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Description of key information

Oral: NOAEL (rat): 750 mg/kg bw/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:
07-01-2014 to 12-09-2014
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: April 2014
Limit test:
yes
Species:
rat
Strain:
Crj: CD(SD)
Remarks:
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 38 to 44 days.
- Weight at study initiation: males 175 - 229 g and females 145 - 189 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 No. 1 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: 9 days.

DETAILS OF FOOD AND WATER QUALITY: Feed: Rodent No. 1 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: 2014-06-13 To: 2014-07-11
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

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 (2-8 ºC) for up to 15 days.
Formulations were therefore prepared weekly during the treatment period, divided into daily aliquots and stored at approximately 2-8 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 ±5% 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 3%. 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 refrigerated storage for at least fifteen (15) days.
- Refrigerated formulations were also analysed after refrigeration on receipt, 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, to provide a solution containing test item at an expected concentration of 40 μg/mL. Solutions also contained internal standard solution at 40 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% 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.:
30 mg/kg bw/day (nominal)
Remarks:
Low - Group
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Intermediate - Group
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
High - Group; Dosage was reduced from 1000 mg/kg bw/day to 750 mg/kg bw/day from Day 2
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
Recovery High - Group ; Dosage was reduced from 1000 mg/kg bw/day to 750 mg/kg bw/day from Day 2
No. of animals per sex per dose:
10 per sex per dose (5 male / 5 female); 5 per sex per dose for recovery phase groups
Control animals:
yes
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: 0 mg/kg/day (Corn Oil) Group 2: 30 mg/kg/day, Group 3: 300 mg/kg/day Group 4: 1000 mg/kg/day (regulatory limit dose). Initial high dose of 1000 mg/kg bw/day was reduced to 750 mg/kg bw/day from day 2 due to unexpected toxicity and poor prognosis for survival.
In the 7-day range finder (administered consecutively, for 14-days) determined: no significant adverse effects detected at up to 1000 mg/kg/day using dose levels of 250, 500 and 1000 mg/kg/day. 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/behavioral 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. A possible intergroup difference was detected during, therefore water consumption was continued during weeks 3 and 4 by quantitative measurement until the termination.

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).
Additionally: Prothrombin time (CT) 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 28) for all non-recovery test and control group individuals. End of recovery period (day 42) for all recovery group individuals.
- Animals fasted: No.
- How many animals: All animals
- Parameters checked: Urea, Aspartate aminotransferase (ASAT), Glucose, Alanine aminotransferase (ALAT), Total protein (Tot. Prot.), Alkaline phosphatase (AP), Albumin, Creatinine (Creat), Albumin/Globulin (A/G) ratio (by calculation), Total cholesterol (Chol), Sodium (Na+), Total bilirubin (Bili), Potassium (K+), Triglycerides (Tri), Chloride (Cl-), Bile acids, Calcium (Ca++), Gamma glutamyltranspeptidase, Inorganic phosphorus (P)

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/behavioral 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.
Other examinations:
- Thyroid Hormone Assessment: blood samples were taken at exsanguination and the plasma was stored frozen at approximately -20 °C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples were discarded.
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:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day dose level: Signs observed on Day 1 comprised swaying and elevated gait, body tremors, uncoordinated behaviour, piloerection and abnormally cold to touch in animals receiving 1000 mg/kg/day. Females were affected to a greater extent than males. The signs became apparent from 15 minutes of dosing. One female receiving 1000 mg/kg/day (No. 54) was killed on Day 1 because of its poor clinical condition and continued deterioration. Signs observed for this animal comprised piloerection, partially closed eyelids, impaired locomotion, splayed hind limbs, elevated and swaying gait, decreased activity, uncoordinated behaviour and body tremors. Macroscopic examination at necropsy revealed a distended stomach. Histopathology revealed nothing of significance. All survivors had recovered by the end of day 1, with exception of two females: uncoordinated with elevated gait. Due to unexpected mortality – the high dose was reduced from 1000 mg/kg bw/day to 750 mg/kg bw/day from day 2.

At 750 mg/kg bw/day dose level: Following the reduction in dose level, elevated gait, piloerection, eating of bedding, decreased activity, and partially closed eyelids were seen for a small number of animals on Days 2 and/or 3, occurring shortly after dosing and, for some, persisting to the end of the working day. These signs were not apparent from Day 4 and did not occur again during the study. Salivation and associated chin rubbing were observed during the first five days of treatment for animals receiving 750/1000 mg/kg/day; chin rubbing was also observed for two females receiving 300 mg/kg/day. Chin rubbing and salivation generally appeared as the animals were returned to their cage after dose administration and were no longer apparent 1 to 2 hours later. These clinical signs are considered to be typically related to the taste of the test-article and not of toxicological importance.

At 30 and 300 mg/kg bw/day dose levels: with the exception of chin rubbing seen in two females at 300 mg/kg bw/day there were no clinical signs.

