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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Aug 2006 - 15 Jan 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Aug 2006 - 15 Jan 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 416 (Two-generation reproduction toxicity study)
Deviations:
yes
Remarks:
Food consumption was not determined between days 14 and 21 after parturition
Qualifier:
according to
Guideline:
other: Corrigendum to EC Commission Directive 2004/73/EC, Part B: Methods for the determination of toxicity: Two-Generation Reproduction Toxicity Study; Official Journal of the European Communities; No. L216, pp. 236–246
Version / remarks:
29 Apr 2004
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Version / remarks:
Aug 1998
GLP compliance:
yes (incl. certificate)
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 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 oral exposure:
DIET PREPARATION
The test substance was weighed and thoroughly mixed with a small amount of food. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer. Test diets were prepared at intervals, which guaranteed that the test substance in the diet remained stable throughout the feeding period.

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

- During the remaining premating period, the dietary concentrations of EAH were adjusted weekly for each group and sex based on body weight and food consumption measurements from the preceding week.
- During the mating period of the F0 parental animals, each group and sex received the concentrations of EAH used during the last week of the premating period. This concentration was maintained throughout the mating period with the following exception: During cohabitation, both sexes received the test substance preparation for females as soon as the male was placed in the cage of its female partner. Both sexes returned to their normal test diet when they were separated the following morning. This test diet cycle remained in effect until there was evidence of successful mating. At that time, the mated animals received the test substance preparations described below at the first opportunity in the specific week.
- During the gestation period, dietary concentrations of EAH for the F0 males were again adjusted weekly on the basis of body weight and food consumption data from the preceding week. The EAH concentrations in the diet of the F0 females were the same as those used during the last week of the premating period.
- During the lactation period, dietary concentrations of EAH for the F0 males continued to be adjusted weekly on the basis of body weight and food consumption data from the preceding week. The EAH concentrations in the diet of the F0 females were 50% of those used during the last week of the premating period. This dietary adjustment, derived from historical body weight and food consumption data, maintained the dams at the desired doses of EAH during this period of increased food intake.
- Post weaning, dietary EAH levels for parental male animals awaiting necropsy were adjusted weekly based on body weight and food consumption data from the preceding week. The EAH concentration of parental female diets was the same as those used during the last week of the preceding premating period.
- Until all litters were weaned (when the last selected F1 pup reached age of day 21 p.p.), the food for the weaned F1 pups selected as F1 parental animals was prepared with EAH concentrations on the basis of historical body weight and food consumption data for rats of similar age.
- During the first week of the premating period of F1 parental animals, dietary EAH concentrations were formulated on the basis of actual body weight on day 0 and historical food consumption data. Subsequently, dietary EAH levels for each F1 dose group and sex were adjusted as described for F0 parental animals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of EAH in the diet over 32 days at room temperature was investigated analytically before the beginning of the study. Homogeneity and concentration control analyses were carried out at the beginning and toward the end of the premating periods. At least one analysis of test substance preparations for female animals was carried out during the gestation and lactation periods.

The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
Duration of treatment / exposure:
semichronic duration (> 75 days)
Frequency of treatment:
continuously
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
in diet
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
in diet
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
in diet
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
MORTALITY:
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied. The examinations of these animals were carried out according to the methods established at the pathology laboratory.

CLINICAL OBSERVATIONS:
All parental animals were checked daily for clinically evident signs of toxicity. For technical reasons, however, the clinical observations recorded during the premating periods were printed out on a weekly basis (the daily observations can be found in the raw data). The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only special findings, e.g. disability to deliver, were documented on an individual dam basis. In addition to the evaluations in the mornings, parturition behavior of the dams was also inspected on weekdays (except public holidays) in the afternoons. The day of parturition was considered the 24-hour period from about 3.00 p.m. of one day until about 3.00 p.m. of the following day. Deviations from this procedure were possible on Saturday, Sunday and on public holidays.

FOOD CONSUMPTION:
In general, food consumption was determined once a week (each time for a period of at least 6 days) for the male and female F0 and F1 parental animals. For the females during pregnancy (animals with evidence of sperm), food consumption was determined weekly for days 0-7, 7-14 and 14-20 p.c. During the lactation period (animals with litter), food consumption was determined for days 1-4, 4-7 and 7-14 p.p. Food consumption was not determined between days 14 and 21 after parturition as required in the test guidelines, since during this time pups will begin to consume considerable amounts of solid food offered, and therefore, there was no point in such measurement. Furthermore, food consumption was not determined for females without positive evidence of sperm and for females without litter.

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

BODY WEIGHT DATA:
In general, the body weight of parental animals was determined on the first day of the premating period and then once a week at the same time of day (in the morning). Based on these results, the body weight change of the animals was calculated. The following exceptions are notable for the female parental animals:
a) During each gestation period the F0 and the F1 generation parental females were weighed on the day of positive evidence of sperm (day 0 p.c.) and on days 7, 14 and 20 p.c.
b) Females showing no positive evidence of sperm in vaginal smears were not weighed during the mating interval.
c) Females with litter were weighed on the day after parturition (day 1 p.p.) and on days 4, 7, 14 and 21 p.p.
d) Females without litter were not weighed during the lactation phase.

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.
Sacrifice and pathology:
All F0 and F1 parental animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention was given to the reproductive organs. As soon as possible after termination, one portion of the liver (lobus medialis) of each 10 dams per group was sampled to be analyzed for choline concentration.

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

ORGAN/TISSUE FIXATION:
The following organs or tissues of the F0 and F1 generation parental animals were fixed in 4% neutral buffered formaldehyde solution or in BOUIN’s solution, respectively:
1. Vagina, 2. Cervix uteri, 3. Uterus, 4. Ovaries (fixed in BOUIN´s solution), 5. Oviducts, 6. Left testis (fixed in BOUIN´s solution), 7. Left epididymis (fixed in BOUIN´s solution), 8. Seminal vesicles, 9. Coagulation glands, 10. Prostate, 11. Pituitary gland, 12. Adrenal glands, , 3. Liver, 14. Kidneys, 15. Spleen, 16. Brain, 17. Thyroids (with parathyroids), 18. All gross lesions. After fixation, the organs fixed in BOUIN´s solution were embedded in Paraplast. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings. All gross lesions were examined. Of the fixated organs the organs of all animals in the control group and the high dose group were evaluated. Additionally, the organs for mating pair suspected of reduced fertility were evaluated.
Statistics:
Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
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/d). Intensively yellow discolored urine was recorded in all F0 males and females of test group 03 (1000 mg/kg bw/d) from study week 3 onwards until the end of the treatment period. This urine discoloration mirrored the systemic availability of the test substance rather than being an adverse effect and was most likely caused by the excreted test compound and/or its metabolites.

