Cobalt hydroxide oxide

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Remarks:

based on a 90 day repeated dose toxicity study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-07-03 to 2014-11-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)

Version / remarks:

1998-09-21

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2014-05-14

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

tricobalt tetraoxide

IUPAC Name:

tricobalt tetraoxide

Constituent 2

Reference substance name:

Tricobalt tetraoxide

EC Number:

215-157-2

EC Name:

Tricobalt tetraoxide

Cas Number:

1308-06-1

Molecular formula:

Co3O4

IUPAC Name:

tricobalt tetraoxide

Constituent 3

Reference substance name:

1308-06-1

Cas Number:

1308-06-1

IUPAC Name:

1308-06-1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Cobalt Oxide 72/73F
- Molecular formula: Co3O4
- Physical state: grey-black powder
- Storage condition of test material: cool and well-ventilated in a tightly closed container.
- Surface area (BET): 3.1 m²/g
- Particle size distribution: D10: 0.4 μm; D50: 1 μm; D90: 4 μm

Test animals

Species:

rat

Strain:

other: CD

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, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at first dosing: males and females: 52 days
- Weight at first dosing: males: 269.0 - 321.5 g; females: 183.3 - 227.8 g
- Housing: animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm × 23 cm and a height of approx. 18 cm; Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material.
- Diet (ad libitum): commercial diet (ssniff® R/M-H V1534, ssniff Spezialdiäten GmbH, 59494 Soest, Germany); food residue was removed and weighed.
- Water (ad libitum): tap 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:

other: 0.5% hydroxypropyl methylcellulose gel

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle to the appropriate concnetrations. The administration formulations were freshly prepared daily.
Administration volume: 5 mL/kg bw/day

The dose of the test item was adjusted to each animal's body weight daily up to and including test week 6, and once weekly thereafter.
The control animals received the vehicle orally at a constant volume in the same way once daily.

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 test item-vehicle mixtures, 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 concentration: immediately after preparation of the formulations as well as after 8 and 24 hours at room temperature (3 samples/test item group).
- homogeneity: at the start of administration, during administration (in the middle), and before administration to the last animal of each dose 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 tricobalt tetraoxide in samples was performed by analysis of cobalt with Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES).

Results:
The results of the analysis show that the test item formulations were correctly prepared. The actual tricobalt tetraoxide concentrations in the respective test item formulations ranged from 95.6 % to 103.0 % of the nominal concentrations before administration to the last male animal of the group on test day 1, and from 87.5 % to 101.7 % of the nominal concentrations before administration to the last female animal of the group on test day 90 (last administration day of the study).

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 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 1000 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 were selected taking into account any existing toxicity and toxicokinetic data available for the test item at the time of initiation of the study.
- Recovery groups were included in this study. One recovery group was included for the control group and other recovery group for the 1000 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 and 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 (e.g. self-mutilation, walking backwards).

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

FOOD CONSUMPTION (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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

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

OPHTHALMOSCOPIC EXAMINATION: Yes (main study animals and recovery animals)
- Time schedule for examinations: prior to the start of administration and at main study termination (all main study and recovery animals), and at the end of the recovery period (all recovery animals)(before blood sampling for laboratory examinations)
- Parameters examined: adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body, and fundus.
Prior to examination, mydriasis was produced after instillation of MYDRUM® eye drops into the conjunctival sacs.

HAEMATOLOGY: Yes (main study animals and recovery animals)
- Time schedule for collection of blood: at the end of the treatment period (test day 91) and at the end of the recovery period (all recovery animals; test day 119)
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes
- How many animals: all main study animals and all recovery animals
- Parameters examined: haemoglobin content, erythrocytes, leucocytes, absolute and relative differential blood count (neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes, monocytes and large unclassified cells), reticulocytes, platelets, haematocrit value, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, thromboplastin time, and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes (main study animals and recovery animals)
- Time schedule for collection of blood: at the end of the treatment period (test day 91) and at the end of the recovery period (all recovery animals; test day 119)
- Animals fasted: Yes
- How many animals: all main study animals and all recovery animals
- Parameters examined: albumin, globulin, albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), triglycerides, urea (in blood), calcium, chloride, potassium, sodium, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, and lactate dehydrogenase