During the treatment-free period, no clinical observations were detected for any of the recovery group. Therefore there was evidence of recovery.
Mortality:
mortality observed, treatment-related
Description (incidence):
At 1000 mg/kg bw/day dose level: one female (No. 54) was killed on Day 1 because of its poor clinical condition and continued deterioration.
Due to unexpected mortality – the high dose was reduced from 1000 mg/kg bw/day to 750 mg/kg bw/day from day 2. There was no further mortality.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day dose level: Body weight gain for males receiving 750/1000 mg/kg/day was statistically significantly lower than that of the controls during the last two weeks of treatment, resulting in a slightly low value overall (Day 1 to 28, 0.90X control; although this did not attain statistical significance). Females were unaffected.

At 30 and 300 mg/kg bw/day dose levels: body weight gain was unaffected by treatment.

During the recovery period, body weight gains for previously treated males were similar to or slightly higher than those of the controls indicating recovery had occurred. Therefore there was evidence of recovery.

Therefore, there was limited effects seen on bodyweight post-exposure and evidence of recovery at all dose levels in males and females.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
no effects observed
Description (incidence and severity):
Food consumption during the treatment period for females receiving 750/1000 mg/kg bw/day and, to a lesser extent, females receiving 300 mg/kg bw/day was consistently higher than that of the controls. A similar trend towards higher food consumption was also apparent from Week 2 of treatment for males receiving the high dose level, though to a lesser degree.
During the recovery period, food consumption for previously treated females remained slightly higher than that of the Controls, though some recovery was evident. Food consumption for previously treated males was similar to that of the controls. Applicant assessment is that this is of no toxicological significance.

Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day dose level: Visual observation of fluid intake indicated increased water consumption during the treatment period for animals receiving 300 or 750/1000 mg/kg/day compared with controls. Quantitative water consumption measurements in Week 3 and 4 confirmed that animals receiving 750/1000 mg/kg/day consumed more water than the controls (approximately 1.2X and 1.6X control for males and females respectively).

At 30 and 300 mg/kg bw/day dose levels: 300 mg/kg/day was not clearly affected by treatment with just a marginal increase in water consumption compared with controls by quantitative water measurement. 30 mg/kg bw/day were unaffected.

Water consumption during the recovery period was similar to the controls for previously treated males, and only marginally higher than controls, but less than during the treatment period for previously treated females, indicating recovery.

Therefore, there was limited effects seen on water consumption post-exposure and evidence of recovery at all dose levels in males and females.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 750 and 300 mg/kg bw/day dose level: Following two weeks of recovery, the effect on prothrombin and activated partial thromboplastin times for previously treated animals had resolved. All individual values for activated partial thromboplastin times were within the background range indicating that the degree of change was not adverse. The majority of individual values for prothrombin time, including three of the controls, were above the 90-percentile background range, but when compared with concurrent controls Although only one female was affected, given the degree of effect and a similar effect being seen in males, the change was considered to be treatment-related. Activated partial thromboplastin times were unaffected for females.

A small number of other inter-group differences from controls attained statistical significance, but these were minor, confined to one sex or lacked dose-relationship and were, therefore, attributed to normal biological variation. Such differences included the slightly reduced basophil counts in females receiving 300 or 750/1000 mg/kg/day and increased monocyte counts in females at the high dose at the end of the treatment period, where all individual values for these animals were within the background range, and the difference in basophils arose as a consequence of a high value in the control group that raised the group mean.

At 30 mg/kg bw/day dose levels: No treatment related effects were seen.

Slightly low red blood cell counts which were evident for previously treated females at the end of the recovery period, with all individual values within the background-range and where there was no similar effect at the end of treatment, were also attributed to normal variation.

Therefore, there was limited effects seen on haematology post-exposure and evidence of recovery at all dose levels in males and females.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
At 750 and 300 mg/kg bw/day dose level: Urea and blood urea nitrogen concentrations were statistically significantly high for males receiving 300 or 750/1000 mg/kg/day. Glucose concentration was low for animals receiving 750/1000 mg/kg bw/day and females receiving 300 mg/kg bw/day. Cholesterol and triglyceride concentrations were increased for females receiving 750/1000 mg/kg bw/day. Bile acid concentration was statistically significantly higher than that of the control for females receiving 750/1000 mg/kg bw/day. Sodium and chloride concentrations were slightly low for animals receiving 750/1000 mg/kg bw/day; attaining statistical significance in females only. Albumin concentration was slightly low for males receiving 300 or 750/1000 mg/kg bw/day.

At 30 mg/kg bw/day dose levels: No significant treatment related effects were reported.

On completion of the 14 day recovery period, albumin/globulin ratio was still slightly low for previously treated males, however the effect on albumin concentration had resolved and all individual values were within the background control ranges and, therefore, these differences were, therefore, of no toxicological significance. Triglyceride concentration in previously treated females remained slightly higher than that of the controls, but the difference was no longer statistically significant and all individual values were within the background range and, therefore, considered of no toxicological significance. All other changes had resolved.