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

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

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

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

Clinical observations for females during lactation of F2 litters:
All F1 females of test group 13 showed intensive yellow discolored urine during the entire lactation period for F2 litters. No other clinical findings were observed in the test groups 10-13 (0, 100, 300 and 1000 mg/kg bw/d).
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):
F0: 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/d) 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/d) were similar to the controls throughout the entire premating period. During gestation, these animals gained less weight from gestation day 7 onwards (up to 38%). As a consequence, body weights on gestation day 20 were 8% lower than the control. This effect may have been caused by the statistically significantly increased postimplantation loss and the statistically significantly decreased mean number of delivered pups in test group 03. This is also indicated by the unaffected body weight of the high dose dams on post-delivery day 1. Mean body weights of the high-dose females remained comparable to the controls during entire lactation, whereas the weight gain wavered up and down in the individual lactation sections.

F1: Mean body weights and body weight gain of the F1 parental males in test groups 11-13 (100, 300 and 1000 mg/kg bw/d) 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/d) were comparable to the control throughout premating, gestation and lactation periods. Mean body weights and body weight gains of the F1 females in test group 03 (1000 mg/kg bw/d) were similar to the controls throughout the entire premating period, the statistically significantly increased body weight gain of the high-dose F1 females (1000 mg/kg bw/d) during premating week 1-2 was regarded as incidental variance. The average weight gain of these animals was significantly below control (26%) during gestation days 14-20, which led to an averaged decrease of weight gain for the entire gestation of 17%. This effect may have been caused by the statistically significantly increased postimplantation loss and the statistically significantly decreased mean number of delivered pups in test group 03. Mean body weights of the high-dose females remained comparable to the controls during entire lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
F0: Food consumption of the F0 parental 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/d) was comparable to the control animals during the periods of premating, gestation and lactation. Food consumption of the high-dose F0 females (1000 mg/kg bw/d) was also comparable during premating and gestation periods. However, in these animals, food consumption was statistically significantly below controls (up to 18%) in the individual lactation sections: days 1-4, 4-7, and 7-14 p.p.

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

For all test groups the intake of Ethanolamine hydrochloride correlated well with the desired target doses. For the actual test substance intake see 'Any other information on results incl. tables'.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the top-dose F0 and F1 males the test substance administration led to a decrease of absolute and relative organ weights of cauda epididymidis and epididymides. Furthermore, prostate weight and the number of homogenization resistant caudal epididymal sperm was slightly, but significantly decreased in the F0 males. These findings were considered to be treatment-related effects, whereas histomorphological correlates were missing.
A statistically significant increase of absolute and relative kidney weights was noted in male and female F1 animals of the mid (300 mg/kg bw/day) and top-dose (1000 mg/kg bw/day) groups. Because no histomorphological correlate was detected, the treatment-related weight increase was considered to be of no toxicological concern. 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. Thus this finding was considered to have no toxicological relevance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
F0: All gross lesions observed in test animals occurred singularly. They are considered to be spontaneous lesions in origin and are not related to treatment. The female animal which was not pregnant as well as the male mating partner did not show relevant gross lesions.
F1: All gross lesions observed in test animals occurred singularly. They are considered to be spontaneous lesions in origin and are not related to treatment.
One non-pregnant female animal did show a bilaterally severe reduced size of the ovaries as well as a moderate thickening of the uterus wall. The other three non-pregnant females did not show any gross lesions. The four male mating partners did not show any gross lesions either.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
F0:
- 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 consideredto be incidental and/or spontaneous in origin and without any relation to treatment.
- Fertility: The non-pregnant female and the male mating partner did not show histopathological findings explaining the infertility. Only a minimal focal atrophy of the prostate was present, which is considered an incidental finding.

F1:
- As compared to control animals, the kidneys of low, mid, and top dose 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
Key result
Dose descriptor:
NOAEL
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
Critical effects observed:
no

Test substance stability:

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

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

Toxicokinetic data of 2-aminoethanol (calculated as 2-aminoethanol hydrochloride) from this two-generation reproduction toxicity 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.

 

Tables

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

 

Test group 01
(100 mg/kg bw/d)

Test group 02
(300 mg/kg bw/d)

Test group 03
(1000 mg/kg bw/d)

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/d

02

300 mg/kg bw/d

03

1000 mg/kg bw/d

01

100 mg/kg bw/d

02

300 mg/kg bw/d

03

1000 mg/kg bw/d

Brain

99%

100%

97%*

 

 

 

Cauda epididymis

99%

102%

88%**

 

 

 

Epididymides

100%

101%

92%**

 

 

 

Prostate

92%

99%

86%**

 

 

 

Spleen

 

 

 

105%*

107%

97%

 

*: p≤0.05; **: p≤0.01

 

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

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

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

Absolute organ weights (F1 generation)

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

 

Male animals

Female animals

Group

11

100 mg/kg bw/d

12

300 mg/kg bw/d

13

1000 mg/kg bw day

11

100 mg/kg bw/d

12

300 mg/kg bw/d

13

1000 mg/kg bw/d

Cauda epididymis

96%

99%

88%**

 

 

 

Epididymides

100%

101%

91%**

 

 

 

Kidneys

99%

106%*

111%**

103%

106%**

115%**

Spleen

99%

103%

92%*

 

 

 

Thyroid glands

106%

99%

109%*

110%

118%**

111%*

 

*: p≤0.05; **: p≤0.01

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

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

 

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

 

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

Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
03 Aug 2006 - 15 Jan 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
Jan 2001
Deviations:
yes
Remarks:
Food consumption was not determined between days 14 and 21 after parturition
Qualifier:
according to
Guideline:
other: Corrigendum to EC Commission Directive 2004/73/EC, Part B: Methods for the determination of toxicity: Two-Generation Reproduction Toxicity Study; Official Journal of the European Communities; No. L216, pp. 236–246
Version / remarks:
29 Apr 2004
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Version / remarks:
Aug 1998
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- No.of test material: 1) 05/0372-2; 2) 05/03723; 3) 05/0372-4
- Lot/batch No.: ad 1) JB116/2+3 (from 09 Aug – 04 Oct 2006); ad 2) JB116/4 (from 04 Oct – 29 Nov 2006); ad 3) JB116/9-17 (from 29 Nov 2006 until the scheduled termination of the in life part of the study)
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, under N2
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI (Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 44 (+/- 1) days
- Weight at study initiation: (P) Males: 162.1 (142.5 – 186.5) g; Females: 126.2 (110.6 – 145.1) g;
- Fasting period before study: none
- Housing: rats were housed individually in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
• overnight mating: male and female mating partners were housed together in type DK III cages
• gestation day 18 – lactation day 21: pregnant animals and their litters were housed in Makrolon type M III cages (floor area of about 800 cm²). The M III cages were also supplied by Becker & Co. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
- For enrichment wooden gnawing blocks (Typ NGM E-022, supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria) were added. The cages with the test animals were arranged in racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum
- Acclimation period: 16 days
- Other: According to a written statement from the breeder, male and female animals were derived from different litters. This was necessary to rule out the possibility of sibling mating.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
The test substance was weighed and thoroughly mixed with a small amount of food. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer. Test diets were prepared at intervals, which guaranteed that the test substance in the diet remained stable throughout the feeding period.

