2-methylaminoethanol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-11-25 to 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-methylaminoethanol

EC Number:

203-710-0

EC Name:

2-methylaminoethanol

Cas Number:

109-83-1

Molecular formula:

C3H9NO

IUPAC Name:

2-(methylamino)ethan-1-ol

Details on test material:

- Name of test material (as cited in study report): Methylaminoethanol (Test item No.07/0540-2)
- Substance type: organic
- Physical state: colorless liquid
- Analytical purity: 99.7 area-% (Analytical Report: 07L00323)

- Purity test date: November 06-07, 2007
- Lot/batch No.: from continuous production
- Expiration date of the lot/batch: 24 Oct 2007


- Stability under test conditions: The stability of the test item under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility
- Storage condition of test material: Room temperature, under N2
- Other:

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-11 weeks
- Weight at study initiation: The weight variation of the animals used did not exceed 20 percent of the mean weight of each sex.
- Fasting period before study: no data
- Housing: individually in type M III polycarbonate cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: yes

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was applied as a solution. To prepare the solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then the vehicle (highly deionized water) was filled up to the desired volume, subsequently mixed using a magnetic stirrer. The test-substance solutions were prepared in such intervals that the stability was guaranteed.

VEHICLE
- highly deionized water
- Concentration in vehicle:0.5, 1.5 and 4.5 g/100 mL
- Amount of vehicle (if gavage): 100 mL

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (Pregnant females were provided with nesting material (cellulose wadding) toward the end of pregnancy.)
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF SE. The stability of the test substance in highly deionized water at room temperature for a period of 10 days was proven before the start of the administration period (Project No.: 01Y0540/078008). The concentration control analyses revealed that the values were in the expected range of the target concentration, i.e. were in a range of about 90.1-102.2% of the nominal concentration.

Duration of treatment / exposure:

The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females (35 days for males and 55 days for females).

Frequency of treatment:

daily at the same time in the morning

Details on study schedule:

- Age at mating of the mated animals in the study: 13-14 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Basis: nominal conc.

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day

Remarks:

Basis: nominal conc.

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day

Remarks:

Basis: nominal conc.

Doses / concentrations 4

Dose / conc.:

450 mg/kg bw/day

Remarks:

Basis: nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): randomized

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.
- Time schedule: A cageside examination was conducted before and after treatment for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented for each animal.
- Cage side observations checked in table 1 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 x 37.5 cm with side borders of 25 cm high).
- The parameters examined are listed in the table 1

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no

Generally, food consumption was determined once a week (in a period of 7 days) for male and female parental animals, with the following exceptions:
• Food consumption was not determined during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
• Food consumption of F0 females, which gave birth to a litter, was determined on PND 0 and 4.
• Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations: testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [no data]
- If yes, maximum of [all] pups/litter ; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities
viability index was calculated as follows: (number of live pups on PND4/number of liveborn pups on the day of birth)x100.
The same for sex ratio: (number of live male or female pups on day 0/ 4/number of live male and female pups on day 0/ 4)x100
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.
The pups were weighed one day after birth (PND 1) and on day 4 after birth.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [after approximately 1 week post-mating period]
- Maternal animals: All surviving animals after PND 4

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. All parental animals were sacrificed by decapitation using isoflurane anaesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs. The animals, which died intercurrently or were sacrificed in a moribund state, were necropsied as soon as possible after their death and assessed by gross pathology. Organ weights were recorded (see table 2).

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in table 3 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age. All surviving pups (after sacrifice on PND 4 by means of CO2), all stillborn pups and those pups that died before schedule, were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: all gross laesions, lungs and spinal cord (cervical, thoracic and lumbar cord) were preserved in neutrally buffered 4 % formaldehyde solution and then analyzed.

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

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in table 3 were prepared for microscopic examination and weighed, respectively.

Statistics:

Food consumption, body weight and body weight change (parental animals and pups (for the pup weights, the litter means were used)), number of mating days, duration of gestation, number of pups delivered per litter, implantation sites, post implantation loss: DUNNETT-test (two-sided)
Reproduction indices and urinalysis, except color, turbidity, volume and specific gravity, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy: FISHER'S EXACT test
Proportions of affected pups per litter with necropsy observations: WILCOXON-test (one-sided)
Faeces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity, clinical pathology parameters, urine volume, urine specific gravity and organ weights : KRUSKAL-WALLIS test (two-sided).

Reproductive indices:

Male mating index %: (number of males with confirmed mating* /number of males placed with females)x100; *- defined by a female with vaginal sperm or with implants in utero;
Male fertility index (%): (number of males proving their fertility */number of males placed with females)x100; * - defined by a female with implants in utero;
Female mating index (%): (number of females mated */ number of females placed with males)x100; * - defined as the number of females with vaginal sperm or with implants in utero;
Female fertility index (%): (number of females pregnant */number of females mated **)x100; * defined as the number of females with implants in utero; ** defined as the number of females with vaginal sperm or with implants in utero.
Gestation index (%): (number of females with live pups on the day of birth/number of females pregnant *); * - defined as the number of females with implants in utero;
Live birth index(%): (number of liveborn pups at birth/total number of pups born)x100;
Post implantation loss (%): (number of implantations number of pups delivered/number of implantations)x100

Offspring viability indices:

Viability index (%): (number of live pups on PND4/number of liveborn pups on the day of birth)x100. The same for sex ratio: (number of live male or female pups on day 0/ 4/number of live male and female pups on day 0/ 4)x100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In test group 3 (450 mg/kg bw/d) one male animal (animal no. 37) was found dead within the first week of the study. One male animal (animal no. 32) of test group 3 (450 mg/kg bw/d) was sacrificed in a moribund state in study week 2. In addition, one female animal (animal no. 126) of test group 2 (150 mg/kg bw/d) was sacrificed on GD 23 because of an inability to deliver.

