test group 02 and 97% in test group 03. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
The mean number of implantation sites was 12.3 / 12.2 / 11.4 and 7.8** (**:p<=0.01) implants/dam in test groups 00 - 03, respectively.
The post-implantation loss did not show any statistically significant differences between the groups and all values were well within the historical control range. Corresponding to the lower number of implants the mean number of F1 pups delivered per dam was lower in the high-dose group as well, the numbers were 11.9 / 11.8 / 11.1 and 7.3** (**:p<=0.01) pups/dam in test groups 00 - 03, respectively. The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 98% / 99% / 98% and 96% in test groups 00 - 03, respectively. Despite that the number of litters with stillborn pups was higher at the top dose (2 / 2 / 4 and 8** (**:p<=0.01) in test groups 00 - 03, respectively), the total number of stillborn pups was comparable between the groups (7 / 2 / 5 and 8 in test groups 00 - 03, respectively) and does not indicate an association to treatment. The number of cannibalized pups was 0 / 2 / 2 and 10** (**:p<=0.01) in test groups 00 - 03, respectively.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
100 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
Key result
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive performance
Key result
Critical effects observed:
no
Clinical signs:
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Pup number and status at delivery
Whereas the litter size of the F1 litters at the top dose (1000 ppm) was lower (11.9 / 11.8 / 11.1 and 7.3** (**:p<=0.01) pups/dam in test groups 00 - 03, respectively) the postimplantation loss as well as the rates of liveborn and stillborn F1 pups were evenly distributed about the groups.
Thus the lower litter size in the high-dose group was a consequence of a lower number of implants and not due to prenatal or perinatal mortality.
Mortality are based on stillborn pups, dead pups, pups sacrificed moribund and cannibalized pups.

Pup viability/mortality
The viability index indicating pup mortality during early lactation (PND 0 - 4) varied between 100% / 99% / 99% and 93% in test groups 00 - 03. The lower index in the high-dose group was slightly below the historical control range. This slightly higher pup mortality at the high dose came from a higher number of dead (4 vs. 1 in control) and cannibalized (10** (**:p<=0.01) vs. 0 in control) pups which were distributed across 8 litters. In 2 of those 8 litters (292, 311) none of the pups survived.
The absolute number of pups surviving early lactation (PND 0 - 4) varied between 350 / 349 / 291* (*:p<=0.05) and 191** (**:p<=0.01) in test groups 00 - 03. The significantly lower number in the mid-dose group reflects the incidentally lower number of liveborn litters in this group (27 vs. 30 in the control) and not an effect on pup survival. The lactation index indicating pup mortality on PND 4 - 21 was 100% in all test groups.
Body weight and weight changes:
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
please compare with results for the F1 generation details
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Sperm parameters
Concerning motility of the sperms and sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed. In males of test group 13 (1000 ppm) incidence of abnormal sperms was slightly higher compared to controls, although not statistically significant. This was due to two individuals (no. 474 and 477) having sperm with missing heads, abnormal hook of the head, bent heads and combined morphological changes of head and tail. These morphological changes occurred also in controls. The mean incidence of the morphological changes on the whole was still in the range of historical controls (abnormal sperms 6.0-7.5 %). Therefore, this change was regarded as incidental and not treatment-related.
Reproductive performance:
not examined
Description (incidence and severity):
No F2 generation as been perfomed according to the agreed study protocol.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

F1 rearing animals, Cohort 1A (14R0781A)
Clinical observations for males and females
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the low- and mid-dose males and in any female animal of all dose groups.
Two high-dose (1000 ppm) males (No. 476 and 478) showed high-stepping gait and piloerection recurring in several study sections. In addition, both animals had small testes which were detected in study week 5 for the first time.

F1 rearing animals, Cohort 1B (14R0781B)
Clinical observations for males and females
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the low- and mid-dose male and female animals. Several high-dose male and female animals (Nos. 693, 694, 697, 785, 797, 798) showed highstepping gait and piloerection recurring during several study sections. Affected male (No. 697) had small testes in addition, which were detected for the first time in test week 5. These findings were considered as treatment-related and adverse. Female No. 693 had also a malocclusion, which is considered as a spurious finding.

Detailed clinical observations (DCO)
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the low- and mid-dose male and female animals (100 and 300 ppm). A number of high-dose male and female animals (1000 ppm) exhibited high-stepping gait and piloerection on seveal occasions during the study, as follows:
- Male animal No. 693: high-stepping gait during study weeks 6 - 8 and piloerection on study week 2 and during weeks 6 - 8
- Male animal No. 697: high-stepping gait during study weeks 5 - 6 and piloerection on week 6
- Female animal No. 785: high-stepping gait and piloerection during study weeks 6 - 8
- Female animal No. 797: high-stepping gait on study week 8
- Female animal No. 798: high-stepping gait on study week 1 and during study weeks 5 - 8 as well as piloerection during study weeks 1 - 2 and study weeks 5 – 8.
These clinical observations were considered to be treatment-related and adverse.


F1 rearing animals, Cohort 2A (14R0782A)
Clinical observations for males and females No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the test substance-treated male as well as in low- and mid-dose female animals.
One high-dose (1000 ppm) female (No. 937) showed high-stepping gait during study weeks 6
– 7 and piloerection during study weeks 5 - 7. These findings were considered as treatment-related and adverse. A malocclusion observed in high-dose male animal No. 833 is considered as a spurious finding.

F1 rearing animals, Cohort 2A (14R0782A)
Detailed clinical observations (DCO)
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the test substance treated male and in any low- and mid-dose female animal. One high-dose (1000 ppm) female (No. 937) showed high-stepping gait and piloerection during study week 6.

F1 rearing animals, Cohort 3 (14R0783)
Clinical observations for males and females
One high-dose (1000 ppm) male (No. 1240) showed high-stepping gait and piloerection recurring in several study sections. In addition, the animal had small testes which were detected in study week 4 for the first time. A malocclusion observed in this male was considered as a spurious finding. High-dose female animal No. 1336 showed high-stepping gait on study week 4 and piloerection during study weeks 3- 4.
Detailed clinical observations (DCO)
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the low- and mid- dose male and female animals. One high-dose (1000 ppm) male (No. 1240) showed high-stepping gait on study week 2 and piloerection during study weeks 1 - 2. High-dose female animal No. 1336 showed high-stepping gait and piloerection on study week 4. These findings were considered as treatment-related and adverse.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Pup number and status at delivery
Whereas the litter size of the F1 litters at the top dose (1000 ppm) was lower (11.9 / 11.8 / 11.1 and 7.3** (**:p<=0.01) pups/dam in test groups 00 - 03, respectively) the postimplantation loss as well as the rates of liveborn and stillborn F1 pups were evenly distributed about the groups.
Thus the lower litter size in the high-dose group was a consequence of a lower number of implants and not due to prenatal or perinatal mortality.
Mortality are based on stillborn pups, dead pups, pups sacrificed moribund and cannibalized pups.

Pup viability/mortality
The viability index indicating pup mortality during early lactation (PND 0 - 4) varied between 100% / 99% / 99% and 93% in test groups 00 - 03. The lower index in the high-dose group was slightly below the historical control range. This slightly higher pup mortality at the high dose came from a higher number of dead (4 vs. 1 in control) and cannibalized (10** (**:p<=0.01) vs. 0 in control) pups which were distributed across 8 litters. In 2 of those 8 litters (292, 311) none of the pups survived.
The absolute number of pups surviving early lactation (PND 0 - 4) varied between 350 / 349 / 291* (*:p<=0.05) and 191** (**:p<=0.01) in test groups 00 - 03. The significantly lower number in the mid-dose group reflects the incidentally lower number of liveborn litters in this group (27 vs. 30 in the control) and not an effect on pup survival. The lactation index indicating pup mortality on PND 4 - 21 was 100% in all test groups.

F1 rearing animals, Cohort 1A (14R0781A)
Mortality
There were no test substance-related mortalities in any of the groups up to a dose of 300 ppm. One high-dose male (No. 462, 1000 ppm) was sacrificed moribund in study week 0, after showing lateral position, apathy, hypothermia and diarrhea.

F1 rearing animals, Cohort 1B (14R0781B)
There were no test substance-related mortalities in any of the groups up to 300 ppm. One high-dose male (No. 687) and one high-dose female (No. 781) were found dead on study week 8, respectively.

F1 rearing animals, Cohort 2A (14R0782A)
Mortality
There were no test substance-related or spontaneous mortalities in any of the groups.

F1 rearing animals, Cohort 3 (14R0783)
Mortality
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean birth weights of the pups were comparable across all test groups including control. Mean body weights of the high-dose pups (1000 ppm) were statistically significantly below the concurrent control values from PND 4 until weaning, the difference was about 13% at maximum. A slight decrease of pup weights was still noted in the mid-dose group beginning on PND 14 (male pups only) and definitive on PND 21 (about 6% below control). Mean body weight gain of the high-dose pups was statistically significantly below the concurrent control values during the entire lactation period (about 21 - 25% below control). A similar decrease though less pronounced (7 – 12%) was still noted for the mid-dose pups (300 ppm); the difference to the control became statistically significant during PND 4 - 7, 14 - 21 and 4 - 21. No test compound-related influence on F1 pup body weights/body weight gain were noted in the low-dose group (100 ppm).