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

- During the remaining premating period, the dietary concentrations of EAH were adjusted weekly for each group and sex based on body weight and food consumption measurements from the preceding week.
- During the mating period of the F0 parental animals, each group and sex received the concentrations of EAH used during the last week of the premating period. This concentration was maintained throughout the mating period with the following exception: During cohabitation, both sexes received the test substance preparation for females as soon as the male was placed in the cage of its female partner. Both sexes returned to their normal test diet when they were separated the following morning. This test diet cycle remained in effect until there was evidence of successful mating. At that time, the mated animals received the test substance preparations described below at the first opportunity in the specific week.
- During the gestation period, dietary concentrations of EAH for the F0 males were again adjusted weekly on the basis of body weight and food consumption data from the preceding week. The EAH concentrations in the diet of the F0 females were the same as those used during the last week of the premating period.
- During the lactation period, dietary concentrations of EAH for the F0 males continued to be adjusted weekly on the basis of body weight and food consumption data from the preceding week. The EAH concentrations in the diet of the F0 females were 50% of those used during the last week of the premating period. This dietary adjustment, derived from historical body weight and food consumption data, maintained the dams at the desired doses of EAH during this period of increased food intake.
- Post weaning, dietary EAH levels for parental male animals awaiting necropsy were adjusted weekly based on body weight and food consumption data from the preceding week. The EAH concentration of parental female diets was the same as those used during the last week of the preceding premating period.
- Until all litters were weaned (when the last selected F1 pup reached age of day 21 p.p.), the food for the weaned F1 pups selected as F1 parental animals was prepared with EAH concentrations on the basis of historical body weight and food consumption data for rats of similar age.
- During the first week of the premating period of F1 parental animals, dietary EAH concentrations were formulated on the basis of actual body weight on day 0 and historical food consumption data. Subsequently, dietary EAH levels for each F1 dose group and sex were adjusted as described for F0 parental animals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of EAH in the diet over 32 days at room temperature was investigated analytically before the beginning of the study. Homogeneity and concentration control analyses were carried out at the beginning and toward the end of the premating periods. At least one analysis of test substance preparations for female animals was carried out during the gestation and lactation periods.

The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: over night
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged (how): individual

In general, male and female animals were mated overnight at a 1 : 1 ratio for a maximum of 2 weeks. Each female animal was paired with a predetermined male animal from the same dose group throughout the entire mating period. Mating was accomplished by placing the male in the cage of the female mating partner from about 4.00 p.m. until 7.00 - 9.00 a.m. of the following morning. Deviations from these specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and was examined for the presence of sperm. If sperm were detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "day 0" and the following day "day 1" post coitum (p.c.).
Duration of treatment / exposure:
semichronic duration (> 75 days)
Frequency of treatment:
continuously
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
in diet
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
in diet
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
in diet
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Details on study design:
F0 PARENTAL ANIMALS
After the acclimatization period, the F0 generation parental animals continuously received the test substance at the appropriate concentrations in the diet up to about 16 hours before they were sacrificed. At least 75 days after the beginning of treatment, males and females from the same dose group were mated. The females were allowed to litter and rear their pups (F1 generation pups) until day 4 (standardization) or 21 after parturition. After weaning of F1 pups the F0 generation parental animals were sacrificed.

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

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

PUPS AFTER STANDARDIZATION AND AFTER WEANING
After standardization or weaning, all pups were sacrificed by means of CO2 with the exception of those F1 generation pups, which were chosen as F1 generation parental animals. All sacrificed pups, including stillborn pups and those that died during their rearing period, were subject of a macroscopic (external and visceral) examination. All pups without any notable findings or abnormalities were discarded after their macroscopic
evaluation.
Maternal examinations:
MORTALITY:
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied. The examinations of these animals were carried out according to the methods established at the pathology laboratory.

CLINICAL OBSERVATIONS:
All parental animals were checked daily for clinically evident signs of toxicity. For technical reasons, however, the clinical observations recorded during the premating periods were printed out on a weekly basis (the daily observations can be found in the raw data). The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only special findings, e.g. disability to deliver, were documented on an individual dam basis. In addition to the evaluations in the mornings, parturition behavior of the dams was also inspected on weekdays (except public holidays) in the afternoons. The day of parturition was considered the 24-hour period from about 3.00 p.m. of one day until about 3.00 p.m. of the following day. Deviations from this procedure were possible on Saturday, Sunday and on public holidays.

FOOD CONSUMPTION:
In general, food consumption was determined once a week (each time for a period of at least 6 days) for the male and female F0 and F1 parental animals. For the females during pregnancy (animals with evidence of sperm), food consumption was determined weekly for days 0-7, 7-14 and 14-20 p.c. During the lactation period (animals with litter), food consumption was determined for days 1-4, 4-7 and 7-14 p.p. Food consumption was not determined between days 14 and 21 after parturition as required in the test guidelines, since during this time pups will begin to consume considerable amounts of solid food offered, and therefore, there was no point in such measurement. Furthermore, food consumption was not determined for females without positive evidence of sperm and for females without litter.

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

BODY WEIGHT DATA:
In general, the body weight of parental animals was determined on the first day of the premating period and then once a week at the same time of day (in the morning). Based on these results, the body weight change of the animals was calculated. The following exceptions are notable for the female parental animals:
a) During each gestation period the F0 and the F1 generation parental females were weighed on the day of positive evidence of sperm (day 0 p.c.) and on days 7, 14 and 20 p.c.
b) Females showing no positive evidence of sperm in vaginal smears were not weighed during the mating interval.
c) Females with litter were weighed on the day after parturition (day 1 p.p.) and on days 4, 7, 14 and 21 p.p.
d) Females without litter were not weighed during the lactation phase.

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

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

POST-MORTEM EXAMINATIONTS:
All F0 and F1 parental animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention was given to the reproductive organs.
As soon as possible after termination, one portion of the liver (lobus medialis) of each 10 dams per group was sampled to be analyzed for choline concentration.