In test group 3 (450 mg/kg bw/d), salivation after treatment was observed in study week 1 in one male animal (animal no. 36) and in study weeks 1, 6 and 7 in six female animals. Poor general state was observed in test group 3 (450 mg/kg bw/d) in study weeks 1 and 2 in two male animals (animal nos. 32 and 36) and in study weeks 1, 6 and 7 in two female animals (animal nos. 132 and 135). In test group 3 (450 mg/kg bw/d), apathy was observed in study week 2 in one male animal (animal no. 32). Clonic convulsion was observed in test group 3 (450 mg/kg bw/d) in study week 1 in one male animal (animal no. 39).

The detailed clinical observations on study days 0, 7, 13, 21, 28 in males and females and additionally day 35, 42 and 49 in female animals did not reveal any additional abnormalities in animals of test groups 0-3 (0, 50, 150 and 450 mg/kg bw/d).

Mortality:

mortality observed, treatment-related

Description (incidence):

In test group 3 (450 mg/kg bw/d) one male animal (animal no. 37) was found dead within the first week of the study. One male animal (animal no. 32) of test group 3 (450 mg/kg bw/d) was sacrificed in a moribund state in study week 2. In addition, one female animal (animal no. 126) of test group 2 (150 mg/kg bw/d) was sacrificed on GD 23 because of an inability to deliver.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In test group 3 (450 mg/kg bw/d) male animals’ body weight was significantly lower in week 4 and body weight change was already significantly lower between weeks 1-2 and in summary between weeks 0-4. In test group 2 (150 mg/kg bw/d) male animals’ body weight change was significantly lower between weeks 3-4. Body weights and body weight changes of all female animals treated with 50, 150 or 450 mg/kg bw/d were not significantly changed during premating.
During gestation body weights of female animals of test group 2 (150 mg/kg bw/d) were significantly lower on GD 14 and 20 and of test group 3 (450 mg/kg bw/d) body weight was even decreased on GD 20.
Body weight changes of female animals during gestation were significantly lower between GD 0-7 in test group 1 (50 mg/kg bw/d) as well as between GD 0-7 and GD 7-14 in test group 2 (150 mg/kg bw/d). A body weight loss could be detected between GD 14-20 in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). Consequently, the overall body weight change between GD 0-20 was also significantly lower for these test groups.
Body weights and body weight changes of female animals treated with 50 mg/kg bw/d were not significantly changed during lactation. During lactation, a comparison of body weight data of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) to the control were not meaningful as only one litter consisting of one stillborn pup existed in test group 2 (150 mg/kg bw/d) and no pups were alive in test group 3 (450 mg/kg bw/d).
During the post-weaning period female body weights were significantly lower in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) in study week 6 and 7. The same was true for females of test group 1 (50 mg/kg bw/d) in study week 7. As the terminal mean body weight in this test group was unaffected (see section 4.4.1.1. Absolute organ weights) this change was assessed as incidental and not related to treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Significantly decreased food consumption of the male animals of test group 3 (450 mg/kg bw/d) was observed during the first two study weeks.
Food consumption of the female rats of test group 3 (450 mg/kg bw/d) was significantly decreased during the first study week.
During gestation the food consumption in test group 2 (150 mg/kg bw/d) was significantly decreased between GD 14 and 20.
During lactation food consumption in test group 2 (150 mg/kg bw/d) was significantly lower compared to the control.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period red blood cell counts (RBC), haemoglobin concentrations and hematocrit values were decreased in rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). Additionally, the hematocrit values were significantly decreased in females and males of test group 1 (50 mg/kg bw/d). This decrease compared to the controls was below 10% (males: 5%; females 7%), and it was the only dose-dependently changed red blood cell parameter in this test group. Therefore, the hematocrit decrease in rats of test group 1 (50 mg/kg bw/d) was regarded as treatment-related but not adverse.
The mean corpuscular volume (MCV) was decreased in male rats of all treatment groups (not significantly changed in test group 3 [450 mg/kg bw/d]). The measured MCV and RBC values were used to calculate the hematocrit values. In male rats of test group 1 (50 mg/kg bw/d) the MCV reflected the decreased hematocrit value because the RBC was not changed. Therefore, the decreased MCV in these rats was regarded as treatment-related, but not adverse as mentioned above.
In female rats of test group 3 (450 mg/kg bw/d) the relative reticulocyte counts were increased. No significant change was observed in the total white blood cell counts (WBC) of treated rats. However, some changes in the relative and absolute differential blood cell counts were measured (males: increased relative neutrophil counts and decreased relative eosinophil counts in test group 3 [450 mg/kg bw/d], decreased relative monocyte counts in test group 2 [150 mg/kg bw/d]; females: decreased absolute eosinophil counts in test group 3 [450 mg/kg bw/d], decreased relative neutrophil counts and increased relative lymphocyte counts in test group 2 [150 mg/kg bw/d]). These changes were regarded as being incidental and not treatment-related because they were not dose-dependently changed and not consistent in both sexes.
The prothrombin time was shortened in rats of both sexes of test group 3 (450 mg/kg bw/d) and, additionally, in females of test group 2 (150 mg/kg bw/d).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Liver enzyme activity was not changed in male and female rats of any test substance-treated group.
The urea levels were increased in males of test group 2 (150 mg/kg bw/d) and in rats of both sexes in test group 3 (450 mg/kg bw/d).
The total bilirubin concentrations were significantly higher in rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d).
The total protein and the albumin levels were increased in females of test group 1 (50 mg/kg bw/d) and higher (total protein level was not significantly increased in test group 3 [450 mg/kg bw/d]), although the increases were not dose-dependent.
In males the total protein levels were significantly increased in test groups 1 (50 mg/kg bw/d) and 2 (150 mg/kg bw/d) and the albumin concentrations in test group 2 (150 mg/kg bw/d), only. These parameters were not changed dose-dependently, and the deviated values were within the historical control ranges (total protein: 62.45-69.74 g/L; albumin 36.12-39.76 g/L). Therefore, these deviations were regarded as non-adverse effects.
The sodium concentrations were increased in rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) and, additionally, in males of test group 1 (50 mg/kg bw/d). The sodium mean in males at least of the low dose group was within the historical control range (140.9-147.1 mmol/L). Apart from this, only this electrolyte level was deviated in test group 1 (50 mg/kg bw/d). Therefore, the sodium levels increase at least in males of the low dose group was regarded as a non-adverse effect.
In males of test groups 1 (50 mg/kg bw/d) and 2 (150 mg/kg bw/d) the cholesterol levels were decreased. The parameter was not changed dose-dependently, and such deviation was not observed in females. Therefore, the cholesterol levels decrease in males of test groups 1 (50 mg/kg bw/d) and 2 (150 mg/kg bw/d) was regarded as non-adverse.
In treated females the potassium concentrations were significantly higher in test group 1 (50 mg/kg bw/d), the creatinine levels were higher in test group 2 (150 mg/kg bw/d) and the magnesium concentrations were increased in test groups 1 and 2. These values were not changed dose-dependently, and the deviations of these parameters were not measured in male rats. Therefore, these changes were regarded as incidental rather than treatment-related.