F1 rearing animals, Cohort 1A (14R0781A)
The body weights of the high-dose males were statistically significantly below the concurrent control values during the entire study period, the final weight was about 24% below control. The body weights of the mid-dose males were statistically significantly below the concurrent control during the first week after weaning, and stayed below control until the end of the study (final weight about 6% below control) albeit the difference was not statistically significant. The body weights of the low-dose male rats were comparable to the concurrent control values throughout the study. The body weights of the high-dose females were statistically significantly below the concurrent control values during the entire study period, the final weight was about 15% below control. The body weights of the mid-dose females were below control throughout the study (final weight about 5% below control), although the difference was small and not statistically significant. The body weights of the low-dose females were comparable to the concurrent control during the entire study. The body weight gain of the high-dose males was statistically significantly below the concurrent control throughout the study except of the last study week, the average reduction of weight gain was about 25%. Mid-dose males gained about 5% less weight, the reduction was statistically significantly below the concurrent control during study days 35 - 49 (up to 28%). The body weight change of the low-dose males was comparable to the concurrent control during the entire study. The high-dose females gained statistically significantly less weight than the concurrent control during the study (14% on days 0 – 63), further their weight gain was statistically significantly below control on days 0 – 7 and 42 - 49 (about 13% and 42%, respectively). The body weight change of the low and mid-dose females was comparable to the concurrent control during the study. The statistically significantly increased body weight change in the low dose females during study days 21 - 28 was considered to be spontaneous in nature.

F1 rearing animals, Cohort 2A (14R0782A)
Body weight data
The body weights of the high-dose males were statistically significantly below the concurrent control values during the entire study period, the final weight was about 13% below control. The body weights of the low- and mid-dose male rats were comparable to the concurrent control values throughout the study. The body weights of the high-dose females were statistically significantly below the concurrent control values during the entire study period, the final weight was about 16% below control. The body weights of the mid-dose females were below control throughout the study (final weight about 6% below control), although the difference was small and not statistically significant. The body weights of the low-dose females were comparable to the concurrent control during the entire study. The body weight gain of the high-dose males was statistically significantly below the concurrent control during several sections of the study, the average reduction of weight gain was about 11%. The body weight change of the low- and mid-dose males was comparable to the concurrent control during the entire study. There was no consistent effect on the body weight gain of the high-dose females, although they gained overall less weight than the concurrent control during the study (about 12%).
The body weight change of the low and mid-dose females was comparable to the concurrent
control during the study. The statistically significantly decreased body weight change in the
low-dose females during study days 28 - 35 was considered to be spontaneous in nature.

F1 rearing animals, Cohort 3 (14R0783)
Body weight data
The body weights of the high-dose males were statistically significantly below the concurrent
control values during the entire study period, the final weight was about 19% below control; as
were the body weights of the high-dose females, their final weight was about 14% below
control.
The body weights of the low- and mid-dose male and female rats were comparable to the
concurrent control values throughout the study.
The body weight gain of the high-dose males was statistically significantly below the concurrent
control during several sections of the study, the average reduction of weight gain was about
19%.
The body weight change of the low- and mid-dose males was comparable to the concurrent
control during the entire study.
There was no consistent effect on the body weight gain of the high-dose females, although
they gained overall less weight than the concurrent control during the study (about 8%).
The body weight change of the low and mid-dose females was comparable to the concurrent
control during the study. The statistically significantly increased body weight change in the middose
females during study days 0 - 7 was considered to be spontaneous in nature.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
F1 rearing animals, Cohort 1A (14R0781A)
Food consumption
Food consumption of the high-dose males was statistically significantly below the concurrent control values during the entire study period (up to 22%), the average reduction was about 17%. Food consumption of the mid-dose males was below the concurrent control values throughout the study (average 6%), although the difference was statistically significant only during study days 0 – 7. Food consumption of all low-dose males was comparable to the concurrent control values throughout the study. Food consumption of the high-dose females was statistically significantly below the concurrent control occasionally during the study (up to 11%), however, the average food intake throughout the study was comparable to control.
Food consumption of the low- and mid-dose females was comparable to the concurrent control throughout the study.

F1 rearing animals, Cohort 1B (14R0781B)
Food consumption of the high-dose males was statistically significantly below the concurrent control during the entire study (up to 18%), the average decrease was about 14%. There was no significant effect on food consumption of the low- and mid-dose males throughout the entire study. Food consumption of the high-dose females was statistically significantly below the concurrent control during major parts of the study period (up to 17%), the average dcrease was about 9%.
There was no significant effect on food consumption of the low- and mid-dose females throughout the entire study.

F1 rearing animals, Cohort 2A (14R0782A)
Food consumption
Food consumption of the high-dose males was below the concurrent control during the entire study period (up to 12%), the average reduction was about 11%. Food consumption of the high-dose females was statistically significantly below the concurrent control during study days 0 - 7 and 14 - 21 (about 15% and 10%, respectively), the average reduction was about 8%. Food consumption of the low- and mid-dose males and females was comparable to the
concurrent control throughout the study.

F1 rearing animals, Cohort 3 (14R0783)
Food consumption of the high-dose males was below the concurrent control during the entire study period although the difference was not statistically significant, the average reduction was about 11%. Food consumption of the high-dose females was also below the concurrent control throughout the study, although the difference was slight (about 5%) and not statistically significant. Food consumption of the low- and mid-dose males and females was comparable to the
concurrent control throughout the study.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
F1 rearing animals, Cohort 1A (14R0781A)
Water consumption of the high-dose males was below the concurrent control values throughout the study (average about 13% lower), the difference became statistically significant on study days 0 – 21, 24 - 28 and 49 - 56 (up to 24%, 14% and 19%, respectively). A statistically significantly reduced water consumption was also noted for the mid-dose males during study days 7 - 10 (about 10%). Water consumption of the high-dose females was below the concurrent control values throughout the study (average about 14% lower), the difference became statistically significant during several parts of the study period (up to 25%). Mid-dose females occasionally had statistically significantly lower water consumption during the study period (up to 18% below control). Water consumption of the low-dose male and female animals was comparable to the concurrent control values during the entire study.

F1 rearing animals, Cohort 1B (14R0781B)
Water consumption of the high-dose males was below the concurrent control values throughout the study (average about 13% lower), the difference became statistically significant during major parts of the study (up to 23%). A statistically significantly reduced water consumption was also noted for the mid-dose males towards the end of treatment (study days 52 – 59; about 10 - 17%). Water consumption of the low-dose males was consistently comparable to the concurrent
control during the entire study, except of a decrease on days 52 – 56 which is considered of no toxicological relevance. Water consumption of the high-dose females was below the concurrent control values throughout the study (average about 16% lower), the difference became statistically significant during major parts of the study period (up to 29%). Mid-dose females also had a consistenly lower water consumption than control (average about 10%), the difference becoming
statistically significant during study days 24 – 28, 31 – 38 and 49 - 56 (up to 18% below control). Water consumption of the low-dose females was comparable to the concurrent control during the entire study.

F1 rearing animals, Cohort 2A (14R0782A)
Water consumption of the high-dose males was below the concurrent control values during major parts of the study (average about 7% lower), the difference became statistically significant on study days 0 - 11 (up to 22%). Water consumption of the low and mid-dose males was comparable to the concurrent control during the entire study. Water consumption of the high-dose females was below the concurrent control values throughout the study (average about 13% lower), the difference became statistically significant during several parts of the study period (up to 18%). Mid-dose females occasionally had statistically significantly lower water consumption during the study period (up to 23% below control). Water consumption of the low-dose female animals was comparable to the concurrent control values during the entire study.

