Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
fertility, other
Remarks:
based on repeated dose 90-day oral toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-10-09 to 2015-02-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998-09-21
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2014-05-14
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Cobalt dichloride hexahydrate
- State of aggregation: solid, lilac crystals
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: to be stored cool and well-ventilated in a closed container, preferably under inert atmosphere.

Test animals

Species:
rat
Strain:
other: CD
Details on species / strain selection:
The rat was selected because of its proven suitability in toxicology studies and to comply with regulatory requirements for testing in a rodent animal species.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at first dosing: males: 52 days; females: 65 days
- Weight at first dosing: males: 244.9 - 295.5 g; females: 204.1 - 246.5 g
- Housing: animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages.
- Diet (ad libitum): commercial ssniff®-R/M-H V1534 (ssniff® Spezialdiäten GmbH, 59494 Soest, Germany); food residue was removed and weighed.
- Water (ad libitum): drinking water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The administration formulations were freshly prepared every day by dissolving the test item in the vehicle to the appropriate concentrations.
Administration volume: 2 mL/kg bw/day
The dose of the test item was adapted to the animal's body weight daily up to and including test week 6, and weekly thereafter.
The control animals received the vehicle at a constant volume of 2 mL/kg bw/day orally once daily in the same way.
Details on mating procedure:
not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the administration formulations, samples of approximately 10 mL were taken at the following times and stored at -20°C or colder until analyses:
1) At study initiation (on the first administration day of male animals):
- analysis of stability and concentration: immediately after preparation of the formulations as well as after 8 and 24 hours storage of formulations at room temperature (3 samples/test item group).
- homogeneity: at the start of administration, during (middle) administration and before administration to the last animal of the test item group (3 samples/test item group).

2) At study termination (on the last administration day of female animals):
- analysis of concentration: during treatment always before administration to the last animal of the group (1 sample/test item group).
The determination of the content of the test item cobalt dichloride hexahydrate in samples was performed by analysis of cobalt with Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES).

Results:
The generated results verify the concentration, the homogeneity and the stability of the test item cobalt dichloride hexahydrate in application mixtures during the toxicology study. The actual cobalt dichloride hexahydrate concentrations ranged from 101.4% to 102.1% of the nominal concentrations.
Duration of treatment / exposure:
90 days (except male recovery animals: 91 days)
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
3 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Main study (per group): 10 males/10 females
Recovery group (control group and 30 mg/kg bw/day dose group only; per group): 5 males/5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels for this study have been selected based on available data.
- Recovery groups were included in this study. One recovery group was included for the control group and other recovery group for the 30 mg/kg bw/day dose group. These groups were kept for 28 days after the treatment period without receiving the test item.
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes (main study animals and recovery animals)
- Time schedule:
Clinical signs: before and after dosing at each time of dosing as well as regular daily
Mortality: twice daily
- Cage side observations (included): skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns.

DETAILED CLINICAL OBSERVATIONS: Yes (main study animals and recovery animals)
- Time schedule: once before the first exposure and once a week thereafter
- Observations (included): skin, fur, eyes, mucous membranes, occurrence of secretions, excretions, autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour.

BODY WEIGHT: Yes (main study animals and recovery animals)
- Time schedule for examinations: at the time of group allocation, on the day of commencement of treatment and once a week thereafter throughout the experimental period.

FOOD CONSUMPTION AND COMPOUND INTAKE (main study animals and recovery animals):
- Food consumption for each animal determined and relative food consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes (main study animals and recovery animals)
- Time schedule for examinations: daily

HORMONE LEVELS:
Blood was preferably collected by puncture of the vena jugularis under light ether anaesthesia as follows (all main study and recovery animals):
- predose (before the first administration; all main study and recovery animals)
- during study conduct, at the end of test week 6 (all main study and recovery animals)
- at the end of test week 13 (before necropsy; all main study and recovery animals)
- at the end of the recovery period (before necropsy; all recovery animals)
The following parameters of all animals of the control group and the 30 mg/kg bw/day dose group were examined: testosterone, progesterone, and 17β-estradiol
Oestrous cyclicity (parental animals):
The stages of the oestrous cycle observed were recorded individually for each female rat (all females of the main study group and recovery group)
Time schedule:
- before the first administration (test week 0; monitoring duration: 7 days)
- during study conduct (test weeks 5/6; monitoring duration: 12 days)
- at the end of the treatment period (test weeks 12/12 before necrospy of main study animals; monitoring duration: 12 days)
- at the end of the recovery period (test weeks 16/17 before necropsy of recovery animals; monitoring duration: 12 days)
Sperm parameters (parental animals):
Detailed histopathological examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure) of all male main study and recovery of the control group and the 30 mg/kg bw/day group following staining.
Litter observations:
not applicable
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes (main study animals and recovery animals)
On test day 91, the main study animals were dissected following a randomisation scheme. Animals not dissected on test day 91 were dosed until one day before sacrifice. Necropsy of all animals allocated to the recovery period was performed on test day 119.
The animals were euthanized under ether atmosphere, exsanguinated, weighed, dissected and inspected macroscopically. All superficial tissues were examined visually and by palpation and the cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