URINALYSIS: Yes (main study animals and recovery animals)
- Time schedule for collection of urine: at the end of the treatment period (all main study animals; test day 89) and at the end of the recovery period (all recovery animals; test day 117)
- Animals fasted: Yes
- Parameters examined: colour, turbidity, volume, pH, specific gravity, protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin, nitrite, and microscopic examinations of urine samples (epithelial cells, leucocytes, erythrocytes, organisms, crystalluria, and further constituents (i.e. sperm, casts))

NEUROBEHAVIOURAL EXAMINATION: Yes (main study animals & recovery animals)
- Time schedule for examinations: week 13 (main study groups) and week 17 (recovery groups)
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity / grip strength / motor activity

1) Observational screening:
Righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, pilo-erection, diarrhoea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire manoeuvre, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation, and auditory function

2) Functional tests: grip strength and locomotor activity

HORMONE LEVELS:
Blood was collected by puncture of the vena jugularis under light isoflurane 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, test day 42 (all main study and recovery animals)
- at the end of test week 13 (before necropsy, test day 91; all main study and recovery animals)
- at the end of the recovery period (before necropsy, test day 119; all recovery animals)
The following parameters of all animals of the control group and the 1000 mg/kg bw/day dose group were examined: testosterone, progesterone, and 17 beta-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:
- pre-dose (before the first administration) (monitoring duration: 7 days)
- during study conduct (test weeks 5/6; monitoring duration: 12 days)
- at the end of the treatment period (test weeks 12/13 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 animals of the control group and the 1000 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, all main study animals were dissected following a randomisation scheme. Necropsy of all animals allocated to the recovery period was performed on test day 119.
The animals were euthanized by carbon dioxide, 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), 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 of all animals were fixed in 7% buffered formalin. The eyes were preserved in Davidson’s solution and the testes in Bouin’s solution for optimum fixation.

Organs: adrenal gland (2), aorta abdominalis, bone (os femoris with joint), bone marrow (os femoris), brain (cerebrum, cerebellum, brain stem), epididymis (2), eye with optic nerve (2), gross lesions observed, heart (right and left ventricle, septum), large intestine (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 and oviducts (2), pancreas, pituitary, prostate and seminal vesicles with coagulating glands, salivary glands (mandibular, sublingual and parotid gland), skin (left flank), spinal cord (3 sections), 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 1000 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 prepared and stained with Oil Red O and examined microscopically.
A 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 animals of the control group and the 1000 mg/kg bw/day group following staining.

Postmortem examinations (offspring):

not applicable

Statistics:

Means and standard deviations were calculated on time-point specific data sets for each sex separately.
The test item-treated groups (100, 300, and 1000 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 Comparisons with a control. Biometrics, 482-491 (Sept 1964): body weight, food consumption, absolute and relative organ weights (p ≤ 0.05 and p ≤ 0.01)
- exact test of R. A. FISHER: histology (p ≤ 0.05)
- STUDENT's t-test: all numerical functional tests: body temperature, hormone levels (p ≤ 0.05 and p ≤ 0.01)

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 specified

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

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

Clinical biochemistry findings:

no effects observed

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:

no effects observed

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 (PARENTAL ANIMALS)
1) Treatment period:
- 100, 300 or 1000 mg/kg bw/day: none of the male and female rats treated orally with revealed any test item-related changes in behaviour or external appearance.
- 1000 mg/kg bw/day: two female animals revealed an exophthalmus from test day 79 onwards. This is considered to be a coincidental finding that is not test item-related.
- the faeces of all test item-treated male and female animals were dark stained from test day 48 onwards for the male animals and from test day 41 onwards for the female animals. The intensity of the staining increased with the dose level. The dark staining of the faeces was not recorded before test day 41/48 as only from these time points onwards a clear difference in the colour of the faeces compared to the control animals was evident for all test item-treated animals. The dark staining of the faeces is considered to be related to the black-grey colour of the test item and not to be a toxicological effect. The consistency of all animals' faeces was normal throughout the treatment period.
- no deaths were noted at any dose level.
- all main study animals survived until their scheduled terminal sacrifice.
- detailed clinical observations: none of the animals treated with 100, 300 or 1000 mg/kg bw/day revealed any test item-related changes in external appearance, body posture, movement and coordination capabilities, or behaviour as assessed in test weeks 1 to 13 (all groups).
- detailed clinical observations: all parameters of all animals scheduled for the control or treatment groups were in the normal range at pre-dose examination in test week -1. One male animal treated with 1000 mg/kg b.w./day revealed a haemorrhagic nose in test week 12. Furthermore, one female animal treated with 1000 mg/kg bw/day revealed a movable solid palpable mass with a diameter of approximately 8 to 20 mm near the second mammary gland from test week 9 onwards. Lastly, another female 1000 mg/kg bw/day dose animal revealed areas with thin fur (size approximately 10 mm × 30 mm) on both forelimbs in test weeks 10 to 13. All findings noted are considered as spontaneous changes that are not related to the test item due to the single occurrence in each case.