F1 rearing animals, Cohort 3 (14R0783)
Water consumption of the high-dose males was below the concurrent control values during major parts of the study (average about 9% lower), the difference became statistically significant on study days 0 - 7 (up to 16%). Water consumption of the low and mid-dose males was comparable to the concurrent control during the entire study. Water consumption of the high-dose females was below the concurrent control values throughout the study (average about 11% lower), the difference became statistically significant during several parts of the study period (up to 18%). Water consumption of the low- and mid-dose female animals was comparable to the concurrent control values during the entire study.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In rats of both sexes of the F1 generation (PND92) of test group 13 (1000 ppm) red blood cell (RBC) counts, hemoglobin and hematocrit values as well as red blood cell indices mean corpuscular volume (MCV and mean corpuscular hemoglobin content (MCH) were decreased. In rats of both sexes of test group 12 (300 ppm) hemoglobin and hematocrit values were decreased and additionally in females RBC counts were decreased and in males of the mentioned test group MCV and MCH were lower compared to controls. In females of this test group hemoglobin values and RBC counts were within and hematocrit values were marginally below historical control ranges (females: hemoglobin 8.1-8.9 mmol/L, RBC 7.49-7.93 Tera/L, hematocrit 0.383-0.421 L/L). However, hematocrit mean in females of test group 12 was only 9% lower compared to the controls and this was the only relevantly changed red blood cell parameter. In males of test group 12, the measured red blood cell parameters hemoglobin and hematocrit were within historical control range (males hemoglobin 8.4-9.5 mmol/L, hematocrit 0.400-0.446 L/L) and only the calculated red blood cell indices MCV and MCH were below historical control ranges (males MCV 50.1-54.7 fL, MCH 1.05-1.18 fmol). Therefore, the red blood cell parameter changes in rats of both sexes of test group 12 (300 were regarded as treatment-related but not adverse.
In females of test group 11 (100 ppm) RBC counts, hemoglobin and hematocrit values and in males of the same test group MCH were still lower compared to study controls, but all values apart from MCH in males were within historical control ranges (females: hemoglobin 8.1-8.9 mmol/L, RBC 7.49-7.93 Tera/L, hematocrit 0.383-0.421 L/L). In males of test group 11 MCH was still lower compared to historical controls, but the measured red blood cell parameters (i.e. RBC counts hemoglobin and hematocrit values) were not changed. Therefore, the mentioned red blood cell alterations in both sexes of test group 11 (100 ppm) were regarded as incidental and not treatment-related. In females of test group 13 (1000 ppm) prothrombin time (Hepatoquick’s test, HQT) was reduced. In males of test groups 12 and 13 (300 and 1000 ppm) platelet counts were increased. In females of test group 12 (300 ppm) platelet counts were increased, but they were not dosedependently
changed. Therefore, these platelet count alterations were regarded as incidental and not treatment-related.
Regarding the differential blood cell counts, in females of test group 13 (1000 ppm) absolute and relative monocyte counts were decreased and this is regarded as adverse finding. The following changes in the differential blood cell counts were within the historical control ranges. Therefore, they were regarded as incidental and not treatment-related: decreased absolute and relative monocyte counts in females of test groups 11 and 12 (100 and 300 ppm), decreased relative basophil counts in females of test groups 11, 12 and 13, increased relative lymphocyte counts in males of test group 13, decreased relative monocyte and eosinophil counts in males of test groups 12 and 13, decreased absolute monocyte counts in males of test group 12 (females: relative monocytes 1.2-1.9 %, absolute monocytes 0.05-0.08 Giga/L,
relative basophils 0.1-0.5 %; males: relative lymphocytes 77.7-87.0 %, relative monocytes 0.9-
2.3 %, relative eosinophils 0.8-2.0 %, absolute monocytes 0.07-0.14 Giga/L).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In rats of both sexes of the F1 generation (PND92) of test group 13 (1000 ppm), aspartate aminotransferase (AST) activities were increased. Additionally, in males of test groups 12 and 13 (300 and 1000 ppm) alkaline phosphatase (ALP) activities were higher compared to controls. However, the ALP activity mean in test group 2 was within the historical control range (ALP 1.64-2.80 μkat/L) and therefore, this change was regarded as incidental and not treatment-related. In male and female rats of test group 13 (1000 ppm) urea levels were increased. In females of test groups 11, 12 and 13 (100, 300 and 1000 ppm) creatinine levels were decreased and glucose levels were increased. However, both parameters were not dose-dependently changed regarding means as well as medians. Therefore these alterations were regarded as incidental and not treatment-related. In females of test groups 12 and 13 (300 and 1000 ppm) total protein, albumin and calcium
levels were higher compared to controls. Albumin levels were within historical control ranges and total protein levels of test group 12 were within and those of test group 13 marginally above the historical control range (albumin 38.12-42.64 g/L, total protein 58.83-65.33 g/L). Calcium values were slightly above the historical control range (calcium 2.38-2.63 mmol/L). As already mentioned in the corresponding paragraph in the F0 generation, unbound and protein-bound calcium levels were measured. In order to maintain a constant free calcium level, total calcium level has to increase with higher albumin levels. Therefore, calcium changes were secondary to the albumin level increases and were regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002). Albumin and total protein alterations were regarded as incidental and not treatment-related. The following parameter changes were within historical control ranges and therefore they were regarded as incidental and not treatment-related: increased sodium in both sexes of test groups 12 and 13 (300 and 1000 ppm), increased chloride levels in both sexes of test group 13, increased calcium levels in males of test group 13, decreased levels of cholesterol in females of test groups 12 and 13 (in test group 13 not statistically significant) (males: sodium 140.7- 145.4 mmol/L, chloride 97.9-103.4 mmol/L, calcium 2.42-2.71 mmol/L; females: sodium 140.5-144.1 mmol/L, chloride 100.1-104.9 mmol/L, cholesterol 1.04-1.65 mmol/L). Inorganic phosphate levels were decreased and triglyceride levels were increased in females of test group 12 (300 ppm), but the changes were not dose-dependent and therefore they were regarded as incidental and not treatment-related. Calcium levels were already increased in females of test group 11 (100 ppm), but this value was at the upper border of the historical controls (females, calcium 2.38-2.63 mmol/L) and this was the only changed clinical chemistry parameter among these individuals. Therefore, calcium level increase in females of test group 11 was regarded as maybe treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002).
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In F1 rats of both sexes (PND 92) no treatment-related, adverse changes among urinalysis
parameters were observed. In females of test groups 11, 12 and 13 (100, 300 and 1000 ppm) urine volume was lower and urine specific gravity was higher compared to controls. However, both parameters were not dose-dependently changed. Statistically significance in this case occurred because of a high urine volume and a low specific gravity in the controls. Therefore, the changes were regarded as incidental and not treatment-related.
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
Vaginal opening
Each female F1 pup, which was selected to be raised as F1 female (cohorts 1A, 1B, 2A, 3), was evaluated for commencement of sexual maturity. The first day when vaginal opening was observed was PND 27, the last was PND 38. The mean number of days to reach the criterion in the control and 100, 300 and 1000 ppm test groups amounted to 29.7 / 30.2 / 30.6* (*:p<=0.05) and 32.0** (**:p<=0.01) days. The mean body weight on the day, when vaginal opening was recorded, amounted to 85.1 g / 87.4 g / 84.8 g and 80.4 g in test groups 00-03. The values for pubertal age and weight of control females were both at the lower end of the historical control range; thus the apparent statistical increase in age at attainment in the middose
group is considered to be due to the very low concurrent control values in this study and not treatment-related. In the high-dose group the pubertal age is just above the upper limit of the historical range while the weight at puberty is below the historical control range. This clearly indicates that the later onset of puberty is a consequence of a general developmental delay and not a specific effect on the timing of puberty.

Preputial separation
Each male F1 pup, which was selected to be raised as F1 male (cohorts 1A, 1B, 2A, 3), was evaluated for commencement of sexual maturity. The first day when preputial separation was observed was PND 38, the last was PND 66. The mean number of days to reach the criterion in the control and 100, 300 and 1000 ppm test groups amounted to 41.1 / 41.2 / 41.8 and 43.3** (**:p<=0.01) days. The mean body weight on the day, when preputial separation was recorded, amounted to 168.5 g / 168.6 g / 165.7 g and 145.2 g** (**:p<=0.01) in test groups 00-03. In the high-dose group the pubertal age is well within of the historical range while the weight at puberty is distinctly below the historical control range. This indicates that the apparent later onset of puberty may be a spurious finding, and if at all, is a consequence of a general developmental delay and not a specific effect on the timing of puberty.

F1 rearing animals, Cohort 1A (14R0781A)
Estrous cycle data, generated during a maximum of 3 weeks, revealed regular cycles in the females of control as well as of the low- and mid-dose groups. The mean estrous cycle duration was prolonged in the high-dose group (4.1 / 4.0 / 4.2 and 5.5* (*:p<=0.05) days in test groups 10-13). There was no particular cycle phase prolonged, some high-dose F1A females with a longer cycle (beyond 5 days) had a prolonged diestrous, others had equally longer estrous and/or metestrous phases. One female (No. 562) stayed exceptionally long in estrous (11 days in 2 cycles). One female (No. 597) was in diestrous for 14 days which is usually considered as a sign of pseudopregnancy, without this animal the average cycle duration would have been 4.9 days.

F1 rearing animals, Cohort 1B (14R0781B)
Estrous cycle data, generated during a maximum of 3 weeks, revealed regular cycles in the females of control as well as of the low- and mid-dose groups. The mean estrous cycle duration was prolonged in the high-dose group (4.2 / 4.0 / 4.1 and 5.0*(*:p<=0.05) days in test groups 10-13). A number of females displayed irregular cycles, however they did not show a unique pattern of change. Specifically, there was no particular cycle phase prolonged in all females, some females had a prolonged diestrous, others had equally longer estrous and/or metestrous phases. Three females (Nos. 777, 785, 797) had no complete cycle at all (estrous missing), one of them (No. 797) was in diestrous during almost the entire observation period. Another female (No. 798) stayed in diestrous for 10 days, had then one regular cycle and afterwards remained in estrous for the remaining observation period.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
F1 rearing animals, Cohort 1A
Please see tables (Tables IC 16 – IC 23) of the attachment. The reduction in terminal body weight in males of test group 12 and 13 (300 and 1000 ppm) and females of test group 13 (1000 ppm) was regarded to be treatment-related. The increase
in kidney and liver weight (relative) was partly caused by the terminal body weight decrease but as most of the weights were outside the historical control values (see PART III) they were assessed to be treatment-related. The increased relative adrenal gland weight in test group 13 males was higher than what could be expected due to the terminal body weight decrease. But as there were no histopathologic findings corresponding to the weight increase this finding might be treatment-related but was not assessed to be adverse. The same comes true for the increase in adrenal weight in test group 12 (300 ppm) females. The male reproductive organs in test group 13 (1000 ppm) were mainly decreased by the
reduction in terminal body weight but this change would be less pronounced without the three male animals which revealed histopathologic findings in these organs. All other changed weight parameters were regarded to have been caused by the decrease in terminal body weight.

