2-(methylamino)ethanol, compound with sulphur dioxide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 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

IUPAC Name:

2-(methylamino)ethanol

Details on test material:

- Name of test material: Methylaminoethanol (2-(Methylamino)ethanol)
- Analytical purity: 99.7 area-%
- Batch No.: from continuous production
- Date of production: 24 Oct 2007
- Expiration date of the batch: 24 Oct 2009
- Storage conditions: room temperature, under N2

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11-13 weeks old
- Housing: individually, in type M III polycarbonate cages
- Diet: ground Kliba maintenance diet mouse/rat (GLP), meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-70 %
- Air changes: 10 air changes per hour
- Photoperiod: 12 hours dark / 12 hours light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

highly deionized 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
- Vehicle: highly deionized water
- Concentration in vehicle: 5, 15, 45 mg/mL
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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). Homogeneity was given because the test substance was completely miscible with water and solutions were considered to be homogenous without further analysis. Concentration control analyses of the test substance preparations were performed in samples of all concentrations at the start and at the end of the administration period. The concentrations ranged from 90.1 to 102.2 % of the nominal concentrations.

Details on mating procedure:

- Impregnation procedure: cohoused overnight
- M/F ratio per cage: 1:1 respectively 1:2 ratio
- Length of cohabitation: overnight, from about 16.00 h until 07.00-09.00 h of the following morning, for a maximum of 2 weeks
- Proof of pregnancy: a vaginal smear was prepared after each mating and examined for sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted "gestation day (GD) 0" and the following day "GD 1".

Duration of treatment / exposure:

The duration of treatment covered a 2-week premating 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.

Frequency of treatment:

once daily (at the same time in the morning)

Duration of test:

at least subchronic exposure duration for the parental generation

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 150, 450 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose levels were selected by the Sponsor (no justification provided in the report)

- Other
On study day 32, a functional observation battery and motor activity measurement were carried out in the first 5 male animals per group.
The females were allowed to litter and rear their pups until day 4 after parturition. On postnatal day 4, all pups were sacrificed and examined.
On study day 53, a functional observation battery and motor activity measurement were carried out in the first 5 female animals (with litter) per group.
From the first 5 male animals and the first 5 female animals (with litter) urinalyses were carried out on study days 34 (males) and 50 (females). Hematological and clinico-chemical examinations were carried out on study days 35 (males) and 55 (females).
At the end of the study (study day 35 for males, study day 55 for females), the animals were sacrificed after a fasting period (withdrawal of food) for at least 16-20 hours.

Examinations

Maternal examinations:

MORTALITY
- Time schedule: 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.

CLINICAL OBSERVATIONS
- Time schedule: a cageside examination was conducted before and after treatment for any signs of morbidity, pertinent behavioural changes and signs of overt toxicity. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: 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.

BODY WEIGHT
- Time schedule: once a week at the same time of the day (in the morning). The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
- Females without a litter were weighed weekly. These body weight data were solely used for the calculations of the dose volume.
- Females after weaning (PND 4) until sacrifice were weighed once a week (for the calculation of the administration volume only)

FOOD CONSUMPTION
- Time schedule: 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 1 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).

HEMATOLOGY
Parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany). Furthermore differential blood smears were prepared and stained according to WRIGHT without being evaluated. The clotting analyses were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: yes (Isoflurane)
- Animals fasted: no data
- How many animals: 5 rats/sex and group
- Parameters examined: leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes, prothrombin time.

CLINICAL CHEMISTRY
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinico chemical parameters.
- Time schedule for collection of blood: in the morning
- Animals fasted: no data
- How many animals: 5 rats/sex and group
- Parameters examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), alkaline Phosphatase (ALP), gamma-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), inorganic Phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), total Bilirubin (TBIL), total protein (TPROT), Albumin (ALB), globulins (GLOB), triglycerides (TRIG), Cholesterol (CHOL), Magnesium (MG)

URINALYSIS
With the exception of volume, color, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semiquantitatively using test strips (Combur-9-test M, Roche, Mannheim, Germany) and a reflection photometer (Miditron M; Roche, Mannheim, Germany).
- Time schedule for collection of urine: overnight
- Metabolism cages used for collection of urine: yes
- Animals fasted: yes
- Parameters examined: pH, protein, Glucose, ketones, Urobilinogen, Bilirubin, blood, specific gravity, sediment, color, turbidity, volume

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: a functional observation battery was performed at the end of the administration period starting at about 10:00 h.
- Dose groups that were examined: the first 5 animals/sex and group
- Battery of functions tested: the FOB consisted of home cage observations, open field observations and sensorimotor tests/assessment of reflexes. Motor activity was assessed on the same day as the FOB.

ORGAN WEIGHTS
The following weights were determined in all parental animals sacrificed on schedule: liver, kidneys, adrenal glands, testes, epididymides, seminal vesicle, prostate, ovaries, uterus, thymus, spleen, brain, heart

NECROPSY, MACROSCOPIC EXAMINATION
All parental animals were sacrificed by decapitation using Isoflurane anesthesia (males: study day 35, females: study day 55). 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.

