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Description of key information

Oral: NOAEL (rat): 500 mg/kg body weight per day ; male/female, OECD TG 407, 2015

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29-01-2015 to 26-01-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US EPA OPPTS 870.3050, Repeated Dose 28-Day Oral Toxicity Study in Rodents (July 2000)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labor and Welfare (MHLW) and Ministry of the Environment (MOE) (31 March 2011)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: February 2014; signature: October 2015
Limit test:
yes
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The species and strain was selected in accordance with the OECD TG 407 and the other relevant guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: approximately 44 to 50 days.
- Weight at study initiation: males 207 - 271 g and females 157 - 192 g; individuals were randomly allocated to treatment groups.
- Fasting period before study: None
- Housing: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals. Wood based bedding (certified) which was changed at appropriate intervals per week. Environmental enrichment was provided in the form of wooden chew blocks and plastic shelter. Cage distribution within the holding rack was randomized.
- Diet: Rodent Maintenance Diet (certified supplier), ad libitum (removed overnight before blood sampling for haematology or blood chemistry and during the period of urine collection).
- Water (e.g. ad libitum): ad libitum (except during urine collection)
- Acclimation period: 15 days.

DETAILS OF FOOD AND WATER QUALITY: Feed: Rodent Maintenance Diet – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 2015-04-01 To: 2015-05-28
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly. The test item was prepared at the appropriate concentrations in corn oil vehicle. The required amounts of test material were weighed out into a suitable container. Starting with the lowest concentration, and in ascending concentration order: approximately 50% of the final volume of vehicle was added to the test material. The formulation was magnetically stirred until all test material was thoroughly mixed. The required volume was made up with vehicle and the formulation returned to the container and mixed using a magnetic stirrer until homogenous.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Applicant assessment indicates: Aqueous vehicle was not applicable due to limited solubility. Corn oil was considered as appropriate based on test item solubility. The stability and homogeneity of the test item formulations were determined during the study. Results show the formulations to be homogeneous and stable for at least fifteen days when stored refrigerated. Stability was confirmed at concentrations of 1 and 200 mg/mL following storage at ambient temperature (nominally 21 ºC) for 24 hours and refrigerated (nominally 4 ºC) for up to 15 days. Formulations were therefore prepared weekly during the treatment period, divided into daily aliquots and stored after one day at room temperature (nominally 21 ºC), refrigerated at nominally 4 ºC ºC in the dark.
- Concentration in vehicle: Samples of the test item formulations were taken and analyzed for concentration of test item (method of analysis provided in full study report). The results indicate that the prepared formulations were within ±6% of the nominal concentration. Corn oil formulations was assessed and confirmed at nominal concentrations, during refrigerated storage. The test item concentrations for each group are indicated in table 1.
- Amount of vehicle (if gavage): Treatment volume was 5 mL/kg for control (negative, untreated group) and all treatment groups with applicable test item concentrations per group. For further information see 'Doses / concentrations'.
- Other: Dose-formulations were analysed during the study and were reported as with ± 10 to 15 % applied limits. The precision of the individual results from mean value was less than 2%. Confirming the precision of the analysis.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The homogeneity and stability was confirmed in corn oil formulations, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 day and refrigeration for up to 15 days.
- Refrigerated formulations were also analysed after refrigeration on receipt, Day 1, Day 8 and Day 15, a bottle was removed from storage and equilibrated to ambient temperature. The contents of the bottle were mixed by 20-fold inversion followed by magnetic stirring for 5 minutes and single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the stirred formulation.
- The analysis consisted of GC FID analysis with internal calibration (within a dedicated formulation analysis report attached to the full study report).