F1 rearing animals, Cohort 1B
Please see tables (Tables IC 33 – IC 36) of the attachment.
The significant reduction in terminal body weight of males and females of test group 12 and 13 (300 and 1000 ppm) was regarded to be treatment-related. The terminal body weight of males and females of test group 12 (300 ppm) was within the range of historical controls (see PART III). The liver weight increase in males and females of all treated groups was regarded to be treatment-related.The significant decrease in absolute weight of male sex organs in test group 13 (1000 ppm) was regarded to have been mainly caused by the reduced terminal body weight but it would have been less pronounced without the three male animals which revealed histopathologic findings in these organs. The same comes true for the significant increase of relative weights of pituitary gland in males of test group 13 (1000 ppm) and testes weight in test group 12 and 13 (300 and 1000 ppm). Whereas, for the significantly decreased prostate weight in test group 13 (1000 ppm) a treatment-related effect cannot be excluded, as the relative weight was still decreased. But as it was decreased less than in F1 generation, cohort 1A animals, it was still regarded to be secondary to the body weight reduction.
The significantly changed ovary weights in test group 12 and 13 (300 and 1000 ppm) were regarded to be incidental due to the to a missing dose response relationship and because it cannot be explained by histologic findings. The significantly increased relative uterus weights in test group 12 and 13 (300 and 1000 ppm) were also thought to be caused by the terminal body weight reduction.

F1 rearing animals, Cohort 3 (Immunotoxicity cohort)
Please see tables (Tables IC 40 – IC 47) of the attachment.
Male and female animals of test group 13 (1000 ppm) revealed a significant decrease in terminal body weight which was regarded to be treatment-related. The increase in relative spleen weight in males of this test group was thought to be a consequence to the body weight reduction and therefore secondary.

Cyclophosphamide monohydrate
Absolut and relative organ weights
When compared to the control group 10 (set to 100%), the mean absolute and relative weight parameters of test group 14 (positive control) were significantly decreased A significant decrease in absolute and relative weights of the spleen and thymus occurred in the positive control male and female animals. This result was expected.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The light-brown discoloration in the liver of twelve males and two females of test group 13 (1000 ppm) corresponded in many cases to the diagnosis “fatty change, peripheral” and were regarded to be treatment-related. The size reduction of prostate, seminal vesicle, epididymides and testes in three males (animals No. 476, 478, and 479) of test group 13 (1000 ppm) corresponded to histopathological findings. The foci in the glandular stomach were partly diagnosed as erosion/ulcer, but histopathologically there was not a significantly higher incidence compared to control. Therefore, it was regarded to be incidental.

F1 rearing animals, Cohort 1B (14R0781B)
Gross pathology
(Table IC 37)
In test group 13 (1000 ppm) three males (animal Nos. 693, 694, 697) revealed reduced size of the testes, epididymides, prostate and seminal vesicle. Four females (animal Nos. 777, 785, 797, 798) of test group 13 (1000 ppm) had reduced size of the ovaries. All these male and female animals revealed a severely lower body weight when compared to the average of this group or the control group. The weight decrease was therefore regarded to be treatmentrelated but secondary to the reduced terminal body weight. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

F1 rearing animals, Cohort 3 (Immunotoxicity cohort)
Gross pathology
(Table IC 48 – IC 49)
One male of test group 13 (1000 ppm) revealed reduced size of testes, epididymides, seminal vesicle and prostate. This was regarded to be caused by the severely reduced terminal body weight and therefore regarded to be treatment-related but secondary. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 rearing animals, Cohort 1A
Please see tables (Tables IC 26 – IC 31) of the attachment.
Similar to the findings described for the F0 generation animals, in the proximal tubules of the kidneys signs of degeneration and regeneration (apoptotic cell death, debris in the tubular lumen, increased numbers of large, vesicular nuclei, increased basophilic staining, nuclear crowding) were seen. These findings were regarded to be treatment-related. Within the papilla, females of test group 13 (1000 ppm) showed an increase of multifocal mineral deposition. Males of test group 12 and 13 (300 and 1000 ppm) showed multifocal mineral depositions at the transition between the outer and inner medulla. The mineralization in males and females was regarded to be treatment-related.

Males and females of test group 12 and 13 (300 and 1000 ppm) showed a centrilobular hypertrophy in the liver. Three males of test group 13 (1000 ppm) revealed a peripheral hypertrophy. Furthermore, males of test group 12 and 13 (300 and 1000 ppm) and females of test group 13 (1000 ppm) showed clear, round vacuoles in the peripheral area. Via the ORO stain it could be demonstrated that they representing lipid vacuoles. These findings were regarded to be treatment-related.

In the male mammary gland of test group 13 (1000 ppm) there was an increase of female-like mammary gland structures (tubule-alveolar structure, less amount of cytoplasm, increase in basophilia). One male revealed a moderate diffuse hyperplasia of the mammary gland tissue in addition. Females in test group 13 (1000 ppm) revealed a higher incidence of eosinophilic amorphous material which was secreted into the ducts. These findings were regarded to be treatmentrelated. In males a similar secretion was seen, but there was no significant difference between control and treated animals.

Three males (animal Nos. 476, 478, 479) of test group 13 (1000 ppm) showed the histological picture of juvenile and immature testes which were characterized by lower numbers or not fully developed and differentiated germ cell rows. In addition, there were no or only very few sperm present within testicular tubules. Consequently, these three animals did not have sperm within the epididymides (aspermia) and reduced size of the secondary sexual organs (prostate, seminal vesicle, and coagulating gland). One male (animal No. 479) showed multifocal degeneration of the testicular tubules in addition. The immaturity in these animals was regarded to be a consequence to the reduced body weight. The tubular degeneration in animal No. 479 was assumed to be treatment-related. Only in one male (animal No. 462), which was sacrificed in a moribund state, the reduced size of the ductus deferens did not match a finding in the testes. In this animal, the premature sacrifice was regarded to be responsible for the smaller size. The vacuolation in the ductus deferens in four males of test group 12 (300 ppm) might still be treatment-related but due to the missing findings in all other sexual organs it was not assumed to be adverse.

In test group 13 (1000 ppm) one female revealed a diffuse atrophy of the ovary. Six females revealed luteal cysts. These findings were regarded to be treatment related. The one cyst in a test group 12 female (300 ppm) might be treatment-related, but as luteal cysts sometimes also occur in control animals it might also be an incidental finding (McInnes 2012).

In the pars distalis of the pituitary gland of males and females of all test groups eosinophilic cysts were observed. These cysts differed from the cysts that occur sporadically as background lesion in the pituitary gland, also in this study. Spontaneous cysts which were observed in control and treated animals had a ciliated epithelium and a mucinous content. The treatmentrelated very small eosinophilic cysts revealed a non-ciliated, irregular border with an eosinophilic homogenous content and were multifocally distributed within the pars distalis occasionally revealing clear vacuoles at the border. In the thyroid glands of males of test group 11 and 12 (100 and 300 ppm) there was a slight increase in hypertrophy of the follicular epithelium. As in test group 13 (3000 ppm) no such finding was observed it was regarded to be incidental. Branchiogenic cysts occurred in control and treated animals and are regarded to be incidental findings. In these study, there was a
slight increase in test group 13 animals (1000 ppm) compared to control animals. Nevertheless, it was regarded to be incidental and not related to treatment. All other findings occurred either individually or were biologically equally distributed about
control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Differential ovarian follicle count – F1 rearing animals, Cohorts 1A and 1B
(Table IC 32)
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 – revealed significant differences between the control group 10 and animals of test group 13

F1 rearing animals, Cohort 1B (14R0781B)
Please see tables (Tables IC 38 – IC 39) of the attachment.
Three males (animal Nos. 693, 694, 697) of test group 13 (1000 ppm) showed the histological picture of juvenile and immature testes which were characterized by lower numbers or even none fully developed and differentiated germ cell rows. In addition, there were no or only very single sperm present within testicular tubules. Consequently, these three animals did not have sperm within the epididymides (aspermia) and reduced size of the secondary sexual organs
(prostate, seminal vesicle, and coagulating gland). Two males (animal No. 694 and 697) showed a slight to moderate multifocal degeneration of the testicular tubules in addition. Two males revealed a minimal degeneration in the testicle,
only. These findings were regarded to be treatment-related. In test group 1 (100 ppm) one male also had an immature testis and tubular degeneration.

Four females (animal Nos. 777, 785, 797, 798) of test group 13 (1000 ppm) revealed a diffuse atrophy of the ovaries, represented by lower numbers of functional corpora and reduction in size. Two of these females did not have any corpora lutea present. Furthermore, in the ovaries of six females of this test group and one female of test group 12 (300 ppm) luteal cysts were observed. These were large cystic cavities surrounded by luteinized and non-luteinized granulosa cells. These findings were regarded to be treatment-related.

NEUROPATHOLOGY
Summary tables of the results are to be found in Part D of PART I; individual tables are to be found in Part D of PART II. Abbreviations and histopathological grading used in pathology report and tables can be found in the appendix.