ORGAN WEIGHTS: Yes (main study animals and recovery animals)
The weights of the following organs of all animals were determined: adrenal gland (2), liver, thymus, brain, ovary (2), prostate and seminal vesicles with coagulating glands as a whole, epididymis (2), pancreas, heart, spleen, uterus (incl. cervix), kidney (2), and testicle (2).
Paired organs were weighed individually and identified as left or right.

HISTOPATHOLOGY: Yes (main study animals and recovery animals)
The following organs or parts of organs with the exception of the eyes, epididymides and testicles of all animals were fixed in 7% buffered formalin. The eyes were preserved in Davidson’s solution for optimum fixation. The epididymides and testicles were preserved in Bouin’s fixative.
Organs: adrenal gland (2), aorta abdominalis, bone (os femoris with joint), bone marrow (os femoris), brain (3 levels: cerebrum, cerebellum, medulla/pons), epididymis (2), eye with optic nerve (2), gross lesions observed, heart (3 levels: right and left ventricle, septum), intestine, large (colon, rectum), small intestine (duodenum, jejunum, ileum, incl. Peyer´s patches; Swiss roll method), kidney and ureter (2), liver, lungs (with mainstem bronchi and bronchioles (preserved by inflation with fixative and then immersion)), lymph node (1, cervical), lymph node (1, mesenteric), mammary gland, muscle (skeletal, leg), nerve (sciatic), oesophagus, ovary (2), pancreas, pituitary, prostate and seminal vesicles with coagulating glands, salivary glands (mandibular, sublingual and parotid gland), skin (left flank), spinal cord (3 levels: cervical, mid-thoracic, lumbar), spleen, stomach, testicle (2), thymus, thyroid (2) (incl. parathyroids), tissue masses or tumours (including regional lymph nodes), trachea (incl. larynx), urinary bladder, uterus (incl. cervix), and vagina.

The afore-listed organs of all main study and recovery animals of the control group and the 30 mg/kg bw/day group were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. In addition, frozen sections of the heart, liver and one kidney were made, stained with Oil Red O and examined microscopically.
Detailed histopathological examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure) of all male main study and recovery of the control group and the 30 mg/kg bw/day group following staining.
The organs and tissues listed above were examined.
Postmortem examinations (offspring):
not applicable
Statistics:
The test item-treated groups (3, 10, and 30 mg/kg bw/day dose groups) were compared with the control group.
The following statistical methods were used:
- Multiple t-test based on DUNNETT, C. W. New tables for multiple relative and absolute organ weights comparisons with a control Biometrics, 482 - 491 (September 1964): body weight, food consumption, relative and absolute organ weights (p ≤ 0.05 and p ≤ 0.01)
- STUDENT's t-test: all numerical functional tests: body temperature; hormone levels (p ≤ 0.05 and p ≤ 0.01)
- Exact test of R. A. FISHER: histopathology (p ≤ 0.05)
These statistical procedures were used for all data.
Reproductive indices:
not applicable
Offspring viability indices:
not applicable

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1) Treatment period:
- 10 mg/kg bw/day: body weight of the male animals was reduced by 5 to 7% from test day 50 onwards (not statistically significant). Body weight gain changed accordingly. The body weight at autopsy was reduced by 6% (not statistically significant) for the male animals. No changes were noted for the female animals.
- 30 mg/kg bw/day: body weight of the animals was reduced by 5 to 14% from test day 8 onwards for the males (statistically significant at p ≤ 0.05 or p ≤ 0.01 on test days 8, 22, and 43 to 90) and by 5 to 10% from test day 29 onwards for the females (statistically significant at p ≤ 0.05 or p ≤ 0.01 on test days 50 to 90), respectively, compared to the control group. Body weight gain changed accordingly. The body weight at autopsy was reduced by 11% for the males (statistically significant at p ≤ 0.05) and by 9% for the females (not statistically significant), respectively.
- the reduced body weights at the 10 mg/kg bw/day and 30 mg/kg bw/day dose groups are considered as test item-related.

2) Recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- the differences in body weight between the animals previously treated with 30 mg/kg bw/day and the control group were still present at the end of the recovery period: The body weight of the male and female animals was still reduced by 17% or by 13%, respectively, on test day 118 (statistically significant reductions at p ≤ 0.05 on test day 97 for the males and at p ≤ 0.01 on test days 97 to 118 for the females) compared to the control group.
- the male animals revealed a slightly higher body weight gain than the control group during the recovery period indicating a trend towards recovery, while the body weight gain of the females was in the range of the control group. The body weight at autopsy was reduced by 17% for the males and by 13% for the females (statistically significant at p ≤ 0.01 for the females), respectively.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
1) Treatment period:
- 10 or 30 mg/kg bw/day: the following test item-related changes in haematological parameters were noted for the male and female animals on test day 91 (male and female main study and female recovery animals) and 92 (male recovery animals). In general, the male animals were affected to a higher degree.
10 mg/kg bw/day (test day 91/92 (combined)):
haemoglobin content (males: +11%; p≤0.01)
erythrocytes (males: +10%; p≤0.01)
haematocrit value (males: +12%; p≤0.01)
reticulocytes (males: -33%; p≤0.05)
platelets (males: -13%)
mean corpuscular volume (females: +4%; p≤0.05);

30 mg/kg bw/day (test day 91/92 (combined)):
haemoglobin content (males: +25%; females: +14%; p≤0.01)
erythrocytes (males: +19%; females: +11%; p≤0.01)
haematocrit value (males: +23%; females: +14%; p≤0.01)
thromboplastin time (males: +7%; p≤0.01)
activated partial thromboplastin time (males: +8%; p≤0.05)
mean corpuscular volume (males: +4%; females: +3%; p≤0.05 (males only))
mean corpuscular haemoglobin (males: +5%; p≤0.01)
reticulocytes (males: -24%)
platelets (males: -26%; females: -12%; p≤0.01 (males only))
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment period:
- 10 or 30 mg/kg bw/day: the following test item-related changes in biochemical parameters were noted for the male and female animals on test day 91 (male and female main study and female recovery animals) and 92 (male recovery animals):
10 mg/kg bw/day (test day 91):
bilirubin (males: +17%)

10 mg/kg bw/day (test day 91/92 (combined)):
bilirubin (males: +14%)

30 mg/kg bw/day (test day 91):
bilirubin (males: +34%; p≤0.01; females: +16%; p≤0.05)

10 mg/kg bw/day (test day 91/92 (combined)):
bilirubin (males: +29%; p≤0.01; females: +16%; p≤0.05)
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment and recovery period (full histopathological evaluation restricted to the control group and 30 mg/kg bw/day group):
- microscopic evaluation revealed test item-related changes in the bone marrow (erythroid hyperplasia) of the femur. There was a significant and test item-related increase for erythroid hyperplasia in the bone marrow of the male and female animals treated with 10 or 30 mg/kg bw/day compared to the controls:
4 of 10 males and 7 of 10 females in the 10 mg/kg bw/day dose group and 7 of 10 animals for both sexes in the 10 mg/kg bw/day dose group versus 0 of 10 in the 3 mg/kg bw/day dose group and controls. The bone marrow change (erythroid hyperplasia) attained statistical significance in animals of the 10 or 30 mg/kg bw/day dose groups for both sexes. After cessation of treatment, no test item-related changes were observed for the recovery animals anymore.
- all other microscopic changes seen in all organs in all animals were either coincidental, or were considered to lie within the normal range of background alterations, which may be seen in untreated rats of this age and strain.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not examined

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
1) Treatment period:
- no test item-related changes in behaviour or external appearance were noted for the male and female animals treated with 3, 10 or 30 mg/kg bw/day.
- 10 mg/kg bw/day: one male showed a haemorrhagic left canthus on test days 26 to 32 and one female showed a reddened right eyelid and/or a haemorrhagic canthus on test days 33 to 53.
- 30 mg/kg bw/day: pilo-erection was noted for 2 male recovery animals on test days 30 to 36. No changes in behaviour or external appearance were noted for the female animals.
- these findings are not considered to be test item-related due to the low number of animals affected.
- faeces of all animals were of normal consistency.
- no deaths were noted at any dose level. All main study animals survived until their scheduled terminal sacrifice.
- none of the animals treated with 3, 10 or 30 mg/kg bw/day revealed any test item-related changes in external appearance, body posture, movement and coordination capabilities, or behaviour at the detailed clinical observations.