A representative sample of test formulation (1 mL, accurately weighed) and dissolved using swirling in a suitable volume of acetone. The extract was diluted using acetone, where necessary to provide a solution containing test item at an expected concentration of 40 μg/mL. Solutions also contained internal standard solution at 32 μg/mL, by adding the appropriate amount of internal standard to each sample before making to volume. The concentration of test item in the final solution was quantified by GC using FID detection as detailed in the chromatographic section. The analytical method was validated (details available within the full study report).
- Mean concentrations of dose-formulations analysed during the study were within ± 10 to 15% applied limits and % difference from mean were within 5% nominal confirming accurate test item/vehicle formulation.
Duration of treatment / exposure:
Minimum period 28 days followed by a 14 day recovery period (treatment free). The last dose was administered on Day 28.
Frequency of treatment:
Once daily at approximately the same time each day.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Recovery control group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low - Group
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Intermediate - Group
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
High - Group
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Recovery High - Group
No. of animals per sex per dose:
5 per sex per dose (5 male / 5 female)
Additional 5 animals per sex and group (control and high dose group only) were treated for 28 days and then allowed a 14-day treatment-free recovery period.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a previously conducted 14-day sighting study (Report number attached to and cited in the full study report). Dose levels were selected following 14-day sighting test as: Group 1: 250 mg/kg/day, Group 2: 500 mg/kg/day Group 3: 1000 mg/kg/day (regulatory limit dose).I n the 14-day range finder (administered consecutively, for 14-days) determined: Two male and two female animals receiving 1000 mg/kg/day were humanely terminated on Day 2 due to poor clinical condition. After the second administration, clinical signs included abnormal gait, breathing irregularities, under activity, abnormal posture, abnormal eyes, piloerection and cold to touch. The surviving male/female were terminated on day 4. Necropsy findings included oedematous pancreas, distended stomach, pale liver and kidney depressions. At 500 mg/kg bw/day: abnormal gait, hunched posture and rapid respiration were seen in females on day 2 to 4. Lower than normal bodyweight gain was observed in males in days 1 to 4, although subsequent bodyweight gain was considered to be normal. Bodyweight relative liver weights were slightly higher than normal. Kidney and spleen weight was considered to be normal. Necropsy revealed in males pale kidneys. No significant findings were seen in females. At 250 mg/kg bw/day: there was no significant adverse signs of toxicity. On this basis the 500 mg/kg bw/day dose level was selected as the maximum dose level for further 28-day repeat dose toxicity testing. Basis: other: nominal in vehicle (Corn Oil)
- Rationale for animal assignment (if not random): Randomly assigned
- Post-exposure recovery period in satellite groups: 14 days.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All individuals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one to 2 hours after dosing. During the week 2 to termination and treatment free period, animals were observed daily. All observations were recorded. Additional functional observations were made as ‘additional evaluations’. Days 2, 3 and 4 to more closely monitor the condition of the animals and establish a pattern of signs. Signs were no longer observed at these additional time-points after Day 3 and, therefore, they were discontinued. In week 4, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded prior to dosing on Day 1 and at weekly intervals thereafter. Body weights were also performed prior to termination and, in the case of recovery group animals prior to termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not applicable.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY: No.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily. Water intake was observed daily, for each cage group, by visual inspection.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment period (day 29) for all non-recovery test and control group individuals. End of recovery period (day 15; recovery phase) for all recovery group individuals.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight.
- How many animals: All main study and recovery.
- Parameters checked: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices – including: mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), Total leukocyte count (WBC), Differential leukocyte count – including: neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), Platelet count (PLT). Morphology: Anisocytosis, Macrocytosis, Microcytosis, , Hypochromasia, Hyperchromasia
Additionally: Prothrombin time (PT) was assessed and Activated partial thromboplastin time (APTT) was assessed using samples collected into sodium citrate solution

