Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 June 2012 - 09 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
GLP compliance:
yes
Remarks:
WIL Research Europe B.V., Hambakenwetering 7, 5231 DD, 's-Hertogenbosch, The Netherlands
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 1H-Benzotriazole-1-methanamine, N,N-bis(2-ethylhexyl)-6-methyl- and 2H-Benzotriazole-2-methanamine, N,N-bis(2-ethylhexyl)-5-methyl- and N,N-bis(2-ethylhexyl)-4-methyl-1H-benzotriazole-1-methylamine and 2H-Benzotriazole-2-methanamine, N,N-bis(2-ethylhexyl)-4-methyl- and N,N-bis(2-ethylhexyl)-5-methyl-1H-benzotriazole-1-methylamine
EC Number:
939-700-4
Molecular formula:
Unspecified
IUPAC Name:
Reaction mass of 1H-Benzotriazole-1-methanamine, N,N-bis(2-ethylhexyl)-6-methyl- and 2H-Benzotriazole-2-methanamine, N,N-bis(2-ethylhexyl)-5-methyl- and N,N-bis(2-ethylhexyl)-4-methyl-1H-benzotriazole-1-methylamine and 2H-Benzotriazole-2-methanamine, N,N-bis(2-ethylhexyl)-4-methyl- and N,N-bis(2-ethylhexyl)-5-methyl-1H-benzotriazole-1-methylamine
Details on test material:
- Description: Clear yellowish slightly viscous liquid
- Purity: ~100%
- Storage: At room temperature in the dark
- Stability under storage conditions: Stable

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 11 weeks
- Weight at study initiation: Males: 306-311g, Females: 199-204g
- Housing: Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm). Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm). Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Solution of 0.5% carboxymethyl cellulose and 0.1% Tween-80 in water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for the mean specific gravity of the test substance (0.95 g/cm3). No adjustment was made for the purity of the test substance, since it was considered nearly 100%.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe.
- Amount of vehicle: 5 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at ABL B.V., The Netherlands, where samples were stored at ≤-70°C until analysis. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42-45 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Females 45 (Group 1), 53 (Group 2) and 78 (Group 4) were not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 5 hours difference between the earliest and latest dose.
Doses / concentrations
Remarks:
Doses / Concentrations:
15, 45, 150 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previous dose range finding study (Project 499974; BASF Project 01R0608/11X398) groups of 4 male and 4 female Crl:WI(Han) rats received Irgamet 39 by oral gavage for a period of 14 days at the dose levels of 0, 100, 300, or 1000 mg/kg bw/day. All females and males at 1000 mg/kg bw/day were euthanized on Days 4 and 5, respectively, for humane reasons. At 300 mg/kg bw/day, one female was found dead on Day 7, and the remaining animals were euthanized on Day 8 due to poor general conditions. In both dose groups, the predominant clinical signs consisted of lethargy, hunched posture, uncoordinated movements and piloerection. Partially, the clinical signs showed a dose-relationship with regards to onset and severity. Body weight loss, together with reduced food intake was recorded in a dose-related manner. Gross necropsy findings were noted for two females at 1000 mg/kg bw/day only. These included gelatinous contents in the gastro-intestinal tract, several dark-red foci on the caecum and/or glandular mucosa of the stomach, thickened glandular mucosa of the stomach, reduced size of the caecum and/or dark-red discoloration of the ileo-caecal valve.
- Mating procedures: Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated.
- Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.

Examinations

Observations and examinations performed and frequency:
At dose level allocation, 5 animals/sex/group were randomly selected for functional observations, locomotor activity, clinical pathology, organ weights (full list) and histopathology.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily
Detailed clinical observations were made in all animals, at least immediately (0-15 min) after dosing (based on results of the dose range finding study, Project 499974; BASF Project 01R0608/11X398). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

BODY WEIGHT: Yes
Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE:
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

LABORATORY INVESTIGATIONS
Blood samples were collected from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into untreated tubes for determination of bile acids. Furthermore, from the selected 5 animals/sex/group an additional blood sample (0.5 mL) was collected into serum tubes for possible future measurement of the thyroid hormones triiodothyronine (T3) and thyroxine (T4). After clotting and centrifugation, serum samples were stored at ≤-75°C. Since histopathological examination of the thyroid glands did not reveal any treatmentrelated changes, all samples were discarded at finalization of the study report without further investigation.