F1 rearing animals, Cohort 2A (Developmental Neurotoxicity Cohort, adults)
Weight parameters
(Tables ID 1 – ID 4)
Length and width of brain
(Table ID 5)
All length and width measurements were without any findings. Only the length parameter in females of test group 13 was minimally decreased. As the width of the brain in this test group was comparable with the control animals and the absolute brain weight was unchanged, a treatment-related effect seems unlikely.
Gross lesions
(Table ID 6)
Only the moribund sacrificed male animal No 837 revealed a dilation of jejunum, ileum, cecum and colon. These findings were assessed as incidental and not related to treatment. No other gross findings were recorded.
Histopathology
(Table ID 7 – ID 9)
The medulla oblongata and the spinal cord of treated animals revealed a minimal to marked, multifocal degeneration of nerve fibers. The finding was characterized by disintegrated myelin sheaths, vacuolation of myelin sheaths, pyknotic nuclei of oligodendroglia and spheroids as well as gitter cells within the lesions. The lesions were especially visible in longitudinal sections and less visible in cross sections of the spinal cord.

In the pars distalis of the pituitary gland, multifocally distributed very small eosinophilic cysts with a non-ciliated, irregular border and eosinophilic homogenous content were seen.

Morphometry
(Table ID 10)
All morphometric brain measurements were without any findings. Only some single parameters in male (hippocampus right, base of lobus vermis cerebrelli no 8) or female (parietal cortex left) animals of test group 13 showed a statistical significant increase. As no other values were changed, this minimal width increase is assumed as incidental and not related to treatment.

F1 animals, Cohort 2B (Developmental Neurotoxicity Cohort, weanlings)
Weight parameters
(Tables ID 11– ID 14)
The statistically significant decrease of terminal body weight in male and female animals of test group 12 (females only) and 13 is regarded as treatment-related. The decrease of brain weight in test group 13 males is assumed to be secondary to the delayed development of the animals, nevertheless the length and the width of brains are comparable to control animals. The increased relative brain weights of test group 12 (females only) and 13 (both sexes) animals are secondarily linked to the decreased terminal body weight. The increase in absolute brain weight in test group 11 females is regarded as incidental.

Length and width of brain
(Table ID 15)
All length and width measurements were without any findings.

Gross lesions
(Table ID 16)
No gross findings were recorded.

Histopathology
(Table ID 17 - ID 18)
No treatment-related neurohistopathological findings were recorded. In the pars distalis of the pituitary gland, multifocally distributed very small eosinophilic cysts with a non-ciliated, irregular border and an eosinophilic homogenous content were seen in four males and five females (each out of ten) of test group 13 (1000 ppm). This finding is regarded as treatment-related.

Other effects:
effects observed, treatment-related
Description (incidence and severity):
Thyroid hormones
In F1 generation females of test group 11 and 12 (100 and 300 ppm) at PND 4 and in females at PND 22 of test group 13 (1000 ppm) T4 was higher compared to controls. In test group 13 (1000 ppm) only two males had a sufficient sample volume for the measurement of the thyroid hormones at PND 4, but no female pup was available.
The T4 means in F1 generation males and females of test group 13 at PND 92 were 19%/18% higher compared to controls (medians 18%37% higher), although the values were not statistically significantly changed.

Platelet activating factor (PAF) determination in serum of F0 parents
The platelet activating factor (PAF) concentration in serum of the F0 females is reduced dosedependently when regarding medians with a decrease of 31% in test group 3 (1000 ppm) compared to controls. This decrease in females of test group 3 was statistically significant when the two-sided Jonckheere-Terpstra trend test was applied. The PAF levels decrease could not been observed among the F0 males.

CHOLINE CONCENTRATION IN BLOOD AND LIVER SAMPLES
The analytical results demonstrated the clear presence of choline in all plasma samples from the animals dosed with the test substance 2,2’-iminodiethanol (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 2,2’-iminodiethanol led to a reduction in the content of choline in the plasma samples analyzed. 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. The analytical results demonstrated the clear presence of choline in all liver samples from the animals dosed with the test substance 2,2’-iminodiethanol (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 2,2’-iminodiethanol 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.
Developmental immunotoxicity:
effects observed, non-treatment-related
Description (incidence and severity):
T-cell dependent antibody response (Anti SRBC IgM antibodies)
Six days after immunization, no changes in the SRBC IgM titers were found in male and female rats of the F1 generation (PND 60) dosed with the test substance. SRBC titers were statistically significantly lower in rats of the positive control group (dosed with Cyclophosphamide).

Splenic lymphocyte subpopulations
In females of the F1 generation (PND 92) of test group 13 (1000 ppm) relative T-helper cell (CD4+/CD3+ lymphocytes in spleen tissue) counts were decreased and relative cytotoxic Tcell (CD8+/CD3+ lymphocytes in spleen tissue) counts were increased compared to controls. This led to a decrease of the CD4/CD8 ratio (not statistically significantly). Absolute T-helper cell counts were not altered in the mentioned test group, but absolute cytotoxic T-cells were also increased (although not statistically significantly).
Auditory Startle Response
The maximum amplitude of the high-dose males and females (1000 ppm) was below the concurrent control during the entire measurement, while latency of reaction to a startle stimulus corresponded to the age of these animals. There was also no habituation to the test environment seen in these animals, males slightly more affected by this than females.The difference in the amplitude was statistically significant in measurement block 1 and 1 - 5 (males) as well as block 5 (females). No influence of the test substance on auditory startle habituation (maximum amplitude and latency) was observed in the low- and mid-dose male and female animals during the measurement. Amplitude and latency of reaction to a startle stimulus and habituation to the test environment corresponded to the age of these animals at PND 24, when normal biological variation inherent in the strain of rats used for this experiment was considered.

Functional observational battery (FOB)
Home cage observations:
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
Open field observations:
The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.
Sensorimotor tests/reflexes:
There were no test substance-related findings in male and female animals of all test groups.
Quantitative Parameters:
No test substance-related impaired parameters (number of rearings, grip strength of fore- and hindlimbs and landing foot splay test) were observed in male and female animals of all test groups. This includes a statistically significantly lower grip strength for forelimbs of the highdose females, which is considedered to be an isolated, and thus incidental, finding.

Motor activity measurement (MA)
Motor activity (number of beam interrupts) of male and female animals was not influenced by the test compound at all dose levels (100, 300 and 1000 ppm). Overall activity levels and habituation to the test environment corresponded to the age of these animals at PND 70. Any occasional differences between treated animals of these dose groups and their concurrent control (like lower numbers of beam interrupts in the low- and high-dose females during interval 3), whether statistically significant or not, were regarded as incidental findings and not related to the test compound.

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

Anogenital distance/anogenital index
Anogenital distance and index of all test substance treated pups (100; 300 and 1000 ppm) were comparable to the concurrent control values.

Nipple/ areola anlagen
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 12. However, owing to the high background rate in control animals on this day, we habitually recheck all animals for nipples/areolae on PND 20, one day prior to weaning. During this re-examination no areolae were detected at all in male pups of all test groups.

Key result
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Generation:
F1
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive performance
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity in the F1 progeny
Generation:
F1
Effect level:
100 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity in the F1 progeny
Key result
Dose descriptor:
NOAEL
Remarks:
developmental neurotoxicity
Generation:
F1
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental neurotoxicity
Key result
Dose descriptor:
NOAEL
Remarks:
developmental immunotoxicity
Generation:
F1
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental immunotoxicity
Key result
Critical effects observed:
no
Reproductive effects observed:
no
Effect on fertility: 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 fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

For the endpoint toxicity to reproduction a WoE Approach is conducted using results from the registered substance MDEA and studies performed with the structurally analogous substances Monoethanolamin (MEA, CAS 141-43-5), Diethanolamin (DEA , CAS 111-42-2) and Triethanolamin (TEA, CAS 102-71-6).

For N-Methyldiethanolamin (MDEA) given daily as an aqueous solution to groups of 10 male and 10 female Wistar rats (F0 animals) by stomach tube at doses of 0, 100, 300 and 1000 mg/kg bw/day) effects on implantation are reported. Clinical signs of toxicity were seen at the high dose level (1000 mg/kg bw/day) such as decreased food consumption in females during lactation and decreased body weight gain in males and females, resulting in reduced terminal body weights in both sexes. Reduced terminal body weights were also seen at the mid dose level (300 mg/kg bw/day). Total litter loss in 4 females, undelivered pups, insufficient lactation behavior, increased duration of gestation, decreased number of implantation sites, increased postimplantation loss and decreased number of delivered pups, reduced pup viability, decreased pup body weights and weight gains were seen exclusively at the high dose level (1000 mg/kg bw/day), i.e. only in the presence of parental toxicity. Pathology revealed increased liver weights at all dose levels, however without any morphological correlate. These findings were therefore assessed as being an adaptive phenomenon, but not an adverse effect. There were no further treatment-related lesions detected, especially, there were no weight or substance-related pathomorphological effects on testes, epididymides, and ovaries present. In conclusion, the administration of N-Methyldiethanolamin at dose levels of 1000 and 300 mg/kg bw/day caused toxic effects on body weight. The NOAEL for general, systemic toxicity was therefore 100 mg/kg bw/day for the F0 parental males and females. The NOAEL for reproductive performance and fertility was 300 mg/kg body weight/day for the F0 parental rats based upon finding such as litter loss, insufficient lactation behavior, and increased duration of gestation. The NOAEL for developmental toxicity was 300 mg/kg bw/day, based on findings such as reduced viability index and reduced postnatal offspring weight gain. Thus, effects on reproductive parameters were affected only in the presence of pronounced parental toxicity.  

In a standard screening study similat to OECD TG 421 (BASF, 2010), the structural analogue 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 the smaller litter sizes.