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment period (day 29) for all non-recovery test and control group individuals. End of recovery period (day 15; recovery phase) for all recovery group individuals.
- Animals fasted: Yes, overnight.
- How many animals: All main study and recovery.
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transferase (gGT), Total bilirubin (Bili), Total bile acids (Bi Ac), Urea, Urea nitrogen (BUN), Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K),
Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb), Albumin/globulin ratio (A/G Ratio) was calculated

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalytical investigations were performed on all non-recovery test and control group animals during day 29 and on all recovery group animals during days 14-15.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (food withheld during time of urine collection; overnight)
- Parameters checked: urine volume, urine appearance, urine density, pH, ketones, bilirubin, urobilnogen, blood pigments, protein, sodium, potassium, chloride, creatinine, glucose. Microscopic examination: Epithelial cells, Leucocytes, Erythrocytes, Crystals, Casts, Spermatozoa, Other abnormal components.

NEUROBEHAVIOURAL EXAMINATION: Yes. Was conducted as part of ‘special evaluations’
- Time schedule for examinations: Prior to the start of treatment and on Days 7, 14, 21 and 28, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: All.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER: Additional post-termination observations were made at necropsy.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- organs weighed: Adrenals, Liver, Brain, Ovaries, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Pituitary (post-fixation), Thyroid/Parathyroid (post fixation), Prostate and Seminal Vesicles, Uterus with Cervix (with coagulating glands and fluids)

HISTOPATHOLOGY: Yes
- Organs and tissues preserved in neutral buffered 10% formalin: Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary, Bone & bone marrow (sternum), Prostate, Brain (including cerebrum, cerebellum and Rectum pons), Salivary glands (submaxillary), Caecum, Sciatic nerve, Colon, Seminal vesicles (with coagulating glands and fluids), Duodenum, Epididymides (Preserved in modified Davidson’s fluid), Skin, Esophagus, Spinal cord (cervical, mid thoracic and lumbar), Eyes (fixed in Davidson’s fluid), Gross lesions, Spleen, Heart, Stomach, Ileum ,Testes (Preserved in modified Davidson’s fluid), Jejunum, Thymus, Kidneys, Thyroid/Parathyroid, Liver, Trachea, Lungs (with bronchi) - inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative, Urinary bladder, Lymph nodes (mandibular and mesenteric), Uterus & Cervix, Mammary gland, Vagina, Muscle (skeletal).
Microscopic analysis was conducted thereof. Any macroscopically observed lesions were also processed.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed: Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Where appropriate, data transformations were performed using the most suitable method. Data were analysed using the decision tree from proprietary tables and statistics modules incorporating, homogeneity of variance from mean values was analysed using Bartlett’s test.
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. If Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead. Where there were only two groups, comparisons were made using t-tests.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test was applied.
For grip strength, motor activity and clinical pathology data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed.
For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom) were applied unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights.
Clinical signs:
no effects observed
Description (incidence and severity):
There was no test item related clinical signs in relation to treatment.

Post dose salivation and chin rubbing were evident during the first week of treatment; it was considered that these signs were from test item palatability of the dose administration rather than from toxicity.
Mortality:
no mortality observed
Description (incidence):
There was no test item related mortality.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no test item related effect in bodyweight gain.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
There was no test item related effect in food consumption.
Food efficiency:
no effects observed
Description (incidence and severity):
See body weight and weight changes sections.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg bw/day dose level: males/females water consumption was increased relative to controls during the last 9 days of treatment. On the second day of the recovery period the water consumption of these animals appeared higher than Control although no variation was apparent on subsequent days indicating recovery.
Ophthalmological findings:
not examined
Description (incidence and severity):
There were no toxicologically significant reported effects to the eyes (in life or post termination) in the parameters examined.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Lower than Control haemoglobin and mean cell haemoglobin concentration were apparent in females in all test groups attaining statistical significance, although a dose-relationship was not apparent with respect to haemaglobin concentration. These variations were restricted to a single sex, were without a clear dose-relationship nor microscopic correlates.

The observations were considered to be fortuitous and not test item related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 500 and 300 mg/kg bw/day: a dose-related higher than Control alanine amino transferase activities were evident in females. Lower than Control bile acids concentration was apparent in both sexes for all treated groups although a dose-relationship was not evident statistical significance was attained in males. Two weeks after cessation of treatment there was no apparent variation in alanine amino transferase activities or bile acids concentration in females, however, the in males the bile acid concentration was still lower than Control although statistical significance was not evident.

At 500 mg/kg bw/day: statistically significant lower than Control sodium and chloride ion concentrations and albumin: globulin ratios were evident in males and females. Lower than Control chloride ion concentrations were also apparent in males treated at 100 or 300 mg/kg/day attaining statistical significance. Statistically significant higher than Control phosphorous concentration was noted for males, the cholesterol level was also higher than Control for females at these dose levels. Fifteen days after cessation of treatment, these variations from Control were not evident indicating recovery.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
At 500 and 300 mg/kg bw/day: higher than Control protein, glucose and sodium and chloride ion concentrations were evident in males and females. Slightly more than control urine volume was apparent although without statistical significance.
At 100 mg/kg bw/day: higher than Control glucose concentration was apparent in males.

Fifteen days after cessation of treatment, these variations from Control were not evident indicating recovery.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item related effect on sensory reactivity or grip strength.

Within grip strength, during the fourth week of treatment forelimb grip strength values for all treated females were very slightly high compared with Controls with statistical significance. However, this was set against a atypical control group and/or the values were similar to historic control data. Rather than an effect of treatment.