2) Recovery period (restricted to the control group and 1000 mg/kg bw/day group)
- 1000 mg/kg bw/day: no abnormalities in behaviour or external appearance were observed for the treated male and female animals during the recovery period. Furthermore, the exophthalmus of the female recovery animal previously treated, that is considered not test item-related, was still present until the end of the recovery period. Lastly, the faeces of the male and female animal previously treated was still dark stained until test day 95. From test day 96 onwards, the dark staining of the faeces had subsided and the colour had returned to normal. The consistency of all animals' faeces was normal throughout the recovery period.
- no deaths were noted during the recovery period. All recovery animals survived until the scheduled recovery sacrifice.
- detailed clinical observations: none of the animals treated with 100, 300 or 1000 mg/kg bw/day revealed any test item-related changes in external appearance, body posture, movement and coordination capabilities, or behaviour as assessed in test weeks 14 to 17 (control group and 1000 mg/kg bw/day group only).

BODY WEIGHT (PARENTAL ANIMALS)
1) Treatment period
- 100 or 300 mg/kg bw/day: no test item-related changes were noted for the male and female animals.
- 1000 mg/kg bw/day (males only): body weight of the male animals was slightly reduced by up to 10% compared to the control group as of test day 22 (p ≤ 0.05 or p ≤ 0.01). The body weight gain was reduced by up to 17 percentage points in comparison to the control. The body weight at autopsy of the male animals was reduced by 12% on test day 91 (p ≤ 0.01).
- 1000 mg/kg bw/day (females only): body weight of the female animals was only marginally reduced by up to 5% compared to the control group as of test day 64 (not statistically significant). The body weight gain and the body weight at autopsy were similarly only slightly reduced compared to the control group.
- reduced body weights at the 1000 mg/kg bw/day dose are considered as test item-related.

2) Recovery period (restricted to the control group and 1000 mg/kg bw/day group)
- slight differences in body weight between the animals previously treated with 1000 mg/kg bw/day and the control group had nearly completely subsided at the end of the treatment period in both the male and female animals.
- animals previously treated with the 1000 mg/kg bw/day dose revealed a higher body weight gain than the control group during the recovery period.
- no noteworthy difference was noted for the body weight at autopsy between the animals previously treated with the 1000 mg/kg bw/day group dose and the control group at recovery sacrifice.

FOOD CONSUMPTION (PARENTAL ANIMALS)
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 100, 300 or 1000 mg/kg b.w./day: no test item-related influence was observed on the food consumption of the male and female animals compared to the control animals throughout the treatment period and the recovery period.
- 1000 mg/kg bw/day: food consumption of the male animals appeared to be slightly increased by 5% compared to the control group in test week 5 (period of test days 29 to 36, p ≤ 0.05). This effect is considered to be due to the reduced body weight of these male animals.
- test item-treated female animals appeared to reveal a statistically significant (at p ≤ 0.05 or p ≤ 0.01) increase in the food consumption of up to 9% in test weeks 3, 4 and/or 5 at all dose levels. This is considered to be a coincidental effect as the food intake of the female animals was generally slightly higher in all test item-treated groups than in the control group during the first six test weeks, but no dose-response relationship was noted.
- statistically significant differences in food consumption compared to the control group that are not considered to be test item-related are as follows: increased relative food consumption
- relatively low food consumption noted for the control and the high dose group in test week 17 of the recovery period (test day 111 to test day 118) is due to the overnight fasting of the animals before urine collection on test day 117.

WATER CONSUMPTION (PARENTAL ANIMALS)
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- no 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 1000 mg/kg bw/day group):
- 100, 300 or 1000 mg/kg bw/day: no test item-related changes of the eyes and the optic region were observed in any animal neither at the end of treatment nor at the end of the 4-week recovery.

HAEMATOLOGY
1) Treatment period
- 100 mg/kg bw/day: no test item-related influence was observed on any of the haematological parameters at the end of the treatment period.

