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Administrative data

Description of key information

Repeated dose toxicity - oral: In a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the test substance was administered daily to rats up to a dose level of 30 mg/kg body weight/day (OECD 422; Otterdijk F, 2017). The NOAEL is established as 10 mg/kg body weight/day. The substance is therefore classified as STOT RE 1 according to CLP Regulation.

Repeated dose toxicity - inhalation: A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation route of exposure.

Repeated dose toxicity - dermal: A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the dermal route of exposure

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-08-08 to 2016-12-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
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. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M15BB1451
- Expiration date of the lot/batch: 2021-01-30
- Purity test date: unknown

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability of the test substance in the solvent/vehicle: Stability of formulations for at least 6 hours at room temperature and 14 days in the refrigerator was confirmed over the concentration range 1 to 200 mg/mL during Test Facility Study No. 510921.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Suspension

OTHER SPECIFICS: Correction factor: No correction was made for the purity/composition of the test item.
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 or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Females approx. 11 weeks (at start pretest) and approx. 13 weeks (at start F0-treatment); Males approx. 11 weeks (at start F0-treatment)
- Weight at study initiation: 304-347 g (males) and 205-242 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: 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).
- 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).

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

IN-LIFE DATES:
From: 2016-09-15 (start pretest, females); 2016-09-29 (start treatment, males); 2016-11-05/06/07/08/09/18 (delivery of litters)
To: 2016-10-28 (necropsy males); 2016-11-18/19/22 and 2016-12-01 (necropsy females); 2016-11-17/18/21/30 (necropsy pups)
Route of administration:
oral: gavage
Details on route of administration:
Method: Oral gavage, using a plastic feeding tube.
Frequency: 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.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% Aqueous
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test item, vehicle, and/or formulation. No correction was made for the purity/composition of the test item. Formulations were placed on a magnetic stirrer during dosing

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.
- Concentration in vehicle: 0 mg/mL (group 1); 0.6 mg/mL (group 2); 2.0 mg/mL (group 3); 6.0 mg/ mL (group 4)
- Amount of vehicle (if gavage): 5 mL/kg body weight (Actual dose volumes were calculated according to the latest body weight)
- 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 during the treatment according to a validated method (Test Facility Study No. 510921). Sextuplicate samples (i.e. 3 sets of duplicate samples). Two sets of duplicate samples were stored in the refrigerator as reserve samples. On Day 5 of treatment (03 October 2016), samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability of Group 2 formulations over 5 hours at room temperature was also determined since the concentration of Group 2 was below the range over which stability was confirmed as part of the analytical method development and validation (Test Facility Study No. 510921). A new Group 4 formulation was analyzed on Day 43 of treatment (10 November 2016) since initial accuracy results were outside specifications. In addition to the criteria mentioned in the validated analytical method, each calibration curve was accepted if the average of the retention times and 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% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10.00%. Formulations were considered stable if the relative difference before and after storage was maximally 10.00%. Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed.
Duration of treatment / exposure:
29 days (males); 50-63 days (females that delivered); 42-53 days (females that failed to deliver). Routinely, females that are littering are left undisturbed. In this study, female nos. 41 and 43 (Group 1), and nos. 65, 66 and 70 (Group 3) were not dosed for one day as they were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.

Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.
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
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (Control)
Dose / conc.:
3 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on results of a dose range finding study (Test Facility Study No. 510918) in which animals were dosed for 10 days at 25 and 50 mg/kg. In summary, there were no mortalities for both dose groups. Tremors and piloerection were observed in all animals in both dose groups, while 1/3 females in the 50 mg/kg group exhibited restlessnes and all animals in the high dose group had hunched posture, were fearful and exhibited hypersensitivity to touch; additionally, one animal in the 25 mg/kg group had thickened area of the right ear and salivation. Body weight loss was observed for all animals in the 50 mg/kg group and one animal in the 25 mg/kg group. For the 50 mg/kg group, food consumption was lower than considered normal over Days 5-10 and was not determined over Days 1-5; most likely no or very low consumption of food occurred during this period. For the 25 mg/kg group, food consumption was lower than considered normal over Days 1-5, and normal over Days 5-10. Macroscopic examination of both dose groups showed no abnormalities. Liver and kidney weights were considered to be normal for both dose groups. Based on the results of this range finding study, dose levels for the main study were selected to be 3, 10 and 30 mg/kg body weight in consultation with the Sponsor. The peak effect of occurrence of relevant clinical signs at 25 mg/kg (tremors and piloerection) occurred between 1 and 3 hours after dosing. This time point was taken into account for conduct of clinical observations and functional observation tests in the main study.

- Rationale for animal assignment (if not random): randomized
Positive control:
no
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, between 1 and 3 hours after treatment (i.e. on the anticipated peak period of effects after treatment, based on the dose range finding study. 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.