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

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

BLOOD SAMPLINGS:
Blood samples were taken from all F0 and F1 parental animals of each sex and test group during week 10 of premating treatment and the plasma was analyzed for the concentration of the test substance.
Ovaries and uterine content:
From both ovaries (”ovary 1” and “ovary 2”) of F1 female animals (control and top dose), five sections were taken from the proximal and the distal part of the ovaries, respectively, at least 100 μm apart from the inner third of the ovary. All ovarian sections were prepared and evaluated. Primordial follicles and growing follicles were counted by light microscope (magnification: 100x) on each of these slides, – according to the definitions given by Plowchalk et al. (PLOWCHALK, D. R., B. J. SMITH, and D. R. MATTISON: Assessment of Toxicity to the Ovary Using Follicle Quantitation and Morphometrics. In: Methods in Toxicology, Vol. 3, Part B: Female Reproductive Toxicology (J. J. HEINDEL and R. E. CHAPIN, Editors), p. 57-68, 1993, Academic Press). To prevent multiple counting on serial slides – especially of the growing follicles – only follicles with an oocyte with visible chromatin on the slide were counted. The number of each type of follicle was recorded individually for ovary 1 and ovary 2 of every animal on any of the slide levels (level 1-10), giving in summary the incidence of each type of the follicles by using EXCEL sheets for the reporting of the results. Finally, the results of all types of follicles were summarized for all animals per group in dose groups 10 and 13. As primordial follicles continuously develop into growing follicles, the assessment of the follicles was extended to the combined incidence of primordial plus growing follicles. In general, the fifth slide of the left and right ovary was evaluated for histological findings. A correlation between gross lesions and histopathological findings was performed.
Fetal examinations:
PUP NUMBER AND STATUS AT DELIVERY:
On the day of birth, all pups derived from the F0 parents (F1 litter) and the F1 parents (F2 litter) were examined as soon as possible to determine the total number of pups, and the number of liveborn as well as stillborn pups of each litter. Pups, which died before examination, were designated as stillborn pups.

PUP VIABILITY/MORTALITY:
- In general, a check was made for dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) and once in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated as dexribed in the section 'Postmortem examinations (offspring)'
- The number and percentage of dead pups on the day of birth (day 0) and of pups dying between days 1-4, 5-7, 8-14 and 15-21 of the lactation period were determined; however, pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day of birth, and on lactation days 4, 7, 14, and 21.

SEX RATIO:
- On the day of birth (day 0 p.p.), the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Subsequently, the sex of the pups was assessed by the external appearance of the anogenital region and/or the mammary line and was finally confirmed at necropsy.
- The sex ratio was calculated at day 0 and day 21 p.p. according to the following formula:
Sex ratio = (number of live male or female pups on day 0/day 21 p.p. / number of live male and female pups on day 0/day 21 p.p.) X 100

PUP CLINICAL OBSERVATIONS:
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed any special findings, these were documented with the dam concerned.

PUP BODY WEIGHT DATA:
The pups were weighed on the day after birth (day 1 p.p.) and on days 4 (before standardization), 7, 14 and 21 after birth. Pups' body weight change was calculated based on these results. The individual weights were always determined at about the same time of the day (in the morning) and on day 4 p.p. immediately before standardization of the litters.

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

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

PUP NECROPSY OBSERVATIONS:
All pups with scheduled sacrifice (i.e. pups, which were culled on day 4 p.p., and pups, which were sacrificed on day 21 p.p. or subsequent days) were killed by means of CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically. All stillborn pups and all pups that died up to weaning were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.
Statistics:
see in 'Any other information on materials and methods incl. tables'.
Indices:
For females, mating, fertility and gestation indices were calculated for F1 and F2 litters according to the following formulas:
- Female mating index (%) = (number of females mated / total number of pups born) x 100
- Female fertility index (%) = (number of females pregnant / number of females mated) x 100
- Gestation inex (%) = (number of females with live pups on the day of birth / number of females pregnant) x 100

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

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

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

Viability and lactation indices were calculated according to the following formulas:
- Viability index (%) = (number of live pups on day 4 (before standardization of litters) after birth / number of live pups on the day of birth) x 100
- Lactation index (%) = (number of live pups on day 21 after birth / number of live pups on day 4(after standardization of litters) after birth) x 100
Historical control data:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
F0: No clinical signs or changes of the general behavior, which may be attributed to the test substance, were detected in F0 female parental animals of the test groups 01 and 02 (100 and 300 mg/kg bw/d). Intensively yellow discolored urine was recorded in all F0 females of test group 03 (1000 mg/kg bw/d) from study week 3 onwards until the end of the treatment period. This urine discoloration mirrored the systemic availability of the test substance rather than being an adverse effect and was most likely caused by the excreted test compound and/or its metabolites.

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

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

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

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

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

Clinical observations for females during lactation of F2 litters:
All F1 females of test group 13 showed intensive yellow discolored urine during the entire lactation period for F2 litters. No other clinical findings were observed in the test groups 10-13 (0, 100, 300 and 1000 mg/kg bw/d).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled mortalities of maternal animals in any test group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F0: During premating, gestation and lactation periods, the mean body weights and body weight gains of the F0 parental females in the low- and mid-dose groups were generally comparable to the concurrent control group. During premating, gestation and lactation periods, the mean body weights and body weight gains of the F0 parental females in the low- and mid-dose groups were generally comparable to the concurrent control group. Mean body weights and body weight gains of the F0 females in test group 03 (1000 mg/kg bw/d) were similar to the controls throughout the entire premating period. During gestation, these animals gained less weight from gestation day 7 onwards (up to 38%). As a consequence, body weights on gestation day 20 were 8% lower than the control. This effect may have been caused by the statistically significantly increased postimplantation loss and the statistically significantly decreased mean number of delivered pups in test group 03. This is also indicated by the unaffected body weight of the high dose dams on post-delivery day 1. Mean body weights of the high-dose females remained comparable to the controls during entire lactation, whereas the weight gain wavered up and down in the individual lactation sections.

F1: Mean body weights and body weight gain of the F1 parental females in test groups 11-12 (100 and 300 1000 mg/kg bw/d) were comparable to the control throughout premating, gestation and lactation periods. Mean body weights and body weight gains of the F1 females in test group 03 (1000 mg/kg bw/d) were similar to the controls throughout the entire premating period, the statistically significantly increased body weight gain of the high-dose F1 females (1000 mg/kg bw/d) during premating week 1-2 was regarded as incidental variance. The average weight gain of these animals was significantly below control (26%) during gestation days 14-20, which led to an averaged decrease of weight gain for the entire gestation of 17%. This effect may have been caused by the statistically significantly increased postimplantation loss and the statistically significantly decreased mean number of delivered pups in test group 03. Mean body weights of the high-dose females remained comparable to the controls during entire lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
F0: Food consumption of the F0 parental females of the low- and mid-dose groups (100 and 300 mg/kg bw/d) was comparable to the control animals during the periods of premating, gestation and lactation. Food consumption of the high-dose F0 females (1000 mg/kg bw/d) was also comparable during premating and gestation periods. However, in these animals, food consumption was statistically significantly below controls (up to 18%) in the individual lactation sections: days 1-4, 4-7, and 7-14 p.p.