2) Recovery period (restricted to the control group and 30 mg/kg bw/day group):
- no abnormalities in behaviour, external appearance or faeces were observed for the male and female animals previously treated with 30 mg/kg bw/day.
- no deaths were noted.
All recovery animals survived until the scheduled recovery sacrifice.
- none of the animals revealed any test item-related changes in external appearance, body posture, movement and coordination capabilities, or behaviour at the detailed clinical observations.

BODY WEIGHT AND WEIGHT GAIN
1) Treatment period:
- 3 mg/kg bw/day: body weight, body weight gain and body weight at autopsy were not influenced in the male and female animals in a test item-related way compared to the control group.

FOOD CONSUMPTION AND COMPOUND INTAKE
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- no test item-related influence was noted on the relative food consumption of the male and female animals treated with 3, 10 or 30 mg/kg bw/day during the treatment period and of the male and female animals previously treated with 30 mg/kg bw/day during the recovery period compared to the control group.
- slight but statistically significant (at p ≤ 0.05) increases in food consumption noted for the 30 mg/kg bw/day dosed males and females in test week 4 and for the 30 mg/kg bw/day dosed male animals in test week 14 are considered to be due to the reduced body weight and to be without any biological relevance.

WATER CONSUMPTION AND COMPOUND INTAKE
- visual appraisal of the drinking water consumption did not reveal any test item-related differences between the test item-treated animals and the control animals throughout the treatment and the recovery period.

OPHTHALMOSCOPIC EXAMINATION
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- no changes of the eyes and the optic region, i.e. adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body and fundus were noted in the male and female rats of the animals treated with 3, 10 or 30 mg/kg bw/day at the end of the treatment period.
- no changes were noted for the male and female rats previously treated with 30 mg/kg bw/day at the end of the recovery period.

HAEMATOLOGY
1) Treatment period:
- 3 mg/kg bw/day: no test item-related influence on haematological parameters was noted for the male and female animals at the end of the treatment period.
- no test item-related influence was noted for the number of leucocytes, the relative and absolute differential blood count, and the mean corpuscular haemoglobin concentration.
- statistically significant differences in haematological parameters compared to the control which are not considered to be test item-related were found in the following parameters: leucocytes, absolute lymphocytes, absolute eosinophilic granulocytes, absolute large unstained cells, and absolute basophilic granulocytes

2) Recovery period (restricted to the control group and 30 mg/kg bw/day group):
- all changes in haematological parameters previously observed after repeated treatment with 30 mg/kg bw/day had subsided after 4 weeks of recovery.
- no effects related to the previous treatment were observed on the haemoglobin content, the numbers of erythrocytes, leucocytes and platelets, the relative reticulocyte count), the haematocrit value, the relative and absolute differential blood count, the thromboplastin time, the activated partial thromboplastin time, the mean corpuscular volume, the mean corpuscular haemoglobin and the mean corpuscular haemoglobin concentration at the end of the recovery period.
- statistically significant differences in haematological parameters compared to the control which are not considered to be test item-related were found for the following parameters: absolute eosinophilic granulocytes

Please also refer for results about haematology to "Attached background material" below.

CLINICAL CHEMISTRY
1) Treatment period:
- 3 mg/kg bw/day: no test item-related influence on biochemical parameters was noted for the male and female animals at the end of the treatment period.
- 10 and 30 mg/kg bw/day:
- no test item-related influence was noted for the albumin/globulin ratio, the plasma levels of albumin, globulin, cholesterol, creatinine, glucose, protein (total), triglycerides, urea, calcium, chloride, potassium and sodium and the serum level of bile acids. Further, the plasma activity of alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase and lactate dehydrogenase was not influenced.
- statistically significant differences in biochemical parameters compared to the control which are not considered to be test item-related were found in the following parameters: albumin, globulin, albumin/globulin ratio, cholesterol, creatinine, glucose, protein, triglycerides, calcium, chloride, and alkaline phosphatase

2) Recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- all changes in biochemical parameters previously observed after repeated treatment with 30 mg/kg bw/day had subsided after 4 weeks of recovery.
- no effects related to the previous treatment were noted for the albumin/globulin ratio, the plasma levels of albumin, globulin, bilirubin, cholesterol, creatinine, glucose, protein (total), triglycerides, urea, calcium, chloride, potassium and sodium, and the serum level of bile acids. Further, the plasma activity of alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, and lactate dehydrogenase was not influenced.