Endocrine findings:

not specified

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

In rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) the incidence of blood (haemoglobin) was found higher compared to the controls (in females of test group 3 not significant). Additionally, the incidence of higher leucocyte counts in the urine sediment was significantly increased in males of test group 2 (150 mg/kg bw/d). However, no significantly higher leucocyte counts were found in the urine sediment of rats of both sexes of test group 3 (450 mg/kg bw/d). In males of test group 3 (450 mg/kg bw/d), the incidence of higher transitional cell counts was increased.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Detailed clinical examinations in an open field, detailed observations in a functional observational battery (FOB) and measurements of motor activity did not reveal indications of test substance-induced effects in low, mid and high-dose rats. Therefore, the clonic convulsions were assessed as being incidental.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Kidneys: The graded severity of tubular degeneration was dose-related increased. The statistically significant increase of the relative kidney weights in animals of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) was considered to be caused by the tubular degeneration/ regeneration process.
Testes: The decrease of the absolute testes weight in males of test group 3 (450 mg/kg bw/d) was related to the diffuse tubular degeneration.
Ovaries: In ovaries, vacuoles of different size were observed in the sex cord stroma in females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). Incidence and severity was dose-related increased (see Table 10). In addition, one female of test group 1 (50 mg/kg bw/d), one female of test group 2 (150 mg/kg bw/d) and all females of test group 3 (450 mg/kg bw/d) showed ovarian cysts. The occurrence of cysts in females of test group 3 (450 mg/kg bw/d) was assessed as treatment-related.
The cysts in each one female of test groups 1 (50 mg/kg bw/d) and 2 (150 mg/kg bw/d) were considered to be rather incidental.
Although there was no clear histopathological correlate for the decreased absolute and relative ovarian weights in females of test group 3 (450 mg/kg bw/d), a test substance-related effect cannot be ruled out.
Spleen: Incidence and graded severity of extramedullary hematopoiesis were dose-related increased in males and females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). The increased relative spleen weights in males of test group 3 (450 mg/kg bw/d) as well as in females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) were associated with these findings. (see Table 8)