- 300 mg/kg bw/day (males only):
haemoglobin (+9%; p≤0.01)
erythrocytes (+10%; p≤0.01)
platelets (-10%)
haematocrit (+9%; p≤0.01)

- 1000 mg/kg bw/day:
haemoglobin (males: +26%; females: +14%; p≤0.01)
erythrocytes (males: +23%; females: +11%; p≤0.01)
platelets (males: -32%; females: -13%; p≤0.01 (males only))
haematocrit (males: +25%; females: +13%; p≤0.01)

- no test item-related effects were observed for the number of leucocytes, the relative reticulocyte count, 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 treatment period (test day 91).

2) Recovery period (restricted to the control group and 1000 mg/kg bw/day group)
- 1000 mg/kg bw/day: all changes in haematological parameters previously observed after repeated treatment had subsided after 4 weeks of recovery (test day 119, day 28 of the 4-week recovery period).
- no test item-related effects were observed on the haemoglobin content, numbers of erythrocytes, leukocytes and platelets, relative reticulocyte count, haematocrit value, relative and absolute differential blood count, thromboplastin time, activated partial thromboplastin time, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration at the end of the recovery period (test day 119).
- statistically significant differences in haematological parameters compared to the control animals on test day 91 (end of treatment) or test day 119 (end of recovery) that are not considered to be test item-related are as follows:
Treatment period (1000 mg/kg bw/day):
decreased absolute eosinophilic granulocytes (males; p ≤ 0.05)
decreased absolute large unclassified cells (males; p ≤ 0.05)
increased mean corpuscular haemoglobin (females; p ≤ 0.05)

Recovery period (1000 mg/kg bw/day):
decreased reticulocytes (males; p ≤ 0.01)
decreased absolute large unclassified cells (males; p ≤ 0.05)

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

CLINICAL CHEMISTRY
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 100, 300 or 1000 mg/kg bw/day: no test item-related influence was noted on the biochemical parameters of the male and female animals at the end of the treatment period and at the end of the recovery period. All data are considered to be within the limits of normal biological variability.
- statistically significant changes in biochemical parameters in comparison to the control group listed as follows are considered to be coincidental and not related to the test item:
Treatment period:
decreased total cholesterol (1000 mg/kg bw/day; males; p ≤ 0.01)
decreased urea (in blood)(100, 300, and 1000 mg/kg bw/day; males; p ≤ 0.05 or p ≤ 0.01)
decreased calcium (1000 mg/kg bw/day; males; p ≤ 0.05)
increased chloride (300 mg/kg bw/day; males; p ≤ 0.05)
increased chloride (300 and 1000 mg/kg bw/day; females; p ≤ 0.01)
increased sodium (1000 mg/kg bw/day; females; p ≤ 0.05)

URINALYSIS
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 100, 300 or 1000 mg/kg bw/day: daily treatment did not lead to any test item-related changes of the urinary parameters of the male and female animals compared to the control group at the end of the treatment period and at the end of the recovery period.
- statistically significant differences in the urinary parameters compared to the control animals on test day 89 or 117 that are not considered to be test item related are as follows:
Treatment period:
increased pH value (1000 mg/kg bw/day; females; p ≤ 0.05)
increased relative urine volume (1000 mg/kg bw/day; females; p ≤ 0.05)

Recovery period:
decreased specific gravity (1000 mg/kg bw/day; females; p ≤ 0.05)

NEUROBEHAVIOUR
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 100, 300 or 1000 mg/kg bw/day: no test item-related influence was noted on any of the parameters examined during the functional observation tests, on the fore- and hind limb grip strength, or on the spontaneous motility for any of the male and female animals in test week 13 and in test week 17.
- individual male and female animals of the test item-treated and/or control groups revealed a score different from the normal score for the parameters urination, toe pinch, tail pinch, wire maneuver, and/or positive geotropism in test week 13 and/or in test week 17. No general difference in the frequency and degree was noted for these parameters between the test item-treated groups and the control group. All occurrences of scores different from the normal score for the examined parameters are considered to be within the normal range of biological variation.
- the following statistically significant changes in comparison to the control animals observed for numerical neurological parameters in test week 13 or 17 are considered to be coincidental effects and not to be related to the treatment with the test item: decreased body temperature, decreased hind leg splay, increased/decreased hind limb (grip strength), and increased spontaneous motility