HAEMATOLOGY: Yes
The following haematology parameters were determined in blood prepared with EDTA as an anticoagulant, using the ADVIA® 120 Hematology System (Siemens Healthcare Diagnostics B.V., Breda, The Netherlands): White blood cells, Differential leucocyte count: Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets. The following clotting parameters were determined in plasma prepared with citrate as anti-coagulant, using the STA Compact® (Diagnostica Stago S.A.S., Asnières, France): Prothrombin Time, Activated Partial Thromboplastin Time.

CLINICAL CHEMISTRY: Yes
The following clinical biochemistry parameters were determined using the AU400® Chemistry System (Beckman Coulter Nederland B.V., Woerden, The Netherlands). All parameters were determined in plasma, except for bile acids which were determined in serum: Alanine aminotransferase, Aspartate aminotransferase, Alkaline Phosphatase, Total protein, Albumin, Total Bilirubin, Bile acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

NEUROBEHAVIOURAL EXAMINATION: Yes
The following tests were performed on the selected 5 animals/sex/group: hearing ability, pupillary reflex, static righting reflex, grip strength, locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head. During motor activity monitoring, all animals were caged individually (pups were housed in their home cages and kept warm using bottles filled with warm water). The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling). These tests were performed as soon as possible after observation for clinical signs (incl. arena observation, if applicable), and before blood sampling.

OTHER:
- General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Sacrifice and pathology:
- Necropsy: All males, the selected females/group were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food. Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.

Necropsy was conducted on the following days: Females which delivered: Lactation Days 5-7, Females which failed to deliver: Post-coitum Day 25-26 (females with evidence of mating). Spontaneous deaths: as soon as possible after death and always within 24 hours. Males: Following completion of the mating period (a minimum of 28 days of dose administration).

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The numbers of former implantation sites and corpora lutea were recorded for all paired females (for females which failed to deliver: In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites).

Samples of the following tissues and organs were collected from all animals and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands): Adrenal glands, (Aorta), Brain - cerebellum, mid-brain, cortex, Caecum, Cervix, Clitoral gland, Colon, Coagulation gland, Duodenum, Epididymides, Eyes (with optic nerve (if detectable) and Harderian gland), (Male and Female mammary gland area), Femur including joint, Heart, Ileum, Jejunum, Kidneys, (Lacrimal gland, exorbital), (Larynx), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), (Esophagus), Ovaries, (Pancreas), Peyer's patches [jejunum, ileum] if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid including parathyroid if detectable, (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions. The epididymides, eyes and testes were fixed in modified Davidson's solution and Milli-Ro water and transferred to formalin after fixation for at least 24 hours. Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

- Organ weights: The following organ weights and terminal body weight were recorded from the selected 5 animals/sex/group on the scheduled day of necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus (including cervix), Prostate (weighed when fixed for at least 24 hours), Seminal vesicles including coagulating glands (weighed when fixed for at least 24 hours), Thyroid including parathyroid (weighed when fixed for at least 24 hours). All remaining males: Epididymides, Testes.

- Histotechnology: All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). Of all males of the control and high dose group and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

- Histopathology: The following slides were examined by a pathologist: (1) The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4. (2) The additional slides of the testes of all males of Groups 1 and 4 and all males suspected to be infertile or which died before mating to examine staging of spermatogenesis. (3) All gross lesions of all animals (all dose groups). (4) The preserved organs and tissues of the animals of the two Group 4 females that either died spontaneously or were euthanized due to a total litter loss. (5) The spleen and thymus of all selected 5 females of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4. (6) The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina) of male no. 31 (Group 4) and female no. 71 (Group 4) that had a total litter loss.
Other examinations:
PUPS: Each litter was examined to determine the following, if practically possible:
- Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation.
- Necropsy pups: Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Details on results:
MORTALITY
No mortalities occurred that were considered to be a direct cause of treatment with Irgamet 39 up to 150 mg/kg bw/day.
One female at 150 mg/kg bw/day died during parturition on Day 21 post-coitum (ten healthy pups were already delivered). No clinical signs were noted. Food consumption (absolute and relative to body weight) was only slightly lower from Days 14-20 post-coitum with no effects on body weight. At necropsy, three fully developed fetuses were discovered in the right uterus horn and watery cloudy fluid was found in the lungs which correlated with the finding of alveolar proteinaceous fluid at the microscopic level. These necropsy findings were indicative for parturition difficulties that likely resulted in circulatory failure. In addition, her spleen was reduced in size and many dark red foci were noted on the thymus.
Another female at 150 mg/kg bw/day had to be euthanized due to a total litter loss on Day 2 of lactation. Clinical signs were recorded from the end of the post-coitum phase onwards and consisted of hunched posture, piloerection and pale faeces. This female had the lowest body weight and the lowest food consumption in this group. At necropsy, the only finding was a smaller thymus. No mortality occurred at the lower doses of 45 and 15 mg/kg bw/day or in the control group.

CLINICAL SIGNS
For females at 150 mg/kg bw/day hunched posture, piloerection and pale faeces were noted mainly during the last two weeks of treatment.
No clinical signs of toxicity were noted for females at 15 and 45 mg/kg bw/day and males up to 150 mg/kg bw/day.
Salivation seen after dosing among females of the 150 mg/kg bw/day dose group towards the end of the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance.
Incidental findings that were noted for a single female each at 15, 45 and 150 mg/kg bw/day included hunched posture, salivation and purple discolouration of the ear, respectively. In addition, scabbing was recorded for two males at 150 mg/kg bw/day. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the low incidence observed, these were considered signs of no toxicological relevance.

BODY WEIGHT AND BODY WEIGHT GAIN
There was a trend towards lower body weights as compared to controls for males and females at 150 mg/kg bw/day during the post-coitum phase, reaching statistical significance in females on Day 20 post-coitum and during the subsequent phase of lactation as well as in males at termination. Body weight gains were significantly lower in females from Days 4-20 post-coitum. Body weight gain between Day 0 and 20 post-coitum was 74 g versus 104 g in controls.
No toxicologically relevant changes in body weights and body weight gain were noted up to 45 mg/kg bw/day.

FOOD CONSUMPTION
A lower food consumption (absolute and relative to body weight) was recorded for females at 150 mg/kg bw/day as compared to controls during the entire treatment period, though differences were not always statistically significant.
For females up to 45 mg/kg bw/day and males up to 150 mg/kg bw/day, food consumption was in the same range as for control animals.
For one cage with five males (cage no. 7; male nos. 31-35) at 150 mg/kg bw/day food consumption (absolute and relative to body weight) was slightly lower during the first week of treatment. However, changes were only slight and transient, and no comparable effect was noted for the other cage with
high dose males. Therefore, this finding was not considered to be toxicologically relevant.

HAEMATOLOGY
Haematological parameters of treated rats were considered not to have been affected by treatment up to 150 mg/kg bw/day.
Any statistically significant changes observed were considered to be of no toxicological relevance as no treatment-related distribution could be established, changes occurred in the absence of corroborating effects in related parameters and remained within the range considered normal for rats of this age and strain. These changes included a decreased prothrombin time (PT) for males at 150 mg/kg bw/day and increased values for white blood cell count (WBC), mean corpuscular haemoglobin concentration (MCHC) and activated partial prothrombin time (APTT) for females at 45 and/or 150 mg/kg bw/day.

CLINICAL CHEMISTRY
Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment up to 150 mg/kg bw/day.
Any statistically significant changes observed were considered to be of no toxicological relevance as no treatment-related distribution could be established, changes occurred in the absence of corroborating effects in related parameters, remained within the range considered normal for rats of
this age and strain and/or were the result of relatively low control values. These changes included increased concentrations of potassium for males at 45 mg/kg bw/day and increased concentrations of creatinine, bile acids and inorganic phosphates for females at 15, 45 and/or 150 mg/kg bw/day.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.
For males, there was a trend towards lower total movements with increasing dose (not statistically significant). However, all values remained within the normal range of biological variation. Moreover, there were no corroborative findings during clinical observations and no comparable change was seen for females. Therefore, this finding was considered to be a chance finding rather than a sign of toxicity.

ORGAN WEIGHTS
For females at 150 mg/kg bw/day, mean thymus organ weight (absolute and relative to body weight) was at the lower end of the historical range (0.131 gram and 0.062% versus a P5 of 0.129 gram and 0.0578%), though no statistical significance was reached as compared to controls. This difference was particularly due to animals no. 73, 76 and 78.
The increased brain to body weight ratio recorded for males at 150 mg/kg bw/day was secondary to the lower terminal body weights and was not considered to be toxicologically relevant.
The increased thyroid organ weight (absolute and relative to body weight) recorded for males at 45 mg/kg bw/day as compared to controls was considered not to be a sign of toxicity as no treatment related distribution could be established. Statistical significance is rather due to the relatively low control value. Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

GROSS PATHOLOGY
There were no treatment-related necropsy findings.
The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance. They included pelvic dilation of one or both kidneys, isolated or several reddish foci on the glandular mucosa of the stomach, reddish or dark-red discolouration of mandibular lymph nodes or thymus, yellowish soft nodule on the tail of the epididymides, an isolated tan or greenish focus on the clitoral glands, dark-red content of the vagina (revealed as parturition debris at the microscopic level), fluid-filled uterus, and scabbing.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were treatment-related microscopic findings in the thymus and spleen of females.
Thymus: Lymphoid atrophy (involution) was present in 4/7 (2 minimal, 1 slight, 1 moderate) females treated at 150 mg/kg bw/day, and did correlate with low thymus weights in females 73, 76 and 78.
Spleen: A decreased severity of hemopoietic foci, primarily erythropoiesis was present in 6/6 (4 minimal, 1 slight, 1 moderate) females treated at 150 mg/kg bw/day compared to normal levels in 5/5 control (1 minimal, 1 slight, 2 moderate, 1 marked), 5/5 15 mg/kg bw/day (2 slight, 1 moderate, 2 marked) and 5/5 45 mg/kg bw/day (1 minimal, 3 moderate, 1 marked) treated female rats.
All other microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age.
There were two Group 3 and four Group 4 rats that failed to mate, conceive, sire or deliver healthy offspring and were therefore selected for histopathological examination of the reproductive organs. No cause of infertility was found for these animals.
The total litter loss observed for one female of group 4 (150 mg/kg bw/day) could not be explained based on the microscopic examination. There was proteinaceous fluid (milk) present in the mammary gland of this female.
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.

Effect levels

Dose descriptor:
NOAEL
Effect level:
45 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

ANALYTICS

Formulation analyses revealed that the high concentration was prepared accurately. The low and mid concentrations were slightly higher than nominal (119% and 116%, respectively). The mid and high concentrations were homogeneously (i.e. coefficient of variation ≤ 10%), whereas the low concentration was not homogeneous (i.e. coefficient of variation of 20.9%). No test compound was detected in the vehicle formulation. The inhomogeneity of the formulation for Group 2 (15 mg/kg bw/day) had no impact on the outcome of this study as the No Observed Adverse Effect Levels (NOAEL) derived were at 45 or 150 mg/kg bw/day (see further below). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at

least 5 hours (i.e. relative difference ≤ 10%).

REPRODUCTION DATA

No toxicologically relevant effects on reproductive parameters were noted.

Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were considered to be unaffected by treatment (for details see section 7.8).

DEVELOPMENTAL DATA

No toxicologically relevant effects on gestation index and duration, parturition, maternal care were observed.

However, there were treatment related effects on early postnatal pup development based on mortality, clinical signs and body weight. No changes in sex ratio or macroscopic abnormalities were noted up to 150 mg/kg bw/day (for details see section 7.8).

Applicant's summary and conclusion

Conclusions:
Based on the results presented, a No Observed Adverse Effect Level (NOAEL) of 45 mg/kg bw/day was derived.
Executive summary:

A GLP compliant combined 28-day repeated dose toxicity study with the reproduction / developmental toxicity screening test was performed in male and female Wistar Han rats at dose levels of 15, 45 and 150 mg/kg bw/day. Animals of the control group received the vehicle, 0.5% carboxymethyl cellulose and 0.1% Tween-80 in water, alone. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42-45 days, i.e. during 2 weeks prior to mating, during mating, during postcoitum, and during at least 4 days of lactation. The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (Week 4 (males); end of lactation (females)), body weight and food consumption (at least at weekly intervals), clinical pathology (Week 4 (males); end of lactation (females)), macroscopy at termination, organ weights and histopathology on a selection of tissues; and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio, and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Formulations were analyzed once during the study to assess accuracy, homogeneity and stability.

Toxicity was noted at 150 mg/kg bw/day. For females, it was characterized by clinical signs, reduced body weight gains with lower food consumption, and slightly reduced thymus organ weight. The slightly lower thymus organ weights were in line with the observation of lymphoid atrophy (involution) present in 4 out of the 7 examined females in this high dose group (2 minimal, 1 slight, 1 moderate). Further microscopic findings consisted of lower mean grade of hematopoietic foci in the spleen of females at 150 mg/kg bw/day. Slightly lower body weight gains were also observed for males at 150 mg/kg bw/day. No toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. mortality, functional observations, haematology, clinical biochemistry, macroscopic abnormalities). There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item. In conclusion, treatment with test material by oral gavage in male and female Wistar Han rats at dose levels of 15, 45 or 150 mg/kg bw/day revealed parental toxicity at 150 mg/kg bw/day. Based on these results, the No Observed Adverse Effect Levels (NOAEL) was set at 45 mg/kg bw/day.