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

For all test groups the intake of Ethanolamine hydrochloride correlated well with the desired target doses. For the actual test substance intake see 'Any other information on results incl. tables'.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
F0: The observed increase of absolute kidney weights of female animals in mid (300 mg/kg bw/day) and top dose (1000 mg/kg bw/day) groups, respectively, was statistically significant. Because no histomorphological correlate was detected, a treatment-related weight increase is less likely. The observed increase of thyroid glands in mid and top dose females, is considered incidental and not treatment-related due to a missing dose response relationship and no histopathological correlates.

F1: The observed increase of absolute kidney weights of female animals in mid (300 mg/kg bw/day) and top dose (1000 mg/kg bw/day) groups, respectively, was statistically significant. Because no histomorphological correlate was detected, a treatment-related weight increase is less likely. The observed increase of thyroid glands IN mid and top dose females, is considered incidental and not treatment-related due to a missing dose response relationship and no histopathological correlates.
Gross pathological findings:
no effects observed
Description (incidence and severity):
F0: All gross lesions observed in test animals occurred singularly. They are considered to be spontaneous lesions in origin and are not related to treatment. The female animal which was not pregnant as well as the male mating partner did not show relevant gross lesions.
F1: All gross lesions observed in test animals occurred singularly. They are considered to be spontaneous lesions in origin and are not related to treatment.
One non-pregnant female animal did show a bilaterally severe reduced size of the ovaries as well as a moderate thickening of the uterus wall. The other three non-pregnant females did not show any gross lesions. The four male mating partners did not show any gross lesions either.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
F0:
- All findings noted were single observations either, or were similarly in distribution pattern and severity in control rats compared to treatment groups. All of them are consideredto be incidental and/or spontaneous in origin and without any relation to treatment.
- Fertility: The non-pregnant female and the male mating partner did not show histopathological findings explaining the infertility. Only a minimal focal atrophy of the prostate was present, which is considered an incidental finding.

F1:
- As compared to control animals, the kidneys of low, mid, and top dose female animals revealed a low incidence of basophilic tubules in a slightly higher number of animals. The severity (minimal to slight) was comparable between controls and treated animals and a clear dose-response relationship was missing. The cauda epididymis and epididymides of top dose males showed no histomorphological correlates to the decreased organ weights. All other findings noted were single observations either, or were similarly in distribution pattern and severity in control rats compared to treatment groups. All of them are considered to be incidental and/or spontaneous in origin and without any relation to treatment.
- Fertility: One non-pregnant female showed a bilateral moderate diffuse stromal hyperplasia and a unilateral severe focally extensive chronic inflammation of the ovaries as well as an ovarian cyst. There were still corpora lutea present and the histopathological findings did not correlate with the gross lesion. The gross lesion “thickening of uterine wall” had no corresponding histological finding. The findings on the ovaries might explain the infertility. The male mating partner did not reveal lesions affecting the fertility. One female Animal showed within the uterus a severe multifocal endometrial and glandular degeneration and within the oviducts a severe diffuse epithelial degeneration, which explains the infertility of this animal. The male mating partner did not reveal lesions affecting the fertility. The other two mating pairs did not show any lesions affecting the fertility.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
The results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant deviations between controls and animals of the top dose group.
Number of abortions:
no effects observed
Description (incidence and severity):
F1 pups: The mean number of F1 pups/dam was statistically significantly reduced in test group 03 (6.8** pups/dam [p≤0.01]). However, the number of stillborn F1 pups was comparable between the test groups 00, 01, 02, and 03 (2, 1, 2, and 2 pups/test group, respectively). Every stillborn pup belonged to a different litter.

F2 pups: The mean number of F2 pups/dam was statistically significantly reduced in test group 13 (1000 mg/kg bw/d). However, a comparison of the total amount of liveborn and stillborn F2 pups per test group did not reveal statistically significant differences between test groups 10-13.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
F0: No statistically significant differences were seen between the control (11.8 implants/dam) and test groups 01 and 02 (12.4 and 11.8 implants/dam, respectively). In contrast, the number of implantation sites was statistically significantly reduced in test group 03 (8.6** [p≤0.01]). Furthermore, there were indications for test substance-induced intrauterine embryo-/fetolethality, since the postimplantation loss was statistically significantly increased in the high-dose group (23.0%** [p≤0.01] at 1000 mg/kg bw/d). There were no statistically significantly differences concerning the postimplantation loss between the remaining test groups and the control (5.7%, 6.8% and 9.9% at 0, 100 and 300 mg/kg bw/d, respectively).