No two-generation study is available with MDEA. For the structural analogue Monoethanolamine (MEA) a two generation reproduction toxicity study in Wistar rats with dietary MEA 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.

For the structural analogue DEA, an Extended One-Generation Reproductive Toxicity Study (EOGRTS) according to OECD TG 443 has been performed (Ethanolamine Reach Consortium, 2018). Cohorts 2A and B as well as cohort 3 were included for an investigation of the developmental neurotoxicity module (DNT) and the developmental immunotoxicity module (DIT), but without the extension to an F2-generation. Additionally, measurements of the essential nutrient choline were performed in different tissues of the F0 and F1 pups (plasma and liver, respectively). Furthermore, additional investigation of parameters such as platelet-activating factor (PAF) have been included as another modification to follow-up on a mode-of-action. DEA was administered to groups of 30 male and 30 female healthy young Wistar rats (F0 parental generation) as a solution to the drinking water in different concentrations (0, 100, 300 and 1000 ppm). At least 16 days after the beginning of treatment, F0 animals were mated to produce a litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 5 different cohorts which were continued in dose groups 10 - 13 in the same fashion as their parents and which were subjected to specific post weaning examinations. The study terminated with the terminal sacrifice of the male and female animals of cohort 1B. Test drinking water containing DEA were offered continuously throughout the study.

The overall mean dose of DEA throughout all study phase and across all cohorts was approx. 12.75 mg/kg body weight/day (mg/kg bw/d) in the 100 ppm group, approx. 37.68 mg/kg bw/d in the 300 ppm group and approx. 128.35 mg/kg bw/d in the 1000 ppm group.

Under the conditions of the OECD TG 443 study the NOAEL for general toxicity is 100 ppm for the F0 parental animals, based on evidence for distinct kidney toxicity and stomach irritation, as well as corresponding effects on water consumption, food consumption, body weights and clinicopathological parameters, which were observed at the LOAEL (Lowest Observed Adverse Effect Level) of 300 ppm. Similar toxicity was noted in the adolescent F1 animals, which had no stomach irritation but liver toxicity in addition.

The NOAEL for fertility and reproductive performance for the F0 and F1 rats is 300 ppm, based on a lower number of implants, prolonged/irregular oestrous cycles as well as pathological changes in sexual organs, pituitary and mammary glands of both genders at the LOAEL (Lowest Observed Adverse Effect Level) of 1000 ppm. Most of the reported effects on reproduction and reproductive organs occurred in the range of general and systemic toxicity and have been assessed to be secondary in nature.

The NOAEL for developmental toxicity in the F1 progeny is 100 ppm, based on impaired pup survival at 1000 ppm as well as reduced pup body weights in the F1 offspring, which were observed at the LOAEL (Lowest Observed Adverse Effect Level) of 300 ppm. As these weight reductions were only observed in the presence of maternal toxicity, including lower weight gain during pregnancy, they are not regarded as independent effect of the treatment.

The NOAEL for developmental neurotoxicity for the F1 progeny is 300 ppm, based on adverse clinical observations, impaired auditory startle response and corresponding neuropathological findings at the LOAEL (Lowest Observed Adverse Effect Level) of 1000 ppm. In addition, increased T4 values were noted in adult and adolescent males at 1000 ppm as well as newborn and weanling females at 100 and 300 ppm although there was no associated change in TSH levels.

The NOAEL for developmental immunotoxicity for the F1 progeny is 300 ppm, based on effects on the T-helper cells and cytotoxic T-cells in the spleen in the F1 females at the LOAEL (Lowest Observed Adverse Effect Level) of 1000 ppm. Lower mean and median anti-SRBC IgM antibody titers of the positive control group (4.5 mg/kg bw/day cyclophosphamide, oral) demonstrated that the test system worked properly.

Effects on developmental toxicity

Description of key information

A screening reproduction/developmental toxicity study (OECD TG 421) with MDEA in the rat by oral gavage has been performed. The NOAEL for developmental toxicity was 300 mg/kg bw/day based on findings such as reduced viability index and reduced postnatal offspring weight. The NOAEL for parental toxicity was set at 100 mg/kg bw/day, based on body weight loss in both sexes. Thus, developmental toxicity was only seen in the presence of parental toxicity.

In a prenatal developmental toxicity study similar to OECD TG 414 pregnant female rats were dermally exposed to MDEA on gestation days 6 to 15. The maternal and developmental NOAELs were established to be 250 and 1000 (the highest dose tested) mg/kg bw/day, respectively.

In addition to the available dermal study with MDEA, read across to the structure analogue MEA is proposed to provide information on a second species. In a study similar to OECD TG 414 MEA was administered via the dermal route to rabbits. No treatment-related effects were observed on reproductive and developmental toxicity parameters. NOAEL for developmental toxicity was set at the highest dose level of 75 mg/kg bw/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: CD
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: male: 77 days; females: 70 days;
- Weight at study initiation: Female rats weighed at least 200 g at the time of mating.

Route of administration:
dermal
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: dorsal trunk
- Type of wrap if used: refolded eight-ply sterilized gauze square and a 2.75 x 2.25 inch area of polyvinyl film.
- Time intervals for shavings or clipplings: 3 days prior to the first dose and subsequently as needed throughout the study.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): the dosing site was wiped gently with a paper towel dampened with warm water and blotted dry.
- Time after start of exposure: 6 ha fter the application.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 4 mL
- Concentration (if solution): Range finding study: Definitive study: 250, 500, and 1000 mg/kg/day (Range finding study: 100, 500, 750, and 1000 mg/kg/day).

VEHICLE
- Amount(s) applied (volume or weight with unit): 4 mL
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
MDEA concentrations in dosing solutions were verified using a gas chromatograph equipped with a flame ionization detector.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
days 6 - 15 of gestation
Frequency of treatment:
6 hours per day
Duration of test:
21 days
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Twenty-five plug-positive females were assigned to each group (range finding test: 8 plug-positive females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results of the range finding study where the following concentrations were tested: 0, 100, 500, 750, and 1000 mg/kg bw/day
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on gd 0, 6, 9, 12,15, 18, and 21

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes

WATER CONSUMPTION AND COMPOUND INTAKEy: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: gravid uterus, ovaries including corpora lutea, cervix, vagina, and peritoneal and thoracic cavities were examined grossly

Other: blood was used for erythrocyte, platelet, total and differential leukocyte counts, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration.
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
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
Statistics:
Data from quantitative, continuous variables were intercompared for the dosed groups and the control group by use of Levene's test for equality of variances, analysis of variance, (ANOVA), and t-tests. Nonparametric data were evaluated using the Kruskal- Wallis test, followed by the Mann-Whitney U test when appropriate incidence data were compared using the Fisher's Exact Test. The probability value of p < 0.05 (two-tailed) was used as the critical level of significance.
Dermal irritation (if dermal study):
effects observed, treatment-related
Description (incidence and severity):
Skin reactions at the dosing site were observed in the 500 and 1000 mg/kg bw/day groups. Effects included exfoliation, excoriation, crusting, ecchymoses, and necrosis.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The erythrocyte count and hematocrit were reduced in the 1000 mg/kg bw/day group. Also, while not statistically significant, hemoglobin was reduced by about 6% in the 1000 mg/kg bw/day group. However, there were no effects on mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, and total or differential leukocyte counts.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no effects of dosing with MDEA on gravid uterine weight, or liver and kidney weights.
Other effects:
no effects observed
Description (incidence and severity):
There were no effects on the number of ovarian corpora lutea, the number of total, viable or nonviable implantations, preimplantation loss, and fetal body weights.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Basis for effect level:
dermal irritation
Key result
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
There were no increased incidences of total external, visceral, and skeletal malformations or variations or individual malformations compared to controls. Among individual variations, there was a statistically significant decrease in the incidence of a soft-tissue variation (dilated bilateral renal pelvis) and a skeletal variation (split number 1, 2, 3, and/or 4 cervical centra) at 1000 mg/kg bw/day. These decreases are not considered characteristic of a developmental delay.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Occluded cutaneous applicatation of methydiethanolamine to pregnant rats during organogenesis resulted in maternal toxicity as indicated by skin irritation and mild anaemia from 500 mg/kg bw/day onwards, but no treatment-related developmental effects were observed.

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 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:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11 - 13 weeks
- Weight at study initiation:
male animals: 279.9 g - 315.9 g, female animals: 173.2 g - 202.8 g
- Housing:
- During the study period, the rats were housed individually
- During overnight matings, male and female mating partners were housed together
- Pregnant animals and their litters were housed together until PND 4.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5-6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
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. The volume administered each day was 10 ml/kg bw. The calculation of the administration volume was based on the most recent individual body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration control analyses of the test substance preparations:
The method of analysis was Capilary electrophoresis (CE). All measured values for N-Methyldiethanolamin were in the expected range of the target concentrations (90-110%).
Details on mating procedure:
Males and females from the same dose group were mated 13 days after the beginning of treatment, overnight in a ratio of 1:1.
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
The duration of treatment covered premating period of 2 weeks and a mating period (max. of 2 weeks) in both sexes and the entire gestation period as well as 4 days of lactation in females.
Frequency of treatment:
daily
Duration of test:
N-Methyldiethanolamin was given daily as an aqueous solution to groups of 10 male and 10 female Wistar rats (F0 animals) by stomach tube at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/day). Control animals were dosed daily with the vehicle only (drinking water). The duration of treatment covered premating period of 2 weeks and a mating period (max. of 2 weeks) in both sexes and the entire gestation period as well as 4 days of lactation in females. The females were allowed to deliver and rear their pups until day 4 after parturition. Four days after PND 4 of the female, which delivered last, all parental females were sacrificed and examined. Pups were sacrificed on PND 4 and gross necropsied. The male animals were sacrificed 28 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
Details on study design:
- Dose selection rationale:
In a test study (Project No. 06R0087/01248, BASF SE) N-Methyldiethanolamin was given daily as an aqueous solution to groups of 3 male and 3 female Wistar rats (F0 animals) by stomach tube at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) for 2 weeks. Control animals were dosed daily with the vehicle only (drinking water). Clinical signs, food consumption and body weights were determined. The only clearly substance-related effect was “salivation after treatment” in all males and females at the high dose level. Food consumption and body weights did not show significant differences.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
A check for moribund or dead animals was made twice daily on working days or once daily (weekends or public holidays).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: once a week in males throughout the study and in females during premating and mating. During gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the parturition day and postnatal day (PND) 4.