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

The male mating index was 100% in all test groups. Fertility was proven for most of the F0 parental males of test groups 0 (control) and 1 (50 mg/kg bw/d) within the scheduled mating interval for the F1 litter. One control male and one male of test group 1 did not generate F1 pups. Furthermore, six males of test group 2 and nine males of test group 3 did not generate F1 pups. Thus, the male fertility index ranged between 11% and 90% (see Tab.4 ). For test groups 0 (control) and 1 (50 mg/kg bw/d) these findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data (see PART III, Supplement). With regard to pathological findings in epididymidis and testis (see section 4.4. Pathology) the test substance did adversely affect reproduction of the F0 males in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d).
The female mating index calculated after the mating period for F1 litter was 100% for all test groups. The mean duration until sperm was detected (GD 0) amounted to 2.4, 1.4, 2.5, and 2.9 days (0, 50, 150 and 450 mg/kg bw/d, respectively). Consequently, the differences between the test groups were assessed as being spontaneous in nature and without biological relevance. All sperm-positive rats of test groups 0 (control) and 1 (50 mg/kg bw/d) delivered pups or had implantations in utero with the following exceptions: one female (test group 0) and one female (50 mg/kg bw/d) did not become pregnant. 6 females of test group 2 (150 mg/kg bw/d), and 9 females of test group 3 (450 mg/kgbw/d) did not become pregnant. The fertility index varied between 10% and 90% (Tab. 5).
Implantation was not affected by the treatment in test group 1 (50 mg/kg bw/d) since the mean number of implantation sites was comparable test group 0 (0 mg/kg bw/d). In test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) a significant reduction with only 5 and 1 implantation sites was found.The mean duration of gestation, i.e. 22.1 and 22.2 days, was similar in test groups 0 (control) and 1 (50 mg/kg bw/d). No parturition was seen in test group 2 (150 mg/kg bw/d) except of female No. 126 which was sacrificed on GD 23 because of an inability to deliver. Gestation length was not calculable for test group 3 (450 mg/kg bw/d). The gestation index varied between 89% (control group) and 100% (50 mg/kg body weight/day). All values seen in test groups 0 (control) and 1 (50 mg/kg bw/d) 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 (PART III, Supplement) and did not show a relation to dosing. However, a clear relation to dosing was obtained for test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d).
The mean number of F1 pups delivered per dam was not affected in test group 1 (50 mg/kg bw/d) whereas only one pup was delivered in test group 2 (150 mg/kg bw/d) and none in test group 3 (450 mg/kg bw/d).
The rate of liveborn pups was unaffected in test group 1 (50 mg/kg bw/d) and the live birth index was 96%. The rate of stillborn pups was not significantly different compared to the control group and within the range of the historical control data (PART III, Supplement). In test group 2 (150 mg/kg bw/d) the live birth index was 0 because only one stillborn pup was delivered.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

In test group 3 (450 mg/kg bw/d) one male animal (animal no. 37) was found dead within the first week of the study. One male animal (animal no. 32) of test group 3 (450 mg/kg bw/d) was sacrificed in a moribund state in study week 2. In addition, one female animal (animal no. 126) of test group 2 (150 mg/kg bw/d) was sacrificed on GD 23 because of an inability to deliver.

In test group 3 (450 mg/kg bw/d), salivation after treatment was observed in study week 1 in one male animal (animal no. 36) and in study weeks 1, 6 and 7 in six female animals. Poor general state was observed in test group 3 (450 mg/kg bw/d) in study weeks 1 and 2 in two male animals (animal nos. 32 and 36) and in study weeks 1, 6 and 7 in two female animals (animal nos. 132 and 135). In test group 3 (450 mg/kg bw/d), apathy was observed in study week 2 in one male animal (animal no. 32). Clonic convulsion was observed in test group 3 (450 mg/kg bw/d) in study week 1 in one male animal (animal no. 39).

The detailed clinical observations on study days 0, 7, 13, 21, 28 in males and females and additionally day 35, 42 and 49 in female animals did not reveal any additional abnormalities in animals of test groups 0-3 (0, 50, 150 and 450 mg/kg bw/d).

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

In test group 3 (450 mg/kg bw/d) male animals’ body weight was significantly lower in week 4 and body weight change was already significantly lower between weeks 1-2 and in summary between weeks 0-4. In test group 2 (150 mg/kg bw/d) male animals’ body weight change was significantly lower between weeks 3-4. Body weights and body weight changes of all female animals treated with 50, 150 or 450 mg/kg bw/d were not significantly changed during premating.
During gestation body weights of female animals of test group 2 (150 mg/kg bw/d) were significantly lower on GD 14 and 20 and of test group 3 (450 mg/kg bw/d) body weight was even decreased on GD 20.
Body weight changes of female animals during gestation were significantly lower between GD 0-7 in test group 1 (50 mg/kg bw/d) as well as between GD 0-7 and GD 7-14 in test group 2 (150 mg/kg bw/d). A body weight loss could be detected between GD 14-20 in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). Consequently, the overall body weight change between GD 0-20 was also significantly lower for these test groups.
Body weights and body weight changes of female animals treated with 50 mg/kg bw/d were not significantly changed during lactation. During lactation, a comparison of body weight data of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) to the control were not meaningful as only one litter consisting of one stillborn pup existed in test group 2 (150 mg/kg bw/d) and no pups were alive in test group 3 (450 mg/kg bw/d).
During the post-weaning period female body weights were significantly lower in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) in study week 6 and 7. The same was true for females of test group 1 (50 mg/kg bw/d) in study week 7. As the terminal mean body weight in this test group was unaffected (see section 4.4.1.1. Absolute organ weights) this change was assessed as incidental and not related to treatment.

Significantly decreased food consumption of the male animals of test group 3 (450 mg/kg bw/d) was observed during the first two study weeks.
Food consumption of the female rats of test group 3 (450 mg/kg bw/d) was significantly decreased during the first study week.
During gestation the food consumption in test group 2 (150 mg/kg bw/d) was significantly decreased between GD 14 and 20.
During lactation food consumption in test group 2 (150 mg/kg bw/d) was significantly lower compared to the control.

HEMATOLOGICAL FINDINGS
At the end of the administration period red blood cell counts (RBC), haemoglobin concentrations and hematocrit values were decreased in rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). Additionally, the hematocrit values were significantly decreased in females and males of test group 1 (50 mg/kg bw/d). This decrease compared to the controls was below 10% (males: 5%; females 7%), and it was the only dose-dependently changed red blood cell parameter in this test group. Therefore, the hematocrit decrease in rats of test group 1 (50 mg/kg bw/d) was regarded as treatment-related but not adverse.
The mean corpuscular volume (MCV) was decreased in male rats of all treatment groups (not significantly changed in test group 3 [450 mg/kg bw/d]). The measured MCV and RBC values were used to calculate the hematocrit values. In male rats of test group 1 (50 mg/kg bw/d) the MCV reflected the decreased hematocrit value because the RBC was not changed. Therefore, the decreased MCV in these rats was regarded as treatment-related, but not adverse as mentioned above.
In female rats of test group 3 (450 mg/kg bw/d) the relative reticulocyte counts were increased. No significant change was observed in the total white blood cell counts (WBC) of treated rats. However, some changes in the relative and absolute differential blood cell counts were measured (males: increased relative neutrophil counts and decreased relative eosinophil counts in test group 3 [450 mg/kg bw/d], decreased relative monocyte counts in test group 2 [150 mg/kg bw/d]; females: decreased absolute eosinophil counts in test group 3 [450 mg/kg bw/d], decreased relative neutrophil counts and increased relative lymphocyte counts in test group 2 [150 mg/kg bw/d]). These changes were regarded as being incidental and not treatment-related because they were not dose-dependently changed and not consistent in both sexes.
The prothrombin time was shortened in rats of both sexes of test group 3 (450 mg/kg bw/d) and, additionally, in females of test group 2 (150 mg/kg bw/d).

CLINICAL BIOCHEMISTRY FINDINGS
Liver enzyme activity was not changed in male and female rats of any test substance-treated group.
The urea levels were increased in males of test group 2 (150 mg/kg bw/d) and in rats of both sexes in test group 3 (450 mg/kg bw/d).
The total bilirubin concentrations were significantly higher in rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d).
The total protein and the albumin levels were increased in females of test group 1 (50 mg/kg bw/d) and higher (total protein level was not significantly increased in test group 3 [450 mg/kg bw/d]), although the increases were not dose-dependent.
In males the total protein levels were significantly increased in test groups 1 (50 mg/kg bw/d) and 2 (150 mg/kg bw/d) and the albumin concentrations in test group 2 (150 mg/kg bw/d), only. These parameters were not changed dose-dependently, and the deviated values were within the historical control ranges (total protein: 62.45-69.74 g/L; albumin 36.12-39.76 g/L). Therefore, these deviations were regarded as non-adverse effects.
The sodium concentrations were increased in rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) and, additionally, in males of test group 1 (50 mg/kg bw/d). The sodium mean in males at least of the low dose group was within the historical control range (140.9-147.1 mmol/L). Apart from this, only this electrolyte level was deviated in test group 1 (50 mg/kg bw/d). Therefore, the sodium levels increase at least in males of the low dose group was regarded as a non-adverse effect.
In males of test groups 1 (50 mg/kg bw/d) and 2 (150 mg/kg bw/d) the cholesterol levels were decreased. The parameter was not changed dose-dependently, and such deviation was not observed in females. Therefore, the cholesterol levels decrease in males of test groups 1 (50 mg/kg bw/d) and 2 (150 mg/kg bw/d) was regarded as non-adverse.
In treated females the potassium concentrations were significantly higher in test group 1 (50 mg/kg bw/d), the creatinine levels were higher in test group 2 (150 mg/kg bw/d) and the magnesium concentrations were increased in test groups 1 and 2. These values were not changed dose-dependently, and the deviations of these parameters were not measured in male rats. Therefore, these changes were regarded as incidental rather than treatment-related.

URINANALYSIS FINDINGS
In rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) the incidence of blood (haemoglobin) was found higher compared to the controls (in females of test group 3 not significant). Additionally, the incidence of higher leucocyte counts in the urine sediment was significantly increased in males of test group 2 (150 mg/kg bw/d). However, no significantly higher leucocyte counts were found in the urine sediment of rats of both sexes of test group 3 (450 mg/kg bw/d). In males of test group 3 (450 mg/kg bw/d), the incidence of higher transitional cell counts was increased.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS) not applicable