F1: There were no statistically significant differences in the number of implantation sites between test groups 11 and 12 (11.6 and 11.8 implants/dam, respectively) and the control. Furthermore, there were indications for test substance-induced intrauterine embryo-/fetolethality, since the postimplantation loss was statistically significantly increased in the high-dose group (12.8* [p≤0.05] – 1000 mg/kg bw/d). In addition, the postimplantation loss in this test group was higher than documented in the historical control data. There were no statistically significantly differences concerning the postimplantation loss between the remaining test groups and the control (4.9%, 3.0%, and 4.0% for 0, 100, and 300 mg/kg bw/d, respectively).
Total litter losses by resorption:
not specified
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
In the high-dose F0 and F1 generation females (1000 mg/kg bw/d), decreased numbers of implants and increased resorption rates resulted in significantly smaller litters, giving evidence for an adverse effect of the test compound on fertility and/or reproductive performance at this dose. It has to be noted that a dose of 1000 mg/kg bw/d also caused systemic toxicity in these females, as was indicated by reduced food consumption and/or body weight gain during gestation/lactation.
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of liveborn and stillborn pups was comparable between all groups, and the live birth index varied between 99% and 100%.
Changes in pregnancy duration:
effects observed, non-treatment-related
Description (incidence and severity):
F0: The mean duration of gestation was comparable in all test groups. The respective values were 21.8, 21.8, 21.7 and 22.2** (p≤0.01) days. Although the duration of gestation appeared to be statistically significantly increased in the high-dose group, this value was only slightly above the concurrent control and was still within the historical control range of the test facility (21.5–22.3 days). Therefore, this finding was not considered to be treatment-related.
F1: The mean duration of gestation was comparable in all test groups (10-13) and varied between 21.8 and 22.1.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
F0: The female mating index calculated after the mating period for F1 litter was 100% in all test groups. Nearly all sperm positive females delivered pups or had implants in utero. One high-dose F0 female (No. 185) did not become pregnant. Consequently, the fertility index varied between 96% (test group 03) and 100% (test groups 00-02).
F1: The female mating index for F2 litter was 100% in all test groups. All sperm-positive rats delivered pups with the following exceptions: one control female, one female of test group 11 (100 mg/kg bw/d) and two females of test group 13 (1000 mg/kg bw/d) did not become pregnant. Consequently, the fertility index was 92% for test group 13 (1000 mg/kg bw/d), 96% for test groups 10 and 11 (0 and 100 mg/kg bw/d) and 100% for test group 12 (300 mg/kg bw/d). These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
Other effects:
effects observed, treatment-related
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Remarks:
maternal developmental toxicity
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
pre and post implantation loss
other: less implantation sites, smaller litters
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
F1: Mean body weights of F1 male and female pups in test group 03 (1000 mg/kg bw/d) were statistically significantly increased during the complete lactation period, being marginally outside the range of historical control data. Body weight gain of these animals was statistically significantly increased between lactation days 1-4 (about 19%). Afterwards the weight gain of the high-dose animals was similar to the concurrent control group. The higher pup body weights in test group 03 were rather regarded as a consequence of the reduced number of pups/litter than a direct test substance-related effect. No test compound-related influence on F1 pup body weights was noted in the low- and middose groups (100 and 300 mg/kg bw/d).

F2: Mean body weights of F2 male and female pups in test groups 11-13 (100, 300 and 1000 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire lactation period. The statistically significantly increased body weights of female pups and the statistically significantly increased body weight value for both sexes in test group 13 on lactation day 1 were related to the decreased litter sizes and, given that, regarded to be secondary effects.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
F1: The mean number of F1 pups/dam was statistically significantly reduced in test group 03 (6.8** pups/dam [p≤0.01]). However, the number of stillborn F1 pups was comparable between the test groups 00, 01, 02, and 03 (2, 1, 2, and 2 pups/test group, respectively). Every stillborn pup belonged to a different litter.

F2: The mean number of F2 pups/dam was statistically significantly reduced in test group 13 (1000 mg/kg bw/d). However, a comparison of the total amount of liveborn and stillborn F2 pups per test group did not reveal statistically significant differences between test groups 10-13.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
F1: The sex distribution and sex ratios of live F1 pups on the day of birth and on day 21 p.p. did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

F2: The sex distribution and sex ratios of live F2 pups on the day of birth and on day 21 p.p. did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
F1: The average litter size (F1 pups per dam) was very similar between test groups 00, 01 and 02 (11.1, 11.4, and 11.4 pups/dam, respectively). A statistically significantly lower number of F1 pups per dam were delivered in test group 03 (6.8** pups/dam [p≤0.01]). Since the number of stillborn pups was comparably low in all groups, the live birth index was 99% for test groups 00, 02 and 03, and 100% for test group 01.

F2: The average litter size (F2 pups per dam) was very similar between test groups 10, 11 and 12. A statistically significantly lowered number of F2 pups per dam were delivered in test group 13 (7.7** pups/dam [p≤0.01] versus 10.8, 11.2, and 11.4 in test groups 10, 11 and 12, respectively). The number of liveborn and stillborn pups was comparable between all groups, and the live birth index varied between 99% and 100%.
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
F1:
- The viability index as indicator for pup mortality between days 0-4 p.p. was unaffected and varied between 99% (control group as well as test groups 01 and 03) and 100% (test group 02). However, cannibalized pups were found in test group 01 (3 pups), 02 (1 pup) and 03 (3 pups* [p≤0.05]). In this case, the calculated statistically significant difference for the highdose group was a consequence of the smaller litter sizes there. No pup was cannibalized in the control group.
- The lactation index, indicating pup mortality between days 4-21 p.p., was slightly, but statistically significantly lower in test group 01 (97%* [p≤0.05]). This value is, however, within the historical control range of the test facility. Thus, pup mortality in the low dose group during the lactation period was not considered to be associated to test substance-treatment. For the other test groups, the lactation index was 98% (test group 03) and 100% (test groups 00 and 02).

F2:
- The viability index as indicator for pup mortality between days 0-4 p.p. was 100% in all test groups.
- The lactation index as indicator for pup mortality between days 4-21 p.p. was not affected by test substance-treatment as it reached 100%, 100%, 99%, and 100% at dose levels of 0, 100, 300, and 1000 mg/kg bw/d. Any isolated pup deaths were assessed as incidental.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
F1: One low-dose pup showed a kinked tail on lactation day 21, which was confirmed by skeletal examination (misshapen caudal vertebra, cartilage changed). These indivisual finding were assumed to be incidental and can also be found in the historical control data

F2: no effects reported
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
F1: One low-dose pup showed a kinked tail on lactation day 21, which was confirmed by skeletal examination (misshapen caudal vertebra, cartilage changed). These indivisual finding were assumed to be incidental and can also be found in the historical control data

F2: no effects reported
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
F1: For one high-dose pup microphthalmia of the left eye was recorded on lactation day 21 and was confirmed by visceral examination. These indivisual finding were assumed to be incidental and can also be found in the historical control data

F2: no effects reported
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at highest dose tested
Key result
Abnormalities:
no effects observed
Developmental effects observed:
no

Test substance stability:

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

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

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

 

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

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

 

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

 

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

 

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

 

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

Tables

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

 

Test group 01
(100 mg/kg bw/day)

Test group 02
(300 mg/kg bw/day)

Test group 03
(1000 mg/kg bw/day)

F0 males

94.3 (72.4 / 102.5)

283.2 (218.4 / 309.4)

943.3 (716.7 / 1032.6)

F0 females (premating)

96.7 (80.5 / 100.7)

289.6 (241.2 / 304.9)

964.4 (792.4 / 1017.8)

F0 females
(F1 litter)
- gestation period
- lactation period*



103.5 (92.6 / 111.6)
99.2 (81.6 / 120.2)



315.2 (284.8 / 337.9)
306.7 (249.7 / 370.3)



1043.2 (989.4 / 1084.7)
866.0 (668.6 / 1053.9)

* = Days 1–14 p.p. only

Absolute organ weights (P-generation)

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

 

Male animals

Female animals

Group

01

100 mg/kg bw/day

02

300 mg/kg bw/day

03

1000 mg/kg bw/day

01

100 mg/kg bw/day

02

300 mg/kg bw/day

03

1000 mg/kg bw/day

Brain

99%

100%

97%*

 