ORGAN WEIGHTS
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 100, 300 or 1000 mg/kg bw/day: no test item-related influence was noted on the relative or absolute organ weights of the male and female animals at the end of treatment (test day 91) and at the end of the recovery period (test day 119).
- statistically significant differences in relative and absolute organ weights compared to the control animals on test day 91 or test day 119 that are not considered to be test item-related are as follows:
Treatment period:
decreased adrenal gland weight (right; absolute)(300 mg/kg bw/day; females; p ≤ 0.05)
increased brain weight (relative)(1000 mg/kg bw/day; males; p ≤ 0.01)
decreased liver weight (absolute)(1000 mg/kg bw/day; males; p ≤ 0.05)
decreased ovary weight (right, relative)(300 mg/kg bw/day; females; p ≤ 0.05)
decreased ovary weight (right, absolute)(300 mg/kg bw/day; females; p ≤ 0.05)

Recovery period:
- 1000 mg/kg bw/day: increased relative weight of prostate and seminal vesicles with coagulating glands (p ≤ 0.05)

GROSS PATHOLOGY
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 100, 300 or 1000 mg/kg bw/day: no test item-related changes in the organs and tissues of the animals were found after terminal sacrifice at the end of the treatment period (test day 91). No abnormal findings were noted at recovery sacrifice at the end of the 4-week recovery period (test day 119).

- a few minor macroscopic findings were noted which are considered to be not test item-related but to be of spontaneous nature in various organs of individual test item-treated and control animals, which were as follows:
liver (medial lobe): dark-brown focus or a yellow focus in the
thymus (left side): dark-red discoloured side
cardiac stomach: haemorrhagic focus
intestines: green-brown content
testes/epididymides: reduced in size and revealed a soft consistency
ovary (right): cystic and enlarged, filled with a dark-red liquid
uterus: dilated, filled with a clear liquid
adrenal gland (right): enlarged
axilla (left): subcutaneous, solid and red-yellow coloured tissue enlargement

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- no treatment-related microscopic changes were noted in the male and female animals of the control group and the 1000 mg/kg bw/day dose group.
- all microscopic changes seen in any organ of any animal are considered to be coincidental, or to be within the normal range of background alterations, which may be seen in untreated rats of this age and strain.
- 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.


REPRODUCTIVE FUNCTION: OESTROUS CYCLE (PARENTAL ANIMALS)
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 100, 300 or 1000 mg/kg bw/day: no test item-related difference was noted in the mean number of oestrous cycles of the female animals in the week before start of treatment (test week -1), in test weeks 5 and 6 during the treatment period, at the end of the treatment (test weeks 12/13), and at the end of the recovery period (test weeks 16/17) between the treated groups and the control group.
- 300 or 1000 mg/kg bw/day: the average number of oestrous cycles appeared to be slightly higher in the treated female animals than in the control animals in test weeks 5 and 6 during the treatment period. However, the slight differences are considered to be coincidental and not related to the test item treatment.

Please also refer for results about oestrous cycle to "Attached background material" below

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 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.


HORMONE LEVELS (PARENTAL ANIMALS)
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 1000 mg/kg bw/day: no test item-related influence was noted on the serum levels of the hormones testosterone, progesterone, and 17 beta-estradiol in the treated male and female animals compared to the control group during study conduct, at the end of the treatment period, and at the end of the recovery period.
- the individual data partly vary over a wide range, resulting in a large scatter.
- differences in hormone levels between the 1000 mg/kg bw/day dose and the control group were already noted at pre-dose examination. Therefore, all data obtained are considered to be within the normal range of biological variation. All differences between the test item-treated animals and the control group are considered as coincidental, despite of being statistically significant (at p ≤ 0.05 or p ≤ 0.01).
- The statistically significant changes in hormone levels in comparison to the control group that are not considered to be related to the test item treatment are as follows:
Treatment period:
decreased 17 beta-estradiol (1000 mg/kg bw/day; males; p ≤ 0.01)
increased progesterone (1000 mg/kg bw/day; males; p ≤ 0.01)
decreased testosterone (1000 mg/kg bw/day; males and females; p ≤ 0.05 or p ≤ 0.01)
Recovery period:
decreased progesterone (1000 mg/kg bw/day; males; p ≤ 0.05)

Please also refer for results about hormone levels to "Attached background material" below

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

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

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

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)

Remarks on result:

not measured/tested

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

NOAEL (fertility): 1000 mg/kg bw/day
No test item-related influence on the oestrous cycle was noted. Furthermore, histopathological examination of one testicle and one epididymis with special emphasis on the qualitative stages of spermatogenesis and of the interstitial testicular structure, did not reveal any test item-related effects. Lastly, no test item-related influence on hormone levels was noted.