FOOD CONSUMPTION:
Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4.
Ovaries and uterine content:
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 were detected, and gestational status were recorded for F0 females.
For the females, mating, fertility and gestation indices were calculated .
Fetal examinations:
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.

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.

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; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy.

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.

The pups were weighed on the day after birth (PND 1) and on PND 4.

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: 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:
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%

Female mating index (%)= number of females mated/ number of females placed with males x 100%

Female fertility index (%)= number of females pregnant/number of females mated x 100%

Gestation index (%)= number of females with live pups on the day of birth/number of females pregnant x 100%

Postimplantation loss (%)=number of implantations – number of pups delivered/number of implantations x 100%

Live birth index (%)= number of liveborn pups at birth/total number of pups born x 100%

Viability index (%) = number of live pups on day 4 after birth/number of live pups on the day of birth x 100%
Details on maternal toxic effects:
1000 mg/kg bw/day:
• Decreased food consumption in females (-37%) during lactation days 1-4
• Decreased body weight gain in males in treatment weeks 2-3 (57% below control) and 0-3 (29% below control)
• Decreased body weights in females on gestation days 14 and 20 (up to 14% below control) and lactation day 4 (about 5% below control)
• Decreased body weight gain in females between gestation days 7-20 (up to about 46% below control)
• Total litter loss in 4 females
• Undelivered pups palpable in 2 females
• Insufficient lactation behavior in 2 females (pups had no or less milk in stomach)
• Increased duration of gestation (22.8 days vs. 21.9 days in control)
• Decreased number of implantation sites (6.7 vs. 12.9 in control)
• Increased postimplantation loss (31.0% vs. 6.0% in control) and mean postimplantation loss
• Decreased number of delivered pups (4.6 vs. 12.1 in control)
• Decrease of terminal body weights in males and females (7 and 5 % below controls, respectively).

300 mg/kg bw/day:
• Decreased terminal body weights in males and females (5% below controls in both sexes).

100 mg/kg bw/day:
• No test substance-related adverse findings.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
1000 mg/kg bw/day:
• Reduced viability index (62%), resulting from significantly higher numbers of died and cannibalized pups
• Decreased pup body weights on PND 4, average difference to the control 20%
• Decreased pup body weight gain during PND 1 – 4, average difference to the control 52%

300 and 100 mg/kg bw/day:
• No test substance-related adverse findings
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on findings such as reduced viability index and reduced postnatal offspring weight gain.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
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
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
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
similar to 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 adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
similar to OECD TG 414
Additional information

In a reproduction/developmental screening study (performed according to OECD guideline 421), MDEA was given daily as an aqueous solution to female and male Wistar rats (F0 animals) by oral gavage at doses of 100, 300 and 1000 mg/kg bw/day (BASF, 2010). The duration of treatment covered pre-mating period of 2 weeks and a mating period (max. of 2 weeks) in both sexes and the entire gestation period as well as 4 days of lactation in females. Parental animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the post-implantation loss in all F0 females. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed one PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed and examined macroscopically for external and visceral findings at necropsy. All F0 parental animals were sacrificed and assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

Clinical signs of toxicity were seen at the highest dose level (1000 mg/kg bw/day) such as decreased food consumption in females during lactation and decreased body weight gain in males and females, resulting in reduced terminal body weights in both sexes. Reduced terminal body weights were also seen at the mid dose level (300 mg/kg bw/day).Pathology revealed increased liver weights at all dose levels, however without any morphological correlate. These findings were therefore assessed as being an adaptive phenomenon, but not an adverse effect. There were no further treatment-related lesions detected, especially, there were no weight or substance-related pathomorphological effects on testes, epididymides, and ovaries present.

Concerning reproductive parameters, total litter loss in 4 females, undelivered pups, insufficient lactation behavior, increased duration of gestation, decreased number of implantation sites, increased post implantation loss and decreased number of delivered pups, reduced pup viability, decreased pup body weights and weight gains were seen exclusively at the high dose level (1000 mg/kg bw/day), i.e. only in the presence of parental toxicity.

In conclusion, the NOAEL for general, systemic toxicity was 100 mg/kgbw/day for the F0 parental males and females.The NOAEL for reproductive performance and fertility was 300 mg/kg bw/day for the F0 parental rats based upon litter loss, insufficientlactation behaviorand increased duration of gestation. The NOAEL for developmental toxicity was 300 mg/kg bw/day, based on reducedviability index andreduced postnatal offspring weight gain.

In a prenatal developmental toxicity study, pregnant CD rats (25/dose) were dermally exposed to 0, 250, 500 or 1000 mg/kg bw/day of MDEA on gestation day 6 -15 for 6 hours/day under occlusive conditions (Leung and Ballantyne, 1998). The NOAEL for maternal toxicity was determined to be 250 mg/kg bw/day, above which local irritant reactions at the application site were observed (including exfoliation, excoriation, crusting, ecchymoses, and necrosis). In addition, high dose dams had decreased erythrocyte counts, hemoglobin and hematocrit. There were no effects on any gestational parameters, and no increases in the total number of malformations or variations (external, visceral, or skeletal). Thus, the NOAEL for developmental toxicity was established at 1000 mg/kg bw/day, the highest dose tested.

In addition to the available dermal study with MDEA, read across with the structure analogue MEA is proposed to provide information on a second species. In the rabbit study with MEA exposure was via the dermal route to 0, 10, 25, and 75 mg/kg/day (protocol comparable to OECD 414). 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 et al, 1996).

Toxicity to reproduction: other studies

Description of key information

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 (Moore et al., 2018).

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of the studies reported herein was to investigate the potential role of choline antagonism in the aetiology of Ethanolamine (EA; 2-aminoethanol; CAS RN 141-43-5)-induced implantation loss. The work was conducted in two phases: the first to determine if there was a critical period of sensitivity during the pre- and peri-implantation period; the second to evaluate the impact of choline co-administration on implantation success following treatment with EA.
GLP compliance:
yes
Type of method:
in vivo
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI (Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Housing: housed singly in Makrolon type M III cages, with dust-free wood chipbedding and suitable environmental enrichment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20–24
- Humidity (%): 30–70
- Photoperiod: 12/12
Route of administration:
other: drinking water (1st Experiment) and diet (2nd experiment)
Details on exposure:
1st Experiment:
Groups of twelve time-mated pregnant female rats were administered either EA-HCl (1000 mg/kg bw/day; 10.25 mmol/kg bw/day) or IPEA (100 mg/kg bw/day; 0.97 mmol/kg bw/day) in distilled water, once daily by gastric intubation on GD 1–3, GD 4–5, or GD 6–7. A control group was administered distilled water through-out the period GD 1–7. The IPEA solution was adjusted to pH 7.0 by the addition of hydrochloric acid prior to administration. Blood was collected by retroorbital venous puncture under isoflurane anaesthesia for standard haematological and clinical chemistry analyses. Food consumption and body weight were recorded throughout gestation, and daily cage-side checks were made for clinical signs of toxicity or morbidity. On GD 20, all animals were anaesthetised with isoflurane and sacrificed by cervical dislocation. After grosspathological examination, the ovaries were removed and the corpora lutea were counted, and the uterus was removed and weighed. The uterus was then opened and evaluated for the number of implantations which were differentiated according to live and dead foetuses, and early or late resorptions. Early resorptions in animals that did not appear to be pregnant, or which had single-horn pregnancy, were confirmed by ammonium sulphide staining. Foetuses were sacrificed by subcutaneous injection of sodium pentobarbital.