Within motor activity at 500 mg/kg bw/day: several of the 6-minute intervals for high and low beam scores and the total scores for males were slightly high compared with Controls, with statistical significance attained at the 42-minute interval. The majority of 6-minute intervals for high beam scores and total scores, for females at all dose levels were slightly low compared with Controls with statistical significance being attained for the 12-minute interval for females treated at 500 mg/kg bw/day.

Two weeks after cessation of treatment, group mean activity scores for males remained high compared with Controls with statistical significance being
attained at several 6-minute interval scores for both high and low beams and the total score for high beams only. Control scores for the first 6-minute interval were below the historic control data. In the absence of a treatment related effect at Week 4, this finding at the end of the recovery period is considered not to be related to treatment.

All inter-group differences from control and/or attaining statistical significance were minor or restricted to single sex. Therefore considered to represent biological variation.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg bw/day: statistically significant higher than Control absolute and adjusted liver weights were evident in all treated groups; although at 500 mg/kg bw/day fifteen days after cessation of treatment liver weights remained statistically higher than Control, however, the difference from Control was reduced indicating partial recovery.

Higher than Control absolute and adjusted adrenal weights and absolute and adjusted uterus and cervix weights were evident in females treated with the adjusted weights attaining statistical significance; absolute uterus and cervix weights were also increased in females treated at 300 mg/kg bw/day although statistical significance was not attained.

At 500 or 300 mg/kg bw/day: statistically significant higher than Control absolute and adjusted kidney and thyroid/parathyroid weights were evident in males. A dose-relationship was not demonstrated for kidneys. Higher than Control thyroid/parathyroid adjusted weights were apparent in males treated at 100 mg/kg bw/day and in females treated at 500 mg/kg bw/day (adjusted weights).

Fifteen days after cessation of treatment, these variations from Control were not evident indicating recovery.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg bw/day: dark content was seen in the caecum of two males. One male that received 500 mg/kg bw/day (M18) had pale kidneys with a dark renal medulla. Pallor of the liver was seen in two males and all females.

At 300 mg/kg bw/day: dark content was seen in the caecum of two males. Two males (M23 and M25) had pale kidneys with a pale renal medulla. Pale kidneys were seen in one female correlated with microscopic minimal tubular vacuolation, this was not considered treatment related and was not seen in any other males/females. Pallor of the liver was seen in two males and three females.

At 100 mg/kg bw/day: Pallor of the liver was seen in two females.

The nature and incidence of all other findings were consistent with the commonly seen background of macroscopic changes and were considered to be unrelated to treatment.

After two weeks recovery: at 500 mg/kg bw/day: pale areas in the renal medulla of the kidney were observed in one male.

Macroscopic findings and increased kidney weights in males correlated with tubular basophilia/vacuolation and granular cysts. Tubular basophilia/vacuolation is considered to be an early degenerative change. Although two weeks after cessation of treatment the weights of the kidneys were similar to Control values, tubular basophilia/vacuolation and granular casts were still evident at a similar incidence and severity indicating that recovery had not occurred.

Correlation with histopathology is indicated and further discussed in 'histopathological findings: non-neoplastic'.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg bw/day: kidneys: treatment related changes were seen in the kidneys of males included tubular basophilia/vacuolation, granular casts and minimal accumulation of hyaline droplets. Liver: centrilobular hypertrophy was seen in the liver of males and females. Minimal to moderate periportal vacuolation was seen in one male and in females. Thyroid: minimal follicular cell hypertrophy in the thyroid gland was evident in males and females.

After two weeks recovery: kidneys: hyaline droplets were absent but tubular basophilia/vacuolation and granular casts were still present. Liver: Periportal vacuolation and centrilobular hypertrophy were reduced in incidence or absent. Thyroid: Follicular cell hypertrophy was recorded in both sexes at an incidence similar to the main
Study.

At 300 mg/kg bw/day: kidneys: treatment related changes were seen in the kidneys of males included tubular basophilia/vacuolation, granular casts and minimal accumulation of hyaline droplets (with lower incidences than at 300 and 100 mg/kg bw/day than 500 mg/kg bw/day). Liver: centrilobular hypertrophy was seen in the liver of males and females.. Minimal to moderate periportal vacuolation was seen in females. Thyroid: minimal follicular cell hypertrophy in the thyroid gland was evident in females.