HISTOLOGICAL ASSESSMENT
After the organs were fixed, histotechnical processing and examination by light microscopy was performed on following organs: trachea, lungs, liver, kidneys, spleen, adrenal glands, heart, all gross lesions, brain, spinal cord (cervical, thoracic, lumbar), sciatic nerve, thyroid glands/parathyroid glands, testes, epididymides, ovaries, uterus, vagina, prostate gland, seminal vesicles, coagulation glands, thymus, lymph nodes (axillary), lymph nodes (mesenteric), stomach (forestomach and glandular stomach), duodenum, jejunum (with Peyer’s patches), ileum, cecum, colon, rectum, urinary bladder, bone marrow (femur)

Ovaries and uterine content:

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

Fetal examinations:

PUP NECROPSY OBSERVATIONS
All surviving pups (after sacrifice on PND 4 by means of diluted nacoren and exsanguination via vena cava), all stillborn pups and those pups that died before schedule, were examined externally, eviscerated and their organs were assessed macroscopically.

Statistics:

- Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means. Parameters analysed:
food consumption, body weight and body weight change, number of mating days, duration of gestation, number of pups per litter, implantation sites, post implantation loss

- Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions. Parameters analysed:
mating index, fertility index, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy

- Non-parametric oneway analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians. Parameters analysed:
feces, rearing, grip strength, landing foot-splay, motor activity, organ weights

Indices:

Male reproduction data: mating and fertility index (%).
Female reproduction and delivery data: mating, fertility, gestation and live birth indices and post implantation loss (%)

Pups:
Viability index (%) and sex ratio

Historical control data:

Historical control data were included in the report.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
Mortality
In test group 3 (450 mg/kg bw/d) 1 male animal was found dead within the first week of the study. One male animal of test group 3 (450 mg/kg bw/d) was sacrificed in a moribund state in study week 2. In addition, 1 female animal of test group 2 (150 mg/kg bw/d) was sacrificed on GD 23 because of an inability to deliver.

Clinical signs
In test group 3 (450 mg/kg bw/d), salivation after treatment was observed in study week 1 in 1 male animal and in study weeks 1, 6 and 7 in 6 female animals. Poor general state was observed in test group 3 (450 mg/kg bw/d) in study weeks 1 and 2 in 2 male animals and in study weeks 1, 6 and 7 in 2 female animals. In test group 3 (450 mg/kg bw/d), apathy was observed in study week 2 in a single male animal. Clonic convulsion was observed in test group 3 (450 mg/kg bw/d) in study week 1 in 1 male animal. The detailed clinical observations on study days 0, 7, 13, 21 and 28 in males and females and additionally on days 35, 42 and 49 in females did not reveal any additional abnormalities in animals of all test groups.

BODY WEIGHT AND BODY WEIGHT GAIN
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 this change was assessed as incidental and not related to treatment.

FOOD CONSUMPTION
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.

HEMATOLOGY
At the end of the administration period red blood cell counts (RBC), hemoglobin 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 CHEMISTRY
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 (50 mg/kg bw/d) and 2 (150 mg/kg bw/d). 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.

URINALYSIS
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.

The urine was discolored almost in all the males and females of test group 3 (450 mg/kg bw/d) from study week 1 onwards.

NEUROBEHAVIOUR
No test substance-related or spontaneous findings in male and female animals of all test groups during the home cage observation were observed. The open field observations did not reveal any test substance-related findings in male and female animals of all test groups. In sensorimotor test/assessment of reflexes, there were no test substance-related findings in male and female animals of all test groups. Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore, these observations were considered as being incidental. There were no significant deviations concerning the overall motor activity (summation of all intervals) in the male and female animals of all test groups in comparison to the concurrent control group. Regarding single intervals, in males of test groups 1 and 2 (50 and 150 mg/kg bw/d) two isolated significantly increased values were measured at interval 4. These findings were considered as being incidental since the overall motor activity was not changed and no findings were observed for female animals.

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, i.e. in decreased absolute adrenal and brain weights in females of test group 3 (450 mg/kg bw/d), in decreased absolute and relative thymus weights in males of test group 3 (450 mg/kg bw/d), in decreased absolute thymus weights in females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d), in increased relative brain and heart weights in males and females of test group 3 (450 mg/kg bw/d), in increased relative weights of adrenal glands and seminal vesicles in males of test group 3 (450 mg/kg bw/d) as well as in increased relative brain weights in females of test group 2 (150 mg/kg bw/d). The increased relative weights of heart and seminal vesicles in males of test group 2 (150 mg/kg bw/d) and the increased relative brain weight in females of test group 1 (50 mg/kg bw/d) were related to the slightly but not significantly decreased terminal body weights in these animals (-4 % and -5 %, respectively).

The increased relative kidney weights in males and females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d), the decrease of the absolute and relative ovarian weight in females of test group 3 (450 mg/kg bw/d), the increased liver weights of males and females in all test substance-treated test groups, and 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 considered to be treatment-related.