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 AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g fo od/ kg body weight/day: Yes
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.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

HAEMATOLOGY
- Blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group under anesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived from food overnight (with a maximum of 24 hours) before blood sampling; but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with K3-EDTA for hematology parameters and with citrate for clotting tests.
- Parameters assessed: white blood cells, differential leukocyte counts, red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothombin time, activated thromboplastin time

CLINICAL BIOCHEMISTRY
- Blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane 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 available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with Li-heparin.
- Parameters assessed: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate
- Thyroid hormone analysis

FUNCTIONAL OBSERVATIONS
- Time schedule: Between 1 and 3 hours after dosing on the selected 5 animals/sex/group. 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 (incl. arena observation if applicable)
- Parameters: hearing ability, pupillary reflex, static righting reflex, fore- and hindlimb grip strength recorded as the mean of three measurements per animal, locomotor activity

Sacrifice and pathology:
SACRIFICE:
- Male animals: All surviving animals, following completion of the mating period (a minimum of 28 days of dose administration).
- Female animals: All surviving animals, on PND 14-16 (females that delivered) or on post-coitum days 25-27 (females that failed to deliver, with evidence of mating).

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. Descriptions of all macroscopic abnormalities were recorded.
- Necropsy was conducted as soon as possibe after spontaneous death and always within 24 hours.
- 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), Clitoral gland (F), Colon (M/F), Coagulation gland (M) , (Cowper’s gland) (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area (M/F), Femur including joint (M/F), (Glans penis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), 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), Preputial gland (M), Prostate gland ( M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve (M/F), Seminal vesicles (F), Skeletal muscle (M/F), (Skin) (M/F), Spinal cord - cervical, midthoracic, 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 mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
- Samples of the following tissues and organs of all remaining animals, and females which failed to deliver, were collected and fixed in 10% buffered formalin:
Cervix (F), Clitoral gland (F), Coagulation gland (M), Cowper’s glands (M), Epididymides (M), Glans penis (M), Levator ani plus bulbocavernosus muscle complex (LABC) (M), Mammary gland area (M/F), Ovaries (F), Preputial gland (M), Prostate gland (M), Seminal vesicles (M), Testes (M), Thyroid including parathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (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 of Groups 1 and 4 males and of Group 1, 3 and 4 females; The additional slides of the testes of all males of Groups 1, 2, 3 and 4 and all males that failed to sire to examine staging of spermatogenesis; All gross lesions of all animals (all dose groups); Kidneys of all selected 5 animals of Group 2 (males and females) and Group 3 (males), and liver of all selected 5 males of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4 (males) or Group 3 (females); The reproductive organs of all animals of Group 4 (in this group, all males failed to sire and all females failed to deliver healthy pups).
- All abnormalities were described and included in the study report. An attempt was made to correlate gross observations with microscopic findings.
- 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 were 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, Testes, Thyroid
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.
- 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.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 30 mg/kg, all females showed piloerection during one or more days in the post-coitum period. Two of these females (nos. 75 and 78) showed red discharge from the vagina on Day 14 or 13 of the post-coitum phase, respectively.

One female at 10 mg/kg (no. 68) showed hunched posture, abnormal gait and piloerection on four subsequent days during the first week of treatment of the premating phase and swelling of the right axillary region from the second week of the premating phase onwards. At necropsy gross examination of this animal revealed a nodule/mass (25 x 20 mm), which was confirmed histopathologically as a subcutaneous abscess. Since these findings were not recorded for the other animals of this dose group nor for animals at the highest dose group, these findings were not considered to be related to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 30 mg/kg, body weight gain of males was statistically significantly lower than controls throughout treatment. For most females of this dose group, weight loss (up to 9% of Day 1 premating body weights) was recorded during the first week of treatment (resulting in a statistically significantly lower mean body weight on Day 8), which recovered as treatment progressed. During the post-coitum phase, statistically significantly lower body weights (generally throughout this phase) and lower body weight gain (during the last week), was attributed to the fact that these animals did not deliver any pups.

Body weights and body weight gain of animals at 3 and 10 mg/kg remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 30 mg/kg, food consumption before or after correction for body weight of females at 30 mg/kg was statistically significantly lower during the first week of the premating phase. This recovered during the subsequent treatment week.

The statistically significantly lower absolute food consumption and higher relative food consumption of females at 30 mg/kg over Days 14-17 and/or 17-20 of the post-coitum phase was attributed to the fact that these animals did not deliver any pups. Food consumption levels of these females were within the range expected for females that would not deliver pups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 3 and 10 mg/kg, prothrombin time (PT) and activated partial thromboplastin time (APTT) of females were statistically significantly delayed compared to controls. Mean prothrombin time levels were approximately 12% and 7% higher than controls, and mean activated partial thromboplastin time levels were approximately 23% and 21% higher than controls at 3 and 10 mg/kg, respectively.

Note: No haematology data were available for females at 30 mg/kg.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 10 mg/kg, cholesterol of females was statistically significantly lower than controls (mean was approximately 26% lower than the control mean).

Note: No clinical biochemistry data were available for females at 30 mg/kg.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Motor activity of females at 30 mg/kg was approximately a factor 3 higher than mean motor activity of the control group and was also distinctly higher than would be expected for non-mated females. For males at this dose level and for other female dose groups (trend), a much less pronounced and statistically non-significant higher mean motor activity was recorded. This higher motor activity was not accompanied by supportive changes in daily detailed clinical observations that would point towards an increased activity, or any other findings that would indicate that this higher motor activity had an adverse effect on functional integrity of these animals. However, given the degree of change in motor activity at 30 mg/kg and since this change could be indicative of neurotoxic potential of the test item, this was considered to be an adverse effect.