At 100 mg/kg bw/day: kidneys: treatment related changes were seen in the kidneys of males included tubular basophilia/vacuolation and minimal accumulation of hyaline droplets. Liver: centrilobular hypertrophy was seen in the liver of females. Minimal to moderate periportal vacuolation was seen in one male and females. Thyroid: minimal follicular cell hypertrophy in the thyroid gland was isolated in males and including controls were considered to represent background variation.

Hyaline droplets were evident in the kidneys of males treated at all dose levels after 28 days of administration; it was not present after two weeks of recovery. The presence of hyaline droplets, also known as intra-cytoplasmic protein droplets, in the cortical tubules of male kidneys is the early indicator of hydrocarbon neuropathy. The droplets are considered to be the result of reversible binding of test item to α2-microglobulin leading to accumulation within the tubular cell lysoma in the kidney. α2-microglobulin is not present in humans. This finding is not considered relevant to human health.

Pale areas evident macroscopically in the liver were considered to be correlated with microscopic observations of periportal vacuolation due to intracytoplasmic lipid accumulation. The increased alanine amino transferase activities seen in females treated at 300 or 500 mg/kg bw/day were considered to be due to leakage caused by hepatocellular periportal vacuolation. The increase in liver weights was associated with hepatocyte hypertrophy and due to hepatic enzyme induction. Which correlates with thyroid findings and/or increased organ weight and follicular cell hypertrophy. The presence of both changes together in males treated with 100 mg/kg bw/day and males and females treated at 300 or 500 mg/kg bw/day is indicative of a relationship between the liver and thyroid changes. Which is a species specific mechanism in rodents and/or considered to reflect an adaptive change. Two weeks after cessation of treatment follicular cell hypertrophy was still apparent at the same incidence and severity indicated that recovery had not occurred.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related abnormalities detected.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Remarks:
The liver, kidney and thyroid findings were considered either adaptive or have no relevance to human health.
Key result
Critical effects observed:
no
Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for males and females is defined as 500 mg/kg body weight per day in males and females; since the liver, kidney and thyroid findings were considered either adaptive or have no relevance to human health.
Executive summary:

The study was performed according the requirements of OECD TG 407, EU method B.7, US EPA OPTTS 870.3050 and Japan MHLW, METI and MOE guidelines under GLP conditions. Following a previously conducted 14-day sighting study, the systemic toxic potential of the test item was assessed orally in a 28 day gavage study in Crl:CD(SD) rats. Recovery from any effects was evaluated during a subsequent 14 day recovery period. Three groups, each comprising five male and five female CD rats, received test item at doses of 100, 300 or 500 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Corn Oil) at a dose volume of 5 mL/kg. Two recovery groups, each of five males and five females, were treated with the high dose (500 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Clinical signs, functional observations including sensory reactivity, grip strength and motor activity were performed, body weight change, food and water consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment free period. All individuals were subjected to gross necropsy examination and at termination. Histopathological examination of selected tissues was performed. There was no test item related effect on mortalities, clinical signs, effect on sensory reactivity, grip strength or motor activity, bodyweight gain or food consumption. There was an increased water consumption was evident at 500 mg/kg bw/day during the last week of treatment. There was no effect on hematology. In clinical chemistry: higher than Control alanine amino transferase activities were evident in females at 300 or 500 mg/kg bw/day and lower than Control bile acids concentration was apparent in all treated groups; two weeks after cessation of treatment there was no apparent variation in alanine amino transferase activities nor in bile acids concentration in females, however, in males the concentration was still lower than Control. Lower than Control sodium and chloride ion concentrations and albumen: globulin ratios were evident in males and females at 500 mg/kg bw/day; lower than Control chloride ion concentrations were also apparent in males at 100 or 300 mg/kg bw/day. Fifteen days after cessation of treatment, these variations from Control were not evident indicating recovery. In urinalysis: greater than Control protein, glucose and sodium and chloride ion concentrations were evident in males and females at 300 or 500 mg/kg bw/day; higher than control glucose concentration was apparent in males at 100 mg/kg bw/day. Fifteen days after cessation of treatment, these variations from Control were not evident indicating recovery. In organ weights: higher than Control absolute and adjusted liver weights were evident in all treated groups; fifteen days after cessation of treatment, the liver weights of animals previously treated at 500 mg/kg bw/day remained higher than Control, however, the difference form Control had reduced since Week 4 indicating partial recovery. Greater than Control absolute and adjusted kidney and thyroid/parathyroid weights were evident in males at 300 or 500 mg/kg bw/day; higher than Control thyroid/parathyroid weights were apparent in males treated at 100 mg/kg bw/day and in females treated at 500 mg/kg bw/day (adjusted weights). Higher than Control absolute and adjusted adrenal weights and absolute and adjusted uterus and cervix weights were evident in females treated at 500 mg/kg bw/day; absolute uterus and cervix weights were also increased in females treated at 300 mg/kg bw/day. Fifteen days after cessation of treatment, these variations from Control were not evident indicating recovery. Macroscopic changes related to treatment with test item were seen in the caecum, kidney and liver after four weeks treatment. Two weeks after cessation, treatment related macroscopic findings were evident in the kidney of one male. Microscopic changes related to treatment with test item were seen in the kidney of males and liver and thyroid of males and females. Two weeks after cessation of treatment, treatment-related changes were still present in the kidneys, liver and thyroids, with evidence of partial recovery in all organs. Macroscopic findings and increased kidney weights in males correlated with tubular basophilia/vacuolation and granular cysts. Tubular basophilia/vacuolation is considered to be an early degenerative change. Although two weeks after cessation of treatment the weights of the kidneys were similar to Control values, tubular basophilia/vacuolation and granular casts were still evident at a similar incidence and severity indicating that recovery had not occurred. Hyaline droplets were evident in the kidneys of males treated at all dose levels after 28 days of administration; it was not present after two weeks of recovery. The presence of hyaline droplets, also known as intra-cytoplasmic protein droplets, in the cortical tubules of male kidneys is the early indicator of hydrocarbon neuropathy. The droplets are considered to be the result of reversible binding of test item to α2-microglobulin leading to accumulation within the tubular cell lysoma in the kidney. α2-microglobulin is not present in humans. This finding is not considered relevant to human health. Pale areas evident macroscopically in the liver were considered to be correlated with microscopic observations of periportal vacuolation due to intracytoplasmic lipid accumulation. The increased alanine amino transferase activities seen in females treated at 300 or 500 mg/kg bw/day were considered to be due to leakage caused by hepatocellular periportal vacuolation. The increase in liver weights was associated with hepatocyte hypertrophy and due to hepatic enzyme induction. Which correlates with thyroid findings and/or increased organ weight and follicular cell hypertrophy. The presence of both changes together in males treated with 100 mg/kg bw/day and males and females treated at 300 or 500 mg/kg bw/day is indicative of a relationship between the liver and thyroid changes. Which is a species specific mechanism in rodents and/or considered to reflect an adaptive change. Two weeks after cessation of treatment follicular cell hypertrophy was still apparent at the same incidence and severity indicated that recovery had not occurred.

The oral (gavage) administration of the test item to males/females at dose levels of 100, 300 or 500 mg/kg bw/day resulted in liver, kidney and thyroid findings that were considered either adaptive or have no relevance to human health. Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 500 mg/kg bw/day for males/females.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is GLP compliant and of a high quality (Klimisch 1); The available information as a whole meets the tonnage driven information requirements of REACH.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose - Oral:

Key study: OECD TG 407, 2015 : The study was performed according the requirements of OECD TG 407, EU method B.7, US EPA OPTTS 870.3050 and Japan MHLW, METI and MOE guidelines under GLP conditions. Following a previously conducted 14-day sighting study, the systemic toxic potential of the test item was assessed orally in a 28 day gavage study in Crl:CD(SD) rats. Recovery from any effects was evaluated during a subsequent 14 day recovery period. Three groups, each comprising five male and five female CD rats, received test item at doses of 100, 300 or 500 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Corn Oil) at a dose volume of 5 mL/kg. Two recovery groups, each of five males and five females, were treated with the high dose (500 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Clinical signs, functional observations including sensory reactivity, grip strength and motor activity were performed, body weight change, food and water consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment free period. All individuals were subjected to gross necropsy examination and at termination. Histopathological examination of selected tissues was performed. There was no test item related effect on mortalities, clinical signs, effect on sensory reactivity, grip strength or motor activity, bodyweight gain or food consumption. There was an increased water consumption was evident at 500 mg/kg bw/day during the last week of treatment. There was no effect on hematology. In clinical chemistry: higher than Control alanine amino transferase activities were evident in females at 300 or 500 mg/kg bw/day and lower than Control bile acids concentration was apparent in all treated groups; two weeks after cessation of treatment there was no apparent variation in alanine amino transferase activities nor in bile acids concentration in females, however, in males the concentration was still lower than Control. Lower than Control sodium and chloride ion concentrations and albumen: globulin ratios were evident in males and females at 500 mg/kg bw/day; lower than Control chloride ion concentrations were also apparent in males at 100 or 300 mg/kg bw/day. Fifteen days after cessation of treatment, these variations from Control were not evident indicating recovery. In urinalysis: greater than Control protein, glucose and sodium and chloride ion concentrations were evident in males and females at 300 or 500 mg/kg bw/day; higher than control glucose concentration was apparent in males at 100 mg/kg bw/day. Fifteen days after cessation of treatment, these variations from Control were not evident indicating recovery. In organ weights: higher than Control absolute and adjusted liver weights were evident in all treated groups; fifteen days after cessation of treatment, the liver weights of animals previously treated at 500 mg/kg bw/day remained higher than Control, however, the difference form Control had reduced since Week 4 indicating partial recovery. Greater than Control absolute and adjusted kidney and thyroid/parathyroid weights were evident in males at 300 or 500 mg/kg bw/day; higher than Control thyroid/parathyroid weights were apparent in males treated at 100 mg/kg bw/day and in females treated at 500 mg/kg bw/day (adjusted weights). Higher than Control absolute and adjusted adrenal weights and absolute and adjusted uterus and cervix weights were evident in females treated at 500 mg/kg bw/day; absolute uterus and cervix weights were also increased in females treated at 300 mg/kg bw/day. Fifteen days after cessation of treatment, these variations from Control were not evident indicating recovery. Macroscopic changes related to treatment with test item were seen in the caecum, kidney and liver after four weeks treatment. Two weeks after cessation, treatment related macroscopic findings were evident in the kidney of one male. Microscopic changes related to treatment with test item were seen in the kidney of males and liver and thyroid of males and females. Two weeks after cessation of treatment, treatment-related changes were still present in the kidneys, liver and thyroids, with evidence of partial recovery in all organs. Macroscopic findings and increased kidney weights in males correlated with tubular basophilia/vacuolation and granular cysts. Tubular basophilia/vacuolation is considered to be an early degenerative change. Although two weeks after cessation of treatment the weights of the kidneys were similar to Control values, tubular basophilia/vacuolation and granular casts were still evident at a similar incidence and severity indicating that recovery had not occurred. Hyaline droplets were evident in the kidneys of males treated at all dose levels after 28 days of administration; it was not present after two weeks of recovery. The presence of hyaline droplets, also known as intra-cytoplasmic protein droplets, in the cortical tubules of male kidneys is the early indicator of hydrocarbon neuropathy. The droplets are considered to be the result of reversible binding of test item to α2-microglobulin leading to accumulation within the tubular cell lysoma in the kidney. α2-microglobulin is not present in humans. This finding is not considered relevant to human health. Pale areas evident macroscopically in the liver were considered to be correlated with microscopic observations of periportal vacuolation due to intracytoplasmic lipid accumulation. The increased alanine amino transferase activities seen in females treated at 300 or 500 mg/kg bw/day were considered to be due to leakage caused by hepatocellular periportal vacuolation. The increase in liver weights was associated with hepatocyte hypertrophy and due to hepatic enzyme induction. Which correlates with thyroid findings and/or increased organ weight and follicular cell hypertrophy. The presence of both changes together in males treated with 100 mg/kg bw/day and males and females treated at 300 or 500 mg/kg bw/day is indicative of a relationship between the liver and thyroid changes. Which is a species specific mechanism in rodents and/or considered to reflect an adaptive change. Two weeks after cessation of treatment follicular cell hypertrophy was still apparent at the same incidence and severity indicated that recovery had not occurred.

The oral (gavage) administration of the test item to males/females at dose levels of 100, 300 or 500 mg/kg bw/day resulted in liver, kidney and thyroid findings that were considered either adaptive or have no relevance to human health. Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 500 mg/kg bw/day for males/females.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for specific organ toxicity repeated exposure (STOT-RE).

Since there was no reported significant effects relevant to humans reported at guidance related levels (ORAL ≤ 300 mg/kg bw/day) then there is no requirement to classify STOT-RE.

References:

1. ECHA Guidance on Application on the CLP Criteria, (v5.0, July 2017), Section 3.9.2 : Table 3.16 - Equivalent guidance values for 28-day and 90-day studies

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