Although the relative testes weight in males of test group 3 (450 mg/kg bw/d) was only slightly but not significantly decreased (-9 %), the reduction of the absolute testes weight was assessed as treatment-related. The increase of the relative testes weight in males of test group 2 (150 mg/kg bw/d) was related to the slightly but not significantly decreased terminal body weight in these animals.

The decreased absolute and relative weights of epididymides in males of test group 3 (450 mg/kg bw/d) were related to treatment. The decrease of the absolute weight of epididymides in males of test group 2 (150 mg/kg bw/d) was considered to be incidental, because there were no histopathological correlates and the relative weights did not show significant weight changes.

GROSS PATHOLOGY
Three males of test group 3 (450 mg/kg bw/d) showed erosions or ulcers in the glandular stomach. The liver was enlarged in 3 males and 1 female of test group 2 (150 mg/kg bw/d) as well as in 3 males and 5 females of test group 3 (450 mg/kg bw/d). Four males of test group 1 (50 mg/kg bw/d) and 4 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 1 female of test group 2 (150 mg/kg bw/d) and in 2 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.

FERTILITY
At the high dose level of 450 mg/kg bw/day, the male fertility index was reduced to 11 % and the female fertility index was reducet to 10 %. Further a reduced number of implantation sites was observed, and no pups were delivered. Mating (male and female mating indices) was not influenced.
At 150 mg/kg bw/day, fertility indices were reduced to 50 % in males and females. Further a reduced number of implantation sites was observed and only one stillborn pup and no liveborn pups were delivered. Mating (male and female mating indices) was not influenced.
No impairment of fertility was observed in the 50 mg/kg bw/day dose group.

HISTOPATHOLOGY
Target organs were the kidney, testes, epididymides, ovaries, liver and spleen.
In kidneys, a multifocal degeneration was observed in proximal tubular cells at the transition of cortex to medulla in males of all treatment groups as well as in females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). The severity increased dose-dependently. The tubular degeneration resulted in increased kidney weights in males and females of test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d). The occurrence of tubular degeneration in kidneys was related to treatment and assessed as an adverse effect. In addition, 9/10 females of test group 3 (versus two control females) showed a mostly minimal or slight multifocal mineralization in the papillae. A treatment-related effect could not be ruled out, but was considered to be non-adverse.
In the testes, a diffuse tubular degeneration was observed in 7 males (minimal) of test group 2 (150 mg/kg bw/d) and in all males (mostly moderate or severe) of test group 3 (450 mg/kg bw/d). The tubular degeneration was associated with the decreased absolute weight of testes in males of test group 3 (450 mg/kg bw/d). The occurrence of diffuse tubular degeneration in testes was considered to be treatment-related and adverse. In 9 males of test group 3 (450 mg/kg bw/d), the diffuse tubular degeneration of testes caused a mostly severe oligospermia in the epididymides. The oligospermia was linked to the decreased absolute and relative weights of epididymides in these males.
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) showing dose-response relationship in incidence and severity. In addition, all females of test group 3 (450 mg/kg bw/d) showed ovarian cysts. The occurrence of cysts and vacuolization of sex cord stroma was related to treatment and was considered to be adverse. Although there were no clear histopathological correlates for the decreased absolute and relative ovarian weights in females of test group 3 (450 mg/kg bw/d), a test substance-related effect could not be ruled out. 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 hematopoiesis occurred in males and females of test groups 2 (150 mg/kg bw/d) and 3 (450mg/kg bw/d). In addition, in females of these test groups the severity of hemosiderin storage was increased. These findings were 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 3 males and 1 female of test group 2 (150 mg/kg bw/d) as well as in 3 males and 5 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 5 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 2 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.
In test group 3 (450 mg/kg bw/d), erosions or ulcers occurred in the forestomach of 3 males and of 1 female. In the glandular stomach, erosions/ulcers were observed in 3 males of test group 3 (450 mg/kg bw/d) in comparison to 1 control male. A test substance related effect could not be ruled out.
Intrasinusoidal erythrocytes (sinus erythrocytosis) occurred in the mesenteric lymph nodes of 1 female (minimal) in test group 2 (150 mg/kg bw/d) and of 1 male and 3 females (slight) in test group 3 (450 mg/kg bw/d). Intrasinusoidal erythrocytes (sinus erythrocytosis) can result from a lymph node, which drains a region of hemorrhage. This can also be an artifact that results from tissue dissection during necropsy. In this study, 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.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
VIABILITY
The viability index as indicator for pup mortality between PND (postnatal day) 0-4 was 100 % for test groups 0 (control) and 1 (50 mg/kg bw/d). No viable pups were observed in test groups 2 (150 mg/kg bw/d) and 3 (450 mg/kg bw/d).

PUP NUMBER AND STATUS AT DELIVERY
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.

SEX RATIO
The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between test groups 0 (control) and 1 (50 mg/kg bw/d).

CLINICAL SIGNS
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, 50 mg/kg bw/d) one pup showed a papilloma-like skin flap. This single observation was considered to be spontaneous in nature and not to be adverse.

BODY WEIGHT
Mean pup body weights/pup body weight changes of all pups in test group 1 (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.

GROSS PATHOLOGY
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.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified