Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-10-10 to 2018-01-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Version / remarks:
July 2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
October 2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EU Method B.7 (Repeated Dose (28 days) Toxicity (oral)).
Version / remarks:
May 2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)
Version / remarks:
July 2000
Deviations:
no
Principles of method if other than guideline:
No testing guidelines were applicable for the pilot phase, as this part of the study was intended for dose level selection purposes only.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study reports): JNJ-39722280-AAA (4-Nitrobenzene-1-sulfonyl chloride)
- Physical state: solid (powder)
- Appearance: Slightly yellow to yellowish brown crystalline powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M16LB4523
- Expiration date of the batch: 2018-04-18
- Purity : 99.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability in vehicle: Stability of formulations bracketing the concentration range used in this study were determined as part of the analytical method development and validation study (Test Facility Study No. 520060)
- Stability under test conditions: Homogeneity and stability of the test item under test conditions was demonstrated in the analytical method development and validation study (Test Facility Study No. 520060)

FORM AS APPLIED IN THE TEST : yellow suspension (groups 2-4)

OTHER SPECIFICS:
Correction factor: 1.00 based on purity

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males approx. 10 weeks (at start F0-treatment); Females 11weeks (at start pretest) and approx. 13 weeks (at start F0-treatment)
- Weight at study initiation: 253-303 g (males); 203-244 g (females)
- Fasting period before study: no
- Housing:
Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Males and females were cohabitated on a 1:1 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).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet. During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
- Water (e.g. ad libitum): Free access to tap-water. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

DETAILS OF FOOD AND WATER QUALITY:
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24 °C
- Humidity (%): 40 to 70%
- Air changes (per hr): at least 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle. The light/dark cycle was interrupted for study related activities.

IN-LIFE DATES: From: 2017-10-10 To: 2018-01-24

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 3 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. A correction was made for the purity/composition of the test item. A correction factor of 1.00 was used.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations and on results of the analytical Method Development and Validation performed at the test facility.
- Concentration in vehicle: 0 mg/mL (Group1), 10 mg/mL (Group2), 30 mg/mL (Group3), 20 mg/mL (Group4)
- Amount of vehicle (if gavage): 5 mL/kg (Group1), 1 mL/kg (Group2), 1 mL/kg (Group3), 5 mL/kg (Group4)
- Lot/batch no. (if required): no data
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (2017-11-23, Day 1 of treatment) according to a validated method (Test Facility Study No. 520060). Three sets of duplicate samples were collected. Two sets of duplicate samples were stored in the refrigerator as reserve samples.
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
In addition to the criteria mentioned in the validated analytical method, each calibration curve was accepted if the average of the response factors of the data points used to construct the calibration line were within a range of ±10.00% compared to those obtained during the method validation. The accuracy of preparation was considered acceptable if the mean measured concentrations were 85.00-115.00% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10.00%. Stability of formulations bracketing the concentration range used in this study were determined as part of the analytical method development and validation study (Test Facility No. 520060). Formulations were considered stable if the relative difference before and after storage was maximally 10.00%.
Duration of treatment / exposure:
Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females that delivered were treated for 54-63 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy.
Females which failed to deliver healthy offspring were treated for 38-41 days.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control, Group 1
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 animals/sex/dose level
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of a 10-day dose range finding study (Test Facility Study No. 520059). During the DRF phase, oral (gavage) administration of the test substance to female Wistar rats was given for 10 days at 500 or 200 mg/kg bw/day. At 500 mg/kg bw/day, all 3 animals were sacrificed in extremis on Day 3. All animals had shown flat/hunched posture, uncoordinated movements, rales, pallor, piloerection, and hypothermia on Day 3 (rales also observed for one animals on day 2). No abnormalities were noted at macroscopic examination of the 500 mg/kg bw/day group. At 200 mg/kg bw/day, no mortality was observed. All animals showed occasional hunched posture, rales and/or piloerection on 2 or 3 days across the treatment period. These clinical signs were not persistently noted with continuing treatment. No effects were observed in body weight or food consumption. At macroscopic examination, irregular surface of the stomach was noted for one female, but no abnormalities in the other 2 animals. A higher liver weight, exceeding the range considered normal for rats of this strain and similar age was observed. Kidney weights were considered to be normal. Based on the results of the range finding study, dose levels for the main study were 10, 30 and 100 mg/kg bw/day.
- Rationale for animal assignment (if not random): randomized
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals, at least after dosing at no specific time point, but within a similar time period after dosing for the respective animals. 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.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. Body weight gain was calculated and reported.

FOOD CONSUMPTION
- Time schedule for examinations: 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 post-coitum and during lactation on PND 1, 4, 7 and 13. Relative food consumption was calculated and reported.

WATER CONSUMPTION: No
- Time schedule for examinations: Subjective appraisal was maintained during the study. For the purpose of collecting historical control data, water consumption was measured daily in control animals only from 01 December 2017 onwards during the entire treatment period. water consumption was measured for males and females which were housed together for the mating period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with K3-EDTA for haematological parameters, with citrate for clotting tests and tubes treated with Li-heparin for clinical biochemistry parameters. An additional blood sample was collected into serum tubes for determination of bile acids.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Animals were not fasted overnight
- How many animals: 5 animals/sex/group
- Parameters examined : white blood cells, differential leukocyte counts (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, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood : Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with K3-EDTA for haematological parameters, with citrate for clotting tests and tubes treated with Li-heparin for clinical biochemistry parameters. An additional blood sample was collected into serum tubes for determination of bile acids.
- Animals fasted: Animals were not fasted overnight
- How many animals: 5 animals/sex/group
- Parameters examined: alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), Gamma glutamyl transpeptidase (GGT), total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate

OTHER:
FUNCTIONAL OBSERVATIONS
- functional observations tests were performed on each individual animal of the selected 5 animals/sex/group.
- The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation. These tests were performed after observation for clinical signs
- Parameters: hearing ability, pupillary reflex, static righting reflex, fore and hind-limb grip strength recorded as mean of three measurements per animal, locomotor activity.

THYROID HORMONE ANALYSIS
-Time schedule: End of study
- How many animals: all animals at planned necropsy (including: females on Day 14-16 of lactation, all females that failed to deliver healthy pups and all males after at least 4 weeks of treatment)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted:
Males: Yes, overnight with a maximum of 24 hours before blood sampling but water was available)
Males sacrified in extremist: No
Females: No
- Blood samples (0.9 mL) were drawn from the retro-orbital sinus
After clotting and centrifugation, serum was used as:
Males: 1 aliquot of 150 µL serum was used for measurement of thyroxine (T4), and the remaining volume of serum was stored for possible future measurement of thyroidstimulating hormone (TSH).
Females: The serum was stored for possible future measurement of thyroxine (T4) and/or thyroid-stimulating hormone (TSH).
Serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable for 6 months.
Sacrifice and pathology:
SACRIFICE:
Animals were not fasted overnight.
All animals surviving to the end of the observation period and all moribund animals were deeply anaesthetized using isoflurane and subsequently exsanguinated.
Necropsy was conducted on the following days:
- Males: Following completion of the mating period (a minimum of 28 days of dose administration).
- Females which delivered: PND 14-16.
- Females which failed to deliver: Post-coitum Days 25 (females with evidence of mating) or approximately 26 days after the last day of the mating period (females without evidence of mating).
- Female with total litter loss: Within 24 hours of litter loss.
- Euthanized in extremis: When pain, distress or discomfort was considered not transient in nature or was likely to become more severe.

GROSS PATHOLOGY: Yes
- All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated.
- After sacrifice, all animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
- Samples of the following tissues and organs of the selected 5 animals/sex/group were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution): Adrenal glands (M/F), Aorta* (M/F), Brain - cerebellum, mid-brain, cortex (7 levels) (M/F), Caecum (M/F), Cervix (F), Colon (M/F), Coagulation gland (M), Duodenum (M/F), Epididymides 3 (M), Eyes (with optic nerve (if detectable) and Harderian gland)4 (M/F), Mammary gland area, inguinal region with skin (M/F), Femur with bone marrow, including joint (M/F), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidneys (M/F), Lacrimal gland*, exorbital (M/F), Larynx* (M/F), Liver (M/F), Lung, infused with formalin (M/F), Lymph nodes (mandibular, mesenteric) (M/F), Nasopharynx* (M/F), Esophagus (M/F), Ovaries (F), Pancreas* (M/F), Peyer's patches [jejunum, ileum] if detectable (M/F), Pituitary gland (M/F), Prostate gland (M), Rectum (M/F), Salivary glands - mandibular, sublingual* (M/F), Sciatic nerve (M/F), Seminal vesicles (M), Skeletal muscle (M/F), Skin (M/F), Spinal cord -cervical, mid-thoracic, lumbar (M/F), Spleen (M/F), Sternum with bone marrow (M/F), Stomach (M/F), Testes (M), Thymus (M/F), Thyroid including parathyroid if detectable (M/F), Tongue* (M/F), Trachea (M/F), Urinary bladder (M/F), Uterus (F), Vagina (F), All gross lesions (M/F).

*Tissues/organs was not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

- All remaining animals, males that failed to sire, females which failed to deliver and females with total litter loss : Cervix (F), Coagulation gland (M), Epididymides4 (M), Mammary gland area, inguinal region with skin (M/F), Ovaries (F), Prostate gland (M), Seminal vesicles (M), Testes 6 (M), Thyroid including parathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F), Identification marks: not processed (M/F).

HISTOPATHOLOGY: Yes
- All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin
- The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4,
- Additional slides of the testes of the selected 5 males and all males that failed to sire for detailed qualitative examination, taking into account the tubular stages of the spermatogenic cycle,
- The preserved organs and tissues of the animals of all dose groups which were euthanized in extremis,
- The mammary gland of the female with total litter loss,
- All gross lesions of all animals (all dose groups),
- Stomach of all selected 5 animals of Groups 2 and 3 (males and females), based on (possible) treatment-related changes in this organ in Group 4,
- Nasal cavity level 3, 4 and caudal to 4 Larynx of intercurrently sacrificed male and female, based on (possible) treatment-related changes in these organs,
- The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.
- A peer review on the histopathology data was performed by a second pathologist
Other examinations:
ORGAN WEIGHTS
- Absolute organ weights and organ to body weight ratios are reported.
- 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 Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid including parathyroid if detectable, Uterus including cervix.
- The following organ weights and terminal body weight were recorded from all remaining animals on the scheduled day of necropsy: Epididymides, Prostate, Seminal vesicles, including coagulation glands, Testes, Thyroid, including parathyroid if detectable.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-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 was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
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

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- Animals surviving until scheduled necropsy:
Rales were observed in males at 30 and 100 mg/kg and in females across all groups (including the control group). This finding generally occurred in a few animals and on a few days of treatment only. Among these high dose animals, one female (no. 78) presented with gasping on a single day and one male (no. 38) showed laboured respiration on 5 consecutive days.
- Salivation seen among all animals across all groups (including the control group) was considered to be a physiological response rather than a sign of systemic toxicity considering the occurrence of this finding in the control groups at similar frequency to test item treated groups, the nature and minor severity of the effect and its time of occurrence (after dosing).
- Any other clinical signs noted during the treatment period 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
- No findings were noted during the weekly arena observations in this study.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- No mortality occurred during the study that was considered to represent a systemic test item effect.
- A total of two females at 100 mg/kg (nos. 72 and 80) and two males at 30 mg/kg (nos. 21 and 26) were sacrificed in extremis between Days 16 and 38 of the study period. Respiratory distress was the main clinical sign for these 4 animals resulting in early sacrifice.
- Female no. 72 (100 mg/kg) was sacrificed in extremis on Day 38 (Day 21 post-coitum). This animal presented with respiratory distress (squeaking and gasping), piloerection, pale skin,
and hypothermia. Necropsy showed gastrointestinal tract distended with gas. Microscopic examination showed marked erosion/ulceration of the trachea and moderate erosion/ulceration of the larynx and nasopharynx together with a marked obstructive granulocytic inflammation of the larynx. Other microscopic findings, such as lymphoid depletion in the spleen and decreased cellularity and increased adipocytes in the bone
marrow, were considered to be related to the poor condition of the animal. The inflammatory and degenerative lesions in the respiratory tract were considered to be the cause of moribundity for this animal.
- Female no. 80 (100 mg/kg) was sacrificed in extremis on Day 16. This animal showed respiratory distress (laboured respiration and gasping), squeaking, hunched posture and weight loss of 6% over the last week of premating. Necropsy showed cecum distended with gas, accentuated lobular pattern of the liver and enlarged adrenal glands. Microscopic examination revealed massive erosion/ulceration of the trachea and nasopharynx, and moderate erosion/ulceration and marked obstructive granulocytic inflammation of the larynx. The nasal cavity was noted with erosion/necrosis of the respiratory epithelium and a marked degree of luminal exudate consisting of mucous with granulocytic inflammatory cells. The inflammatory and degenerative lesions in the respiratory tract were considered to be the cause
of moribundity for this animal.
- Male no. 21 (Group 3) was sacrificed in extremis on Day 18. This animal presented with breathing problems (laboured respiration and rales), hunched posture, piloerection, ptosis and a 15% weight loss over the treatment period. Necropsy showed cecum distended with gas, and reduced size of the spleen and thymus. Microscopic examination showed slight lymphogranulocytic inflammation of the forestomach and glandular stomach and slight erosion/ulceration of the forestomach. Other microscopic findings considered to be related to the poor condition of the animal were lymphoid depletion in the thymus and mesenteric
lymph nodes together with a decreased cellularity and increased adipocytes in the bone marrow. Although histopathologically no clear cause of moribundity could be established, the distended intestinal tract and inflammatory lesions in the stomach were considered to have contributed to the moribund state of this animal.
- Male no. 26 (Group 3) was sacrificed in extremis on Day 29. This animal showed respiratory distress (rales and gasping), squeaking and a 20% weight loss over Days 8-15 of the mating period. Necropsy revealed gastrointestinal tract distended with gas, non-collapsed lungs and reduced size of the spleen. Main findings at microscopic examination consisted of slight erosion/ulceration of the larynx. Other microscopic findings considered to be related to the poor condition of the animal were lymphoid depletion in the thymus and decreased cellularity and increased adipocytes in the bone marrow. No clear cause of moribundity could be established from the sections examined, but the distended intestinal tract and the slight erosion/ulceration of the larynx were regarded to have contributed to the moribund state of this animal.
- Respiratory tract lesions recorded for these four animals (nos. 21, 26, 72 and 80) were indicative of a dosing procedure-related occurrence and/or gavage-related reflux (Ref. 6, Ref.7).
- One control female (no. 44) was sacrificed on Day 2 of lactation due to total litter loss. One female at 100 mg/kg (no. 74) was found dead on Day 3 (this animal was replaced by a reserve female). No relevant clinical signs were noted for female no. 74 prior to death.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- At 100 mg/kg, mean absolute body weight and body weight gain of females was slightly lower than controls from Day 17 post-coitum onwards (not statistically significant).
- Mean body weight and body weight gain during lactation was not considered to be affected by treatment. The statistically significantly higher mean body weight gain of females at 30 mg/kg on Day 4 of lactation was not considered to be related to treatment as this occurred in the absence of a dose-related trend and was attributed to a higher weight gain of one female (no. 66). One female at 30 mg/kg (no. 69) showed weight loss of 16% over Days 4-7 of lactation (accompanied by a lower food intake over Days 4-7 of lactation). These individual variations in body weight development were not considered to be related to treatment as they occurred in the absence of a dose-related trend.
- Body weights and body weight gain of other treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was not considered affected by treatment. Any statistically significant changes in food consumption were considered to be unrelated to treatment since no dose-related trend was apparent.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Haematological parameters of treated rats were not considered to be affected by treatment. Any statistically significant changes in haematology parameters were not considered to be related to treatment as these occurred in the absence of a dose-related trend.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Clinical biochemistry parameters of treated animals were not considered to be affected by treatment. Any statistically significant changes in clinical biochemistry parameters were not considered to be related to treatment as they occurred in the absence of a treatment-related distribution.
- Thyroid hormone analyses: Serum levels of T4 in F0 males were not considered to be affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
- At 30 and 100 mg/kg, mean forelimb grip strength of selected males was 14 and 20% lower than control values, respectively (statistically significant at 100 mg/kg). Means remained within the range of the historical control data, and hindlimb grip strength of males remained similar to control levels.
- Grip strength in females was considered unaffected by treatment. Hearing ability, pupillary reflex and static righting reflex 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 higher activity in the first interval that decreased over the duration of the test period. The habituation profile of males deviated from that normally encountered, and total movements of all groups (including the control group) were lower than expected for rats of this age and strain. This was attributed to conducted procedures, which did not adversely affect evaluation of the study results.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights and organ to body weight ratios of treated animals were considered to be unaffected by treatment. Any statistically significant changes in organ weights were not considered to be related to treatment as they occurred in the absence of a treatment-related distribution.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations in surviving animals at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
The incidence of the 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, not considered to be toxicologically relevant.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with the test substance for at least 28-days were noted in the stomach of males. There were no test item-related microscopic observations in female rats after the scheduled study period.
In male rats at 100 mg/kg a combination of microscopic findings was recorded for the forestomach, which were considered to be related to the treatment with the test item. These findings included minimal to slight inflammation, slight squamous hyperplasia, slight vacuolation and moderate hyperkeratosis.
The inflammation of the glandular stomach of male rats did not show a dose-related trend and was considered to be within background pathology for rats of this age and strain.

Findings of note in the surviving animals were recorded for male no. 32 (100 mg/kg) in the trachea (marked erosion/ulceration) and lung (slight acute alveolar inflammation) and for female no. 41 (control) in the lung (marked ulceration of the bronchi and lipophilic alveolar contents in one lobe, moderate acute bronchioalveolar inflammation and slight alveolar macrophage aggregation). These findings in the respiratory tract in one high dose male and one control female were considered to be gavage procedure related and not a test item effect.

The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
Analysis of dose preparations:
- Accuracy of preparation: The concentrations analysed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85.00% and 115.00%). No test item was detected in the Group 1 formulation.
- Homogeneity: The formulations of Group 2 and Group 3 were homogeneous (i.e. coefficient of variation ≤ 10.00%).

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 10, 30 and 100 mg/kg. Based on the results, the parental No Observed Adverse Effect Level (NOAEL) was derived to be at least 100 mg/kg. The substance is considered not classified as repeated dose toxicant according to the CLP Regulation.