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS) not examined

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS) not examined

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

The male mating index was 100% in all test groups. Fertility was proven for most of the F0 parental males of test groups 0 (control) and 1 (50 mg/kg bw/d) within the scheduled mating interval for the F1 litter. One control male and one male of test group 1 did not generate F1 pups. Furthermore, six males of test group 2 and nine males of test group 3 did not generate F1 pups. Thus, the male fertility index ranged between 11% and 90% (see Tab.4 ). For test groups 0 (control) and 1 (50 mg/kg bw/d) these findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data (see PART III, Supplement). With regard to pathological findings in epididymidis and testis (see section 4.4. Pathology) the test substance did adversely affect reproduction of the F0 males in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d).
The female mating index calculated after the mating period for F1 litter was 100% for all test groups. The mean duration until sperm was detected (GD 0) amounted to 2.4, 1.4, 2.5, and 2.9 days (0, 50, 150 and 450 mg/kg bw/d, respectively). Consequently, the differences between the test groups were assessed as being spontaneous in nature and without biological relevance. All sperm-positive rats of test groups 0 (control) and 1 (50 mg/kg bw/d) delivered pups or had implantations in utero with the following exceptions: one female (test group 0) and one female (50 mg/kg bw/d) did not become pregnant. 6 females of test group 2 (150 mg/kg bw/d), and 9 females of test group 3 (450 mg/kgbw/d) did not become pregnant. The fertility index varied between 10% and 90% (Tab. 5).
Implantation was not affected by the treatment in test group 1 (50 mg/kg bw/d) since the mean number of implantation sites was comparable test group 0 (0 mg/kg bw/d). In test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) a significant reduction with only 5 and 1 implantation sites was found.The mean duration of gestation, i.e. 22.1 and 22.2 days, was similar in test groups 0 (control) and 1 (50 mg/kg bw/d). No parturition was seen in test group 2 (150 mg/kg bw/d) except of female No. 126 which was sacrificed on GD 23 because of an inability to deliver. Gestation length was not calculable for test group 3 (450 mg/kg bw/d). The gestation index varied between 89% (control group) and 100% (50 mg/kg body weight/day). All values seen in test groups 0 (control) and 1 (50 mg/kg bw/d) 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 (PART III, Supplement) and did not show a relation to dosing. However, a clear relation to dosing was obtained for test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d).
The mean number of F1 pups delivered per dam was not affected in test group 1 (50 mg/kg bw/d) whereas only one pup was delivered in test group 2 (150 mg/kg bw/d) and none in test group 3 (450 mg/kg bw/d).
The rate of liveborn pups was unaffected in test group 1 (50 mg/kg bw/d) and the live birth index was 96%. The rate of stillborn pups was not significantly different compared to the control group and within the range of the historical control data (PART III, Supplement). In test group 2 (150 mg/kg bw/d) the live birth index was 0 because only one stillborn pup was delivered.

ORGAN WEIGHTS (PARENTAL ANIMALS)

Absolute organ weights: When compared to control group 0 (set to 100%), the mean absolute weights of the organs listed in the Table 8 were significantly increased or decreased. All other mean absolute weight parameters did not show significant differences when compared to test group 0 (control).
Relative organ weights: The terminal body weight was significantly decreased in males of test group 3 (450 mg/kg bw/d) and in females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) resulting in significant, secondary weight changes in various organs (Table 9)

GROSS PATHOLOGY (PARENTAL ANIMALS)
Three males of test group 3 (450 mg/kg bw/d) showed erosions or ulcers in the glandular stomach. The liver was enlarged in three males and one female of test group 2 (150 mg/kg bw/d) as well as in three males and five females of test group 3 (450 mg/kg bw/d). Four males of test group 1 (50 mg/kg bw/d) and four males of test group 2 (150 mg/kg bw/d) showed a prominent acinar pattern of the liver. The mesenteric lymph nodes were red discolored in one female of test group 2 (150 mg/kg bw/d) and in two females of test group 3 (450 mg/kg bw/d). All other gross lesions occurred either singly or were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental.

HISTOPATHOLOGY (PARENTAL ANIMALS) (see Table 8)
Kidneys: The graded severity of tubular degeneration was dose-related increased. The statistically significant increase of the relative kidney weights in animals of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) was considered to be caused by the tubular degeneration/ regeneration process.
Testes: The decrease of the absolute testes weight in males of test group 3 (450 mg/kg bw/d) was related to the diffuse tubular degeneration.
Ovaries: In ovaries, vacuoles of different size were observed in the sex cord stroma in females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). Incidence and severity was dose-related increased (see Table 10). In addition, one female of test group 1 (50 mg/kg bw/d), one female of test group 2 (150 mg/kg bw/d) and all females of test group 3 (450 mg/kg bw/d) showed ovarian cysts. The occurrence of cysts in females of test group 3 (450 mg/kg bw/d) was assessed as treatmentrelated.
The cysts in each one female of test groups 1 (50 mg/kg bw/d) and 2 (150 mg/kg bw/d) were considered to be rather incidental.
Although there was no clear histopathological correlate for the decreased absolute and relative ovarian weights in females of test group 3 (450 mg/kg bw/d), a test substance-related effect cannot be ruled out.
Spleen: Incidence and graded severity of extramedullary hematopoiesis were dose-related increased in males and females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). The increased relative spleen weights in males of test group 3 (450 mg/kg bw/d) as well as in females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) were associated with these findings.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

2

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

3

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The F1 pups did not show adverse clinical signs up to scheduled sacrifice on PND 4. In one litter (dam No. 112 of test group 1) one pup showed a papilloma-like a skin flap. This single observation was considered to be spontaneous in nature and not to be adverse.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The mean number of delivered pups per dam and the rate of liveborn and stillborn pups were evenly distributed among test groups 0 (control) and 1 (50 mg/kg bw/d). The respective values reflect the normal range of biological variation inherent in the strain used in this study.
The viability index as indicator for pup mortality between PND 0-4 was 100% for test groups 0 (control) and 1 (50 mg/kg bw/d). No viable pups were observed in test group 2 (150 mg/kg bw/d) and test group 3 (450 mg/kg bw/d).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean pup body weights/pup body weight changes of all pups in test group (50 mg/kg bw/d) were comparable to the concurrent control values. The observable differences between the groups were assessed as being spontaneous in nature and without biological relevance.
One runt of each gender was seen in test group 0 (control) and 5 female runts were seen in test group 1 (50 mg/kg bw/d). Both values were within the range of the biological variation inherent in the strain of rats used for this study.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

One stillborn pup of test group 1 (50 mg/kg bw/d) showed post mortem autolysis. In 3 pups of test group 1 (50 mg/kg bw/d) and in the single stillborn pup of test group 2 (150 mg/kg bw/d) the stomach was found empty.

Histopathological findings:

no effects observed

Description (incidence and severity):

no treatment -related effects in the low dose group

Details on results (F1)

VIABILITY (OFFSPRING)
The mean number of delivered pups per dam and the rate of liveborn and stillborn pups were evenly distributed among test groups 0 (control) and 1 (50 mg/kg bw/d). The respective values reflect the normal range of biological variation inherent in the strain used in this study.
The viability index as indicator for pup mortality between PND 0-4 was 100% for test groups 0 (control) and 1 (50 mg/kg bw/d). No viable pups were observed in test group 2 (150 mg/kg bw/d) and test group 3 (450 mg/kg bw/d).

CLINICAL SIGNS (OFFSPRING)
The F1 pups did not show adverse clinical signs up to scheduled sacrifice on PND 4. In one litter (dam No. 112 of test group 1) one pup showed a papilloma-like a skin flap. This single observation was considered to be spontaneous in nature and not to be adverse.

BODY WEIGHT (OFFSPRING)
Mean pup body weights/pup body weight changes of all pups in test group (50 mg/kg bw/d) were comparable to the concurrent control values. The observable differences between the groups were assessed as being spontaneous in nature and without biological relevance.
One runt of each gender was seen in test group 0 (control) and 5 female runts were seen in test group 1 (50 mg/kg bw/d). Both values were within the range of the biological variation inherent in the strain of rats used for this study.

SEXUAL MATURATION (OFFSPRING) not applicable
ORGAN WEIGHTS (OFFSPRING) not examined

GROSS PATHOLOGY (OFFSPRING)
One stillborn pup of test group 1 (50 mg/kg bw/d) showed post mortem autolysis. In 3 pups of test group 1 (50 mg/kg bw/d) and in the single stillborn pup of test group 2 (150 mg/kg bw/d) the stomach was found empty.

HISTOPATHOLOGY (OFFSPRING) no treatment -related effects in the low dose group
OTHER FINDINGS (OFFSPRING)

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Based on:

test mat.

Sex:

male/female

Remarks on result:

not measured/tested

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

50 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:

yes

Any other information on results incl. tables

Table 4: Reproductive performance (male animals)

	Test group 0 (0 mg/kg bw/d)	Test group 1 (50 mg/kg bw/d)	Test group 2 (150 mg/kg bw/d)	Test group 3 (450 mg/kg bw/d)
Male fertility index [%]	90	90	40	11**

Table 5: Reproductive performance (female animals)

	Test group 0 (0 mg/kg bw/d)	Test group 1 (50 mg/kg bw/d)	Test group 2 (150 mg/kg bw/d)	Test group 3 (450 mg/kg bw/d)
Female fertility index [%]	90	90	40*	10**

* p ≤ 0.05; ** p ≤ 0.01

Table 6: Absolute organ weight (parental animals)

	Male animals			Female animals
Test group (mg/kg bw/day)	1 (50)	2 (150	3 (450)	1 (50)	2 (150	3 (450)
Terminal body weight	101%	96%	86%**	95%	93%**	85%**
Adrenal glands				96%	90%	82%**
Brain				99%	100%	96%*
Epididymides	100%	90%	68%**
Liver	113%*	121%**	129%**	105%	123%**	124%**
Ovaries				97%	99%	74%**
Testes	103%	105%	78%**
Thymus	98%	92%	67%**	88%	83%*	69%

Table 7: Relative organ weight (parental animals)

	Male animals			Female animals
Test group (mg/kg bw/day)	1 (50)	2 (150	3 (450)	1 (50)	2 (150	3 (450)
Adrenal glands	104%	102%	128%*
Brain	98%	104%	114%*	104%*	107%*	113%**
Epydidymides	94%	98%	80%**
Heart	96%	106%*	122%**	98%	105%*	116%**
Kidney	101%	110%*	126%**	108%	116%**	132%**
Liver	111%**	127%**	150%**	111%**	133%**	146%**
Ovaries				102%	106%	86%*
Seminal vesicle	104%	113%*	117%*
Spleen	102%	111%	144%**	102%	112%*	121%**
Testes	101%	110%*	91%
Thymus	97%	96%	79%*

* : p ≤ 0.05; **: p ≤ 0.01

Table 8: Histopathology (parental animals)

	Male animals			Female animals
Test group (mg/kg bw/day)	1 (50)	2 (150	3 (450)	1 (50)	2 (150		3 (450)
Kidneys	Multifocal tubular degeneration				Multifocal tubular degeneration; increase of the kidney weight
		increase of the kidney weight
Testes		diffuse tubular degeneration
Epididymides		Oligospermia
Ovaries				Ovarian cysts incidental			Ovarian cysts
Spleen		extramedullary hematopoiesis			extramedullary hematopoiesis; hemosiderin storage
Liver	Fatty change of hepatocytes				enlarged livers
Fore- and glandular stomach			Erosions or ulcers			Erosions or ulcers
Mesenteric lymph node			Sinus erythrocytosis		Sinus erythrocytosis
Thymus			reduced cellularity of cortex			reduced cellularity of cortex

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance and fertility was 50 mg/kg bw/d for the parental rats. The NOAEL for general, systemic toxicity of the test substance was 50 mg/kg bw/d for females and less than 50 mg/kg bw/d for male animals based on the tubular degeneration in the kidneys of six males.