URINALYSIS
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- 3, 10 or 30 mg/kg bw/day: no test item-related influence on the urinary status was noted for the male and female animals at the end of the treatment period.
- no test item-related influence on the urinary status was noted for the male and female animals previously treated with 30 mg/kg bw/day at the end of the recovery period.
- no test item-related changes were noted for the specific gravity of the urine, the pH value of the urine and the urine volume. The analyte concentrations of nitrite, protein, glucose, ketones, urobilinogen, bilirubin and haemoglobin were not influenced in male and female animals. No test item-related changes were observed in the urine colour and the microscopically analysed urine sediments.
- statistically significant differences in urine parameters compared to the control which are not considered to be test item-related were found in the following parameter: pH

NEUROBEHAVIOUR
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- neurological screening performed at the end of the treatment period on test day 86 and at the end of the recovery period on test day 118 did not reveal any test item-related influence on the male and female rats treated with 3, 10 or 30 mg/kg bw/day, neither on any of the parameters examined during the functional observation tests nor on the fore- and hind limb grip strength or on the spontaneous motility.
- statistically significant differences in neurological parameters compared to the control which are not considered to be test item-related were found in the following parameters: body temperature, forelimb grip strength, and hindlimb grip strength

ORGAN WEIGHTS
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- 3, 10 or 30 mg/kg bw/day: no test item-related changes in relative and absolute organ weights were noted for the male and female rats at the end of the treatment period.
- no test item-related changes were noted for the male and female rats previously treated with 30 mg/kg bw/day at the end of the recovery period.
- statistically significant differences in relative and absolute organ weights compared to the control which are not considered to be test item-related were found in the following parameters: brain (relative), gonads (left testis, relative), spleen (relative), adrenal (left, absolute), brain (absolute), kidney (left, absolute), kidney (right, absolute), and gonads (right ovary, absolute)

GROSS PATHOLOGY
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- 3, 10 or 30 mg/kg bw/day: no test item-related changes were noted for the male and female rats at the end of the treatment period.
- no test item-related changes were noted for the male and female rats previously treated with 30 mg/kg bw/day at the end of the recovery period.
- there were no test item-related abnormalities (gross pathology, tissue masses or tumours) in any tissue, including the adrenal gland, kidney and pancreas, in any of the exposed animals at the end of the treatment period, nor at the end of the recovery period.
- macroscopic changes were noted in the kidney (cyst), spleen (rough surface, adhered to peritoneum), stomach (haemorrhagic foci), uterus (cystic, filled with clear liquid) and testis, epididymis, seminal vesicle and prostate (reduced in size) in individual animals of the control and test item-treated groups at terminal or recovery sacrifice.
- these changes are considered to be incidental findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment and recovery period (full histopathological evaluation restricted to the control group and 30 mg/kg bw/day group):
- there were no histopathological findings in any tissues, including the adrenal gland, kidney and pancreas, in any of the animals exposed p.o. to 30 mg/kg bw/day.

BONE MARROW EXAMINATION
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group).
- 30 mg/kg bw/day: no test item-related changes in the myeloid:erythroid ratio were noted for the male and female rats at the end of the treatment period.
- no test item-related changes were noted for the male and female rats previously treated with 30 mg/kg bw/day at the end of the recovery period.
- the slightly decreased myeloid:erythroid ratio (statistically significant at p ≤ 0.05) noted for the previously high dosed females at the end of the recovery period is considered to be in the normal range of variation and without any biological relevance.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

not applicable

Effect levels (F1)

Generation:
F1
Remarks on result:
not measured/tested

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOAEL (fertility): 30 mg/kg bw/day
No test item-related changes were noted on oestrous cycles. Furthermore, histopathological examination performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure), did not reveal any test item-related effects. Lastly, no effects on the hormone levels of the male and female rats were observed.