 

 

Cauda epididymis

99%

102%

88%**

 

 

 

Epididymides

100%

101%

92%**

 

 

 

Prostate

92%

99%

86%**

 

 

 

Spleen

 

 

 

105%*

107%

97%

 

*: p≤0.05; **: p≤0.01

 

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

 

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

 

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

Absolute organ weights (F1 generation)

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

 

 

Male animals

Female animals

Group

11

100 mg/kg bw/day

12

300 mg/kg bw/day

13

1000 mg/kg bw day

11

100 mg/kg bw/day

12

300 mg/kg bw/day

13

1000 mg/kg bw/day

Cauda epididymis

96%

99%

88%**

 

 

 

Epididymides

100%

101%

91%**

 

 

 

Kidneys

99%

106%*

111%**

103%

106%**

115%**

Spleen

99%

103%

92%*

 

 

 

Thyroid glands

106%

99%

109%*

110%

118%**

111%*

 

*: p≤0.05; **: p≤0.01

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

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

 

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

 

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
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:
other: Corrigendum to EC Commission Directive 2004/73/EC, Part B: Methods for the determination of toxicity: Two-Generation Reproduction Toxicity Study; Official Journal of the European Communities; No. L216, pp. 236–246
Version / remarks:
29 Apr 2004
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Version / remarks:
Aug 1998
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
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

Test animals

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 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

Administration / exposure

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:
sub-chronic 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.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
in diet
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
in diet
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
in diet
No. of animals per sex per dose:
25
Control animals:
yes, plain diet

Examinations

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

CLINICAL OBSERVATIONS:
All parental animals were checked daily for clinically evident signs of toxicity. For technical reasons, however, the clinical observations recorded during the premating periods were printed out on a weekly basis (the daily observations can be found in the raw data). The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only special findings, e.g. disability to deliver, were documented on an individual dam basis. In addition to the evaluations in the mornings, parturition behavior of the dams was also inspected on weekdays (except public holidays) in the afternoons. The day of parturition was considered the 24-hour period from about 3.00 p.m. of one day until about 3.00 p.m. of the following day. Deviations from this procedure were possible on Saturday, Sunday and on public holidays.

FOOD CONSUMPTION:
In general, food consumption was determined once a week (each time for a period of at least 6 days) for the male and female F0 and F1 parental animals. For the females during pregnancy (animals with evidence of sperm), food consumption was determined weekly for days 0-7, 7-14 and 14-20 p.c. During the lactation period (animals with litter), food consumption was determined for days 1-4, 4-7 and 7-14 p.p. Food consumption was not determined between days 14 and 21 after parturition as required in the test guidelines, since during this time pups will begin to consume considerable amounts of solid food offered, and therefore, there was no point in such measurement. Furthermore, food consumption was not determined for females without positive evidence of sperm and for females without litter.

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

BODY WEIGHT DATA:
In general, the body weight of parental animals was determined on the first day of the premating period and then once a week at the same time of day (in the morning). Based on these results, the body weight change of the animals was calculated. The following exceptions are notable for the female parental animals:
a) During each gestation period the F0 and the F1 generation parental females were weighed on the day of positive evidence of sperm (day 0 p.c.) and on days 7, 14 and 20 p.c.
b) Females showing no positive evidence of sperm in vaginal smears were not weighed during the mating interval.
c) Females with litter were weighed on the day after parturition (day 1 p.p.) and on days 4, 7, 14 and 21 p.p.
d) Females without litter were not weighed during the lactation phase.

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 length was evaluated by daily analysis of vaginal smear for all F0 and F1 female parental rats for a minimum of 3 weeks prior to mating. Determination was continued throughout the mating period until the female exhibited evidence of mating. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 and F1 female with 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 female pups on day 0/day 21 p.p.) X 100

PUP CLINICAL OBSERVATIONS:
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed any special findings, these were documented with the dam concerned.

PUP BODY WEIGHT DATA:
The pups were weighed on the day after birth (day 1 p.p.) and on days 4 (before standardization), 7, 14 and 21 after birth. Pups' body weight change was calculated based on these results. The individual weights were always determined at about the same time of the day (in the morning) and on day 4 p.p. immediately before standardization of the litters.

SEXUAL MATURATION:
- Vaginal opening: All female F1 pups selected to become the F1 parental generation females (25/group) were examined daily for vaginal opening beginning on day 27 p.p. On the day of vaginal opening, the body weights of the respective animals were additionally determined.
- Preputial separation: All male F1 pups selected to become the F1 parental generation males (25/group) were examined daily for preputial separation beginning on day 40 p.p. On the day of preputial separation, the body weights of the respective animals were additionally determined.
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. Coagulation glands, 10. Prostate, 11. Pituitary gland, 12. Adrenal glands, , 3. Liver, 14. Kidneys, 15. Spleen, 16. Brain, 17. Thyroids (with parathyroids), 18. All gross lesions. After fixation, the organs fixed in BOUIN´s solution were embedded in Paraplast. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings. All gross lesions were examined. Of the fixated organs the organs of all animals in the control group and the high dose group were evaluated. Additionally, the organs for mating pair suspected of reduced fertility were evaluated.

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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/d). Intensively yellow discolored urine was recorded in all F0 males and females of test group 03 (1000 mg/kg bw/d) from study week 3 onwards until the end of the treatment period. This urine discoloration mirrored the systemic availability of the test substance rather than being an adverse effect and was most likely caused by the excreted test compound and/or its metabolites.

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

Clinical observations for females during lactation of F1 litters:
All F0 females of test group 03 showed intensively yellow discolored urine during the entire lactation period for F1 litter. One high-dose female (1000 mg/kg bw/d) had just one pup (female), which was cannibalized by its mother on lactation day 8. No other clinical findings were observed in the test groups 00-03 (0, 100, 300 and 1000 mg/kg bw/d).
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/d) 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/d) were similar to the controls throughout the entire premating period. During gestation, these animals gained less weight from gestation day 7 onwards (up to 38%). As a consequence, body weights on gestation day 20 were 8% lower than the control. This effect may have been caused by the statistically significantly increased postimplantation loss and the statistically significantly decreased mean number of delivered pups in test group 03. This is also indicated by the unaffected body weight of the high dose dams on post-delivery day 1. Mean body weights of the high-dose females remained comparable to the controls during entire lactation, whereas the weight gain wavered up and down in the individual lactation sections.
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/d) was comparable to the control animals during the periods of premating, gestation and lactation. Food consumption of the high-dose F0 females (1000 mg/kg bw/d) was also comparable during premating and gestation periods. However, in these animals, food consumption was statistically significantly below controls (up to 18%) in the individual lactation sections: days 1-4, 4-7, and 7-14 p.p.