Executive summary:

N-Methylaminoethanol was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 50, 150 and 450 mg/kg bw/d.

The objective of the study was to detect possible effects of the test substance on the integrity and performance of the reproductive system of both sexes. Furthermore, it was intended to obtain information about the general toxicological profile including target organs and the no observed adverse effect level (NOAEL) after repeated oral administration. Control animals were dosed daily with the vehicle (highly deionized water). The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females.

Regarding clinical examinations, signs of general systemic toxicity were only observed at a dose level of 450 mg/kg bw/d as there were significantly lower body weights in male and female parental animals accompanied with reduced food consumption and reduced general condition in single animals in several phases of the study. Reduced food consumption and body weights during gestation in females of test group 2 (150 mg/kg bw/d) were most likely related to implantation losses.

Detailed clinical examinations in an open field, detailed observations in a functional observational battery (FOB) and measurements of motor activity did not reveal indications of test substance-induced effects in low, mid and high-dose rats. Therefore, the clonic convulsions were assessed as being incidental.

Salivation was seen after dosing in all high-dose rats. From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. Urine discoloration was also observed for all high-dose rats which was most likely related to the test compound. However, both types of findings were not considered to be adverse, toxicologically relevant effects.

Fertility was severely impaired by test-substance administration at dose levels of 150 and 450 mg/kg bw/d. Although mating (male and female mating indices) was not influenced no lifeborn pups were delivered for both test groups.

The deviated levels of clinical chemistry and haematology parameters pointed to anaemia and changed liver cell metabolism. The total protein and the albumin levels were significantly higher in female rats starting at test group 1 (50 mg/kg bw/d). As these were the only deviating parameters in females of this test group the changes were regarded as treatment-related, but non-adverse. The reason for the increase of the sodium concentrations in rats of both sexes in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d) remains unclear, but a test substance-related effect could not be excluded. The higher incidences of leucocytes in the urine of rats of both sexes in test group 3 (450 mg/kg bw/d) and, additionally, in males of the test group 2 (150 mg/kg bw/d) as well as the increased incidence of higher transitional cell counts in males of test group 3 (450 mg/kg bw/d) can be regarded as signs of an affection of the urinary tract in treated rats.

Regarding pathology, after administration of the test substance the terminal body weight was significantly lower in females of test group 2 (150 mg/kg bw/d) and in males and females of test group 3 (450 mg/kg bw/d). The body weight reduction resulted in weight changes of adrenal glands, brain, heart, seminal vesicle, and thymus. Target organs were the kidney, testes, epididymides, ovaries, liver, and spleen. In kidneys and testes, tubular degeneration was dose dependent and assessed as an adverse effect. In ovaries, the occurrence of cysts and vacuolization of sex cord stroma was related to treatment and was considered to be adverse. In test group 3 (450 mg/kg bw/d), the infertility was linked to the reduced number of sperms (oligospermia) caused by tubular degeneration in testes. In addition, the occurrence of ovarian cysts and vacuolization of the sex cord stroma in females may have influenced the fertility. In test group 2 (150 mg/kg bw/d), the severity of the findings in testes or ovaries was only minimal or slight and the findings did not occur in all infertile animals. Nevertheless, these lesions may have affected fertility. In the spleen, a dose-related increase in incidence and severity of extramedullary haematopoiesis occurred in males and females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). In addition, in females of these test groups the severity of haemosiderin storage was increased. These findings are associated with the increased relative spleen weights in females of test group 2 (150 mg/kg bw/d) as well as in males and females of test group 3 (450 mg/kg bw/d). They were induced in response to anaemia and related to treatment. The liver weights were dose-related increased in males and females of all treatment groups. The liver was enlarged in three males and one female of test group 2 (150 mg/kg bw/d) as well as in three males and five females of test group 3 (450 mg/kg bw/d). In females, the liver enlargement correlated with a minimal central hepatocellular hypertrophy that was observed in five animals of test group 2 (150 mg/kg bw/d) and in 9 animals of test group 3 (450 mg/kg bw/d). In males, mainly a minimal fatty change of hepatocytes was observed in two animals of test group 1 (50 mg/kg bw/d), in 8 animals of test group 2 (150 mg/kg bw/d), and in 7 animals of test group 3 (450 mg/kg bw/d). The liver findings were related to treatment and considered to be adaptive. Although, there were no clear histopathological correlates for the increased liver weights in males of all treatment groups and in females of test group 1 (50 mg/kg bw/d), a test substance-related effect could not be ruled out. There was no correlation between erosion/ ulcer in the stomach and erythrocytosis of the mesenteric lymph node (findings occurred in different animals). However, a treatment-related effect could not be ruled out but was assessed as non-adverse. All further findings occurred either singly or were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.