2nd Experiment:
After a seven-day acclimatisation period, groups of twelve non-pregnant female rats were given standard diet or diet supplemented with choline chloride to achieve choline intakes of approximately 1.2–1.5 and 6.2–7.5 mmol/kg bw/day respectively. Subsets of these groups were administered EA.HCl (1511 mg/kg bw/day;15.49 mmol/kg bw/day) in distilled water adjusted to pH 7.0 with aqueous sodium hydroxide, once daily by gastric intubation; EA.HCl in the diet at an inclusion level to
achieve an equivalent delivered daily dose; or distilled water by oral gastric intubation. A higher dose of EA was selected compared to that used in Experiment 1, based on the OECD limit dose of 1000 mg/kg/day as EAfree base. Therefore, there were six experimental groups in a 2 × 3 block design: untreated control (group 0); choline-supplemented diet (group 1); EA administered by gastric intubation (group 2); EA administered by gastric intubation with choline-supplemented diet (group 3); EA-su
pplemented diet (group 4); and diet supplemented with both EA and choline (group 5). After fourteen days of treatment each female was paired overnight with an untreated male in the male’s home cage. In cases where females were treated via the diet, the males’ diet was replaced by that of the females. The pairing procedure continued until evidence of copulation, either vaginal plug or sperm in the vaginal smear. This day was denoted GD 0, after which the females remained in their owncages and were treated as before until GD 8. Blood was collected approximately 1–1.5 hours after the final dose administration, by retroorbital venous puncture under isoflurane anaesthesia, for standard haematological and clinical chemistry analyses as well as the measurement of EA concentration in the plasma. All animals were then given standard untreated diet from GD 9 onwards and gavage treatment was curtailed. Food consumption and bodyweight were recorded throughout gestation, and daily cage-side checks were made for clinical signs of toxicity or morbidity. The experiment was terminated on GD 20 in the same manner as the first.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of EA.HCl and choline chloride in the die twere determined by HPLC/MS, monitoring the protonated [M + H]+ions formed by heated electrospray ionisation. A sample of the treated diet was accurately weighed and suspended in 0.1% aqueous formic acid by shaking for one hour. An aliquot was then diluted further with 0.1% aqueous formic acid and filtered through a Millex-GV 0.45 μm filter prior to direct injection (10 μL) onto the column (Primesep 200, 250 × 3.2 mm, 5 μm). Separation was by means of gradient elution between water and acetonitrile, each containing 0.1% formic acid, at a constant flow rate of 0.5 mL/min. The concentration of EA (as free base) in rat serum was also determined by HPLC/MS. An aliquot (20 μL) of serum was mixed with methanol (980 μL), centrifuged (13000 rpm, 5 min), and an aliquot (10 μL) of the supernatant was applied directly to the column. The mobile phase consisted of 80% acetonitrile and 20%aqueous ammonium formate (50 mM, pH 3.7) at a flow rate of0.5 mL/min. Again, the protonated [M + H]+ions were monitored following heated electrospray ionisation in positive mode. The working limit of quantitation was approximately 50 ng/mL; in comparison, the concentration of EA in serum from an untreated rat was 445 ng/mL.
Duration of treatment / exposure:
see "Details on exposure"
Frequency of treatment:
see "Details on exposure"
Duration of test:
see "Details on exposure"
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
EA treatment in 1st Experiment
Dose / conc.:
1 511 mg/kg bw/day (nominal)
Remarks:
EA treatment in 2nd Experiment
No. of animals per sex per dose:
12
Control animals:
yes
Details on study design:
see "Details on exposure"
Statistics:
If Bartlett’s test for homoscedasticity was not significant, fol-lowing transformation if necessary, continuous data were analysedby unpaired t-test or by ANOVA followed by either Dunnett’stest (experiment 1) or the Tukey-Kramer test (experiment 2); the central tendency is presented as the mean, with dispersion as standard error. Proportional data were analysed by Chi2 and Fisher’s exact tests. Count data, and continuous data for which parametric analysis was not appropriate, were analysed by unpaired U-test or by Kruskal-Wallis ANOVA followed by either Steel’s test (experiment
1) or the Dwass-Steel-Critchlow-Fligner test (experiment 2); the central tendency is presented as the median, with dispersion represented by first and third quartiles. Multiple pair-wise comparisons were correctedwhere necessary. Growth (that isrepeated-measures of body weight) was evaluated with multilevel mixed-effects linear regression. The effects of EA and IPEA administered during the pre- and peri-implantation period (experiment 1) and the effect of EA administered by gastric intubation or diet(experiment 2; comparing groups 0, 2, and 4, and comparing groups 1, 3, and 5) were tested by parametric or Kruskal-Wallis ANOVA followed by appropriate post hoc tests. The interaction between choline and EA (experiment 2; comparing group 3 with group 2, and group 5 with group 4) was tested by unpaired t- or U tests. Preimplantation loss per litter was calculated as the difference between counts of corpora lutea and total implantation sites; post-implantation loss per litter was calculated as the difference between total implantation sites and live foetuses.
Key result
Remarks on result:
not measured/tested
1st Experiment:
Treatment with EA.HCl (1000 mg/kg bw/day; 10.25 mmol/kg bw/day) or with IPEA (100 mg/kg bw/day; 0.97 mmol/kg bw/day) during GD 6-7 had no consistent effect upon food intakeduring the period of treatment or thereafter. Treatmentwith IPEA during GD 1-3 or 4-5, however, resulted in substantially reduced food intake during the final trimester. EA caused aminor reduction in body weight compared to the concurrent control group only when administered during the periods GD 1-3 and GD 4-5; body weight gain was significantly reduced from GD 13, but mean body weight values were within 5% of the control group value and only attained statistical significance from GD 19. IPEA caused a substantial reduction in body weight when administer
ed during the same periods. In both cases, administration during GD 6-7 had little or no effect on body weight gain. There were no dead foetuses resulting from the administration of either EA or IPEA. Furthermore, treatment with EA had no effect upon the number of implantation sites, early resorptions, or live foetuses. Total litter resorption in one dam treated during GD 4-5, which involved a single implant, is considered to be an isolated event and unrelated to treatment. Treatment with IPEA resulted in a strong response, most notably in terms of total pre-implantation loss among animals treated during GD 1–3; of the twelve animals that were mated, eleven were found to be not pregnant, as opposed to one of twelve in the control group. When treatment was delayed to the period GD 4-5, the response was manifested as an early loss of implantations. Treatment later in the peri-implantation period had no effect upon the number of either implants or live foetuses. Total litter resorption of five and eleven implants in one dam from the GD 1-3 and GD 6-7 treatment groups respectively may reflect the variability in actual timing of conception compared to the nominal assignment.
2nd Experiment:
Treatment with EA (1511 mg/kg bw/day, 15.49 mmol/kg bw/day) by gastric intubation had no effect upon food consumption and only marginal effect on body weight gain towards the end of gestation. Dietary administration of EA resulted in slightly reduced food consumption, slight reduction in body weight gain during the latter part of the administration period, and a slight reduction on body weight compared to control during the latter part of gestation. The overall intake of EA by both routes was
closely matched. The inclusion of choline in the diet resulted in an approximately fivefold increase in choline intake, and a reduction in food consumption which was reversed on cessation of treatment at GD 8, the latter of which was reflected in reduced body weight increase. Nevertheless, body weights of all EA-treated groups were within 10% of the respective control group values. Time to successful mating was unaffected by treatment. There were no dead foetuses and, with the exception of one implant in group 1, no late resorptions .The administration of EA resulted in an increase in pre- and postimplantation losses, although this only reached statistical significance for post-implantation loss when EA was administered in the diet. The co-administration of choline ameliorated the pre- and postimplantation loss induced by EA when administered both by gastric intubation and via the diet. Although this was only statistically significant for the induction of pre-implantation loss by dietary EA (p < 0.05), the induction of post-implantation loss by EA dosed as a bolus was reduced by borderline statistical significance (p = 0.054). Gavage administration resulted in a higher plasma concentration than dietary administration. The co-administration of choline significantly reduced EA concentrations in both cases (p < 0.05).

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. 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 are 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.  For MDEA given daily as an aqueous solution to female Wistar rats by stomach tube effects on implantation are reported. Clinical signs of toxicity were seen at the high dose level such as decreased food consumption in females during lactation and decreased body weight gain in males and females, resulting in reduced terminal body weights in both sexes. Reduced terminal body weights were also seen at the mid dose level. Total litter loss in 4 females, undelivered pups, insufficient lactation behavior, increased duration of gestation, decreased number of implantation sites, increased postimplantation loss and decreased number of delivered pups, reduced pup viability, decreased pup body weights and weight gains were seen exclusively at the high dose level, i.e. only in the presence of parental toxicity. There were no weight or substance-related pathomorphological effects on testes, epididymides, and ovaries present. For the structurally similar Ethanolamines Mono-, Di- and Triethanolamine effects on pre- and/or post-implantation losses were observed as well while most of the reported effects on reproduction and reproductive organs occurred in the range of general and systemic toxicity and have been assessed to be secondary in nature.

2. Major 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. 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 are 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.”

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 ethanolamins 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 the OECD TG 443 study performed with DEA described in this section 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 significant 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 significant 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).

For TEA it is reported that it decreases the hepatic levels of Phosphatidylcholine and Betaine, the primary oxidation product, when TEA is given dermally to female B6C3F1mice (Stott, 2004) at the high dose of 1000 mg/kg bw up to 26-42% indicating a disturbance. 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 (5 days/week). Higher doses of TEA applied orally as it has been done in the available OECD 421 described above 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.

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 cholinedeficiency 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 occurs 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).

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 MDEA and its structurally analogues substances MEA, DEA and TEA 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. Furthermore, effects of MDEA on reproduction were only observed in the presence of parental toxicity and thus were regarded as secondary effects.

Therefore, MDEA is not subjected for classification on toxicity to reproduction or developmental toxicity according to Regulation (EC) No 1272/2008.

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

Hoffbauer FW and Zaki FG (1965).Choline Deficiency in Baboon and Rat Compared. Arch Path 79: 364369

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). SAdenosylmethionine: 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

Additional information