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
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 / performance (P0)

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 significantly 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/d) 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.

Details on results (P0)

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.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive function (sperm measures)
Remarks on result:
not determinable
Remarks:
no clear evidence of treatment-related effects
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Remarks on result:
other: occurring together with systemic toxicity

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

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/d).
Intensively yellow discolored urine was recorded in all F1 males and F1 females of test group 13 (1000 mg/kg bw/d) from study week 0 onwards until the end of the treatment period. This urine discoloration mirrored the systemic availability of the test substance rather than being an adverse effect and was most likely caused by excreted test compound and/or its metabolites. Furthermore, one F1 male animal of test group 11 (100 mg/kg bw/d) 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/d).

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

Clinical observations for females during lactation of F2 litters:
All F1 females of test group 13 showed intensive yellow discolored urine during the entire lactation period for F2 litters. No other clinical findings were observed in the test groups 10-13 (0, 100, 300 and 1000 mg/kg bw/d).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
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/d) 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/d) were comparable to the control throughout premating, gestation and lactation periods. Mean body weights and body weight gains of the F1 females in test group 03 (1000 mg/kg bw/d) were similar to the controls throughout the entire premating period, the statistically significantly increased body weight gain of the high-dose F1 females (1000 mg/kg bw/d) during premating week 1-2 was regarded as incidental variance.
- The average weight gain of these animals was significantly below control (26%) during gestation days 14-20, which led to an averaged decrease of weight gain for the entire gestation of 17%. This effect may have been caused by the statistically significantly increased postimplantation loss and the statistically significantly decreased mean number of delivered pups in test group 03. Mean body weights of the high-dose females remained comparable to the controls during entire lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of F1 male and female animals in test groups 11-12 (100, and 300 mg/kg bw/d) was generally comparable to the control group throughout the entire treatment period, covering premating, gestation and lactation periods. Food consumption of the high-dose F0 females (1000 mg/kg bw/d) was also comparable during premating and gestation periods. However, in these animals, food consumption was statistically significantly below controls (-11%) on lactation days 1-4, and remained below control on lactation days 4-7 and 7-14 (-7%), although not statistically significant.

For all test groups the intake of Ethanolamine hydrochloride correlated well with the desired target doses. For the actual test substance intake see 'Any other information on results incl. tables'.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/d: 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 / performance (P1)

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/d). 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/d), 96% in test groups 10 and 11 (0 and 100 mg/kg bw/d) and 100% in test group 12 (300 mg/kg bw/d). 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/d0 and two females of test group 13 (1000 mg/kg bw/d) did not become pregnant. Consequently, the fertility index was 92% for test group 13 (1000 mg/kg bw/d), 96% for test groups 10 and 11 (0 and 100 mg/kg bw/d) and 100% for test group 12 (300 mg/kg bw/d). These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
- 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/d). In addition, the postimplantation loss in this test group was higher than documented in the historical control data. There were no statistically significantly differences concerning the postimplantation loss between the remaining test groups and the control (4.9%, 3.0%, and 4.0% for 0, 100, and 300 mg/kg bw/d, respectively).
- 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%.

Effect levels (P1)

open allclose all
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

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

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/d). However, a comparison of the total amount of liveborn and stillborn F2 pups per test group did not reveal statistically significant differences between test groups 10- 13.
- The viability index as indicator for pup mortality between days 0-4 p.p. was 100% in all test groups.
- The lactation index as indicator for pup mortality between days 4-21 p.p. was not affected by test substance-treatment as it reached 100%, 100%, 99%, and 100% at dose levels of 0, 100, 300, and 1000 mg/kg bw/d. Any isolated pup deaths were assessed as incidental.mortality between days 4-21 p.p. was not affected by test substance-treatment as it reached 100%, 100%, 99%, and 100% at dose levels of 0, 100, 300, and 1000 mg/kg bw/d. Any isolated pup deaths were assessed as incidental.
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/d) were generally comparable to the concurrent control group throughout the entire lactation period. The statistically significantly increased body weights of female pups and the statistically significantly increased body weight value for both sexes in test group 13 on lactation day 1 were related to the decreased litter sizes and, given that, regarded to be secondary effects.
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/d). However, this calculated incidence of 5.7% was clearly within the range of historical control data.
Histopathological findings:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

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

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

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

Any other information on results incl. tables

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

 

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

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

 

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

 

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

 

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

 

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

Tables

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

 

Test group 01
(100 mg/kg bw/day)

Test group 02
(300 mg/kg bw/day)

Test group 03
(1000 mg/kg bw/day)

F0 males

94.3 (72.4 / 102.5)

283.2 (218.4 / 309.4)

943.3 (716.7 / 1032.6)

F0 females (premating)

96.7 (80.5 / 100.7)

289.6 (241.2 / 304.9)

964.4 (792.4 / 1017.8)

F0 females
(F1 litter)
- gestation period
- lactation period*



103.5 (92.6 / 111.6)
99.2 (81.6 / 120.2)



315.2 (284.8 / 337.9)
306.7 (249.7 / 370.3)



1043.2 (989.4 / 1084.7)
866.0 (668.6 / 1053.9)

* = Days 1–14 p.p. only

Absolute organ weights (P-generation)

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

 

Male animals

Female animals

Group

01

100 mg/kg bw/day

02

300 mg/kg bw/day

03

1000 mg/kg bw/day

01

100 mg/kg bw/day

02

300 mg/kg bw/day

03

1000 mg/kg bw/day

Brain

99%

100%

97%*

 

 

 

Cauda epididymis

99%

102%

88%**

 

 

 

Epididymides

100%

101%

92%**

 

 

 

Prostate

92%

99%

86%**

 

 

 

Spleen

 

 

 

105%*

107%

97%

 

*: p≤0.05; **: p≤0.01

 

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

 

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

 

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

Absolute organ weights (F1 generation)

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

 

Male animals

Female animals

Group

11

100 mg/kg bw/day

12

300 mg/kg bw/day

13

1000 mg/kg bw day

11

100 mg/kg bw/day

12

300 mg/kg bw/day

13

1000 mg/kg bw/day

Cauda epididymis

96%

99%

88%**

 

 

 

Epididymides

100%

101%

91%**

 

 

 

Kidneys

99%

106%*

111%**

103%

106%**

115%**

Spleen

99%

103%

92%*

 

 

 

Thyroid glands

106%

99%

109%*

110%

118%**

111%*

 

*: p≤0.05; **: p≤0.01

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

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

 

The 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 was less likely.

 

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

Applicant's summary and conclusion