Results of hearing ability, pupillary reflex and static righting reflex that were conducted at the end of treatment were not recorded. It was considered unlikely that treatment-related changes would have occurred in these parameters since there were no signs of toxicity in related observations, such as daily detailed clinical signs (all groups), weekly arena observations (all groups), histopathological evaluation of eyes/neuronal tissues of Group 4 males and Group 3 females, and grip strength (all groups).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Except for thyroid and terminal body weights, no organ weight data were available for females at 30 mg/kg.
Test item-related higher liver weights (absolute and relative to body weights) and lower thymus weights (absolute and relative to body weights) were noted in the 30 mg/kg/day group.
Some organ weight differences were statistically significant when compared to the control group but based on the absence of a dose-relationship were considered not test item-related.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with T000750 were noted in males in the liver (starting at 10 mg/kg/day) and in the kidneys (at 30 mg/kg/day). Hepatocellular hypertrophy was present in males at 10 and 30 mg/kg up to minimal and slight degree, respectively. This was considered to be related to higher liver weights recorded at 30 mg/kg, which were approximately 20% higher than controls. Based on the absence of any additional test item-related degenerative, proliferative or inflammatory changes, the liver lesions are considered to be non-adverse. An increased incidence and severity of hyaline droplet accumulation up to marked degree was present in males at 30 mg/kg. This was considered to represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia. This male rat specific protein is not present in female rats nor in higher mammals, including man. There were no additional degenerative or inflammatory changes in this study and therefore the hyaline droplet accumulation was considered non-adverse.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
- Analysis of dose preparations: The concentrations analysed in the formulations of Group 2 and Group 3 were in agreement with target concentrations (i.e. mean accuracies between 85.00% and 115.00%). For the formulation of Group 4 prepared for use on 03 October 2016, the mean accuracy was below the target concentration (i.e. 68% of target). Therefore, the first reserve sample set was measured. The mean accuracy of this set was also below the target concentration (i.e. 66% of target). It was therefore decided to reanalyze the accuracy of the formulation of Group 4. The concentration analysed in this formulation was in agreement with the target concentration (i.e. 93% of target).

No test item was detected in the Group 1 formulation.
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (nominal)
System:
central nervous system
Organ:
other: adverse changes in motor activity
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
In conclusion, treatment with JNJ-130806-AAA (T000750) by oral gavage in male and female Wistar Han rats at dose levels of 3, 10 and 30 mg/kg resulted in adverse changes in motor activity at 30 mg/kg. Motor activity of females at 30 mg/kg was approximately a factor 3 higher than mean motor activity of the control group and was also distinctly higher than would be expected for nonmated females. For males at this dose level and for other female dose groups (trend), a much less pronounced and statistically non-significant higher mean motor activity was recorded. Based on these results, the No Observed Adverse Effect Level (NOAEL) was concluded to be 10 mg/kg.

Therefore, the substance is classified as a repeated dose toxicant (STOT RE) category 1 according to the CLP Regulation.


Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subacute
Species:
rat
System:
other: Neurobehavioral
Organ:
other: adverse changes in motor activity

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated toxicity: oral

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in rats, in which male and female rats were exposed to 0 (vehicle), 3, 10 and 30 mg/kg bw/day via gavage (OECD 422; Otterdijk F, 2017). The vehicle used was 1% aqueous carboxymethyl cellulose (CMC) and the test solutions were prepared daily and administered within 4 hours after preparation.

At 30 mg/kg, two females showed red discharge from the vagina on a single day of the postcoitum phase, and all females showed piloerection during one or more days in the postcoitum period. It is conceivable that these findings relate to the observation that none of the females at this dose delivered offspring (two were not pregnant and eight had implantation sites only). For most females of this dose group, weight loss (up to 9% of Day 1 premating body weights) along with lower food intake was recorded during the first week of treatment, which recovered as treatment progressed. For males at 30 mg/kg, body weight gain was slightly lower throughout treatment. Food intake levels however remained similar to control levels throughout treatment for both sexes.

Motor activity of females at 30 mg/kg was approximately a factor 3 higher than mean motor activity of the control group and was also distinctly higher than would be expected for nonmated females. For males at this dose level and for other female dose groups (trend), a much less pronounced and statistically non-significant higher mean motor activity was recorded.

There were no other adverse test item-related effects noted on clinical pathology, thyroid hormone T4, macroscopic examination, organ weights or histopathology up to 30 mg/kg.

Based on the abovementioned considerations, the NOAEL was considered to be 10 mg/kg bw/day (nominal dose received).

Repeated toxicity: inhalation

A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation route of exposure.

Repeated toxicity: dermal

A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the dermal route of exposure.

Justification for classification or non-classification

Based on the available data and according to the criteria of the CLP Regulation, T000750 should be classified as STOT RE 1 via the oral route.