Therefore, there was limited effects seen on clinical chemistry post-exposure and at least partial evidence of recovery at all dose levels in males and females.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
At 750 and 300 mg/kg bw/day dose level: Urinalysis investigations after 28 days of treatment revealed low pH for animals receiving 750/1000 mg/kg/day; high total protein and creatinine output for females receiving 750/1000 mg/kg/day and high specific gravity for males receiving 750/1000 mg/kg/day.

At 30 mg/kg bw/day dose levels: No significant treatment related effects were reported.

All effects had reversed after the 14-day recovery period.

Therefore, there was limited effects seen on urinalysis post-exposure and evidence of recovery at all dose levels in males and females.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
At 750 and 300 mg/kg bw/day dose level: During Week 4 of treatment motor activity for males receiving 1000/750 mg/kg/day was slightly higher than that of the Controls, though there was high individual variation. During Week 2 of recovery, the same pattern occurred with high and low beam scores for males previously treated at 750/1000 mg/kg/day being higher than Controls and with the two animals that had shown the highest activity after four weeks of treatment similarly showing the highest activity during Week 2 of recovery. These changes were considered of doubtful toxicological significance. Sensory reactivity responses and grip strength were unaffected by treatment with no significant difference from controls.

At 30 mg/kg bw/day dose levels: No significant treatment related effects were reported.

All effects partially or fully reversed after the 14-day recovery period, with none of the differences attaining statistical significance.

Therefore, there was limited effects seen on behaviour and sensory/functional performance post-exposure and evidence of recovery at all dose levels in males and females.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 750 and 300 mg/kg bw/day dose level: Higher liver weights for animals which received 300 or 750/1000 mg/kg/day were seen. In males, just the body weight adjusted weights were high, whilst in females, both the absolute and body weight adjusted weights were increased (maximum effect 1.2X and 1.3X in males and females, respectively); attaining statistical significance. In addition, body weight-adjusted kidney weights were also statistically significantly high, compared with controls, for males which received 300 or 750/1000 mg/kg/day (maximum effect 1.1X control). There was no similar effect in females.

Absolute and body weight-adjusted ovary weights for females which received 750/1000 mg/kg/day were low when compared with the controls (0.68X 0.66X control for
absolute and body weight-adjusted weights, respectively); attaining statistical significance. With the exception of one low value (animal No, 53), individual values were within the background data range (90-percentile range (absolutes) 0.062 - 0.113g, n=296).

At 30 mg/kg bw/day dose levels: No significant treatment related effects were reported.

All effects partially or fully reversed after the 14-day recovery period, with none of the differences attaining statistical significance.

Therefore, there was limited effects seen on organ weights post-exposure and evidence of recovery at all dose levels in males and females.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic abnormalities detected.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related abnormalities detected.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related abnormalities detected.
Other effects:
no effects observed
Description (incidence and severity):
1. Thyroid Hormone Assessment: no effects were identified to the thyroid (organ weight) and therefore further analysis was not conducted.
Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
haematology
organ weights and organ / body weight ratios
urinalysis
water consumption and compound intake
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 750 mg/kg body weight per day in males and females; findings were either of limited toxicological significance, adaptive changes or have no relevance for 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 30, 300 or 1000 mg/kg/day. During the definitive test following day 1 and unexpected mortality, the high dose was reduced to 750 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 (750 mg/kg bw/day; reduced from 1000 mg/kg bw/day on day 2) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Clinical signs, 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 unscheduled mortality at 1000 mg/kg bw/day due to poor clinical condition which led to a reduction of dose level to 750 mg/kg bw/day on day 2. Transient clinical signs related to dose including swaying and elevated gait, body tremors, uncoordinated behaviour, piloerection and reduced body temperature at 1000 mg/kg bw/day on Day 1, and to a lesser extent on Days 2 and 3 following a reduction in dose level to 750 mg/kg bw/day. These signs were not evident from Day 4. salivation and associated chin rubbing were observed during the first five days of treatment for animals receiving 750 mg/kg bw/day; chin rubbing was also observed for two females receiving 300 mg/kg bw/day. These clinical signs are considered to be typically related to the taste of the test-article and not of toxicological importance. Treatment related motor activity effects in males were considered of doubtful toxicological significance. Sensory reactivity responses and grip strength were unaffected by treatment with no significant difference from controls. Overall body weight gain for males receiving 750 mg/kg bw/day was lower than that of the controls, predominantly due to low gains in the last two weeks of treatment. Recovery was evident following cessation of treatment. Food consumption and water consumption was high during the treatment period for males and females receiving 750 mg/kg bw/day. There was evidence of recovery following cessation of treatment. Haematological examination after 28 days of treatment revealed, when compared with the controls, increased group mean prothrombin and activated partial thromboplastin times for males receiving 300 or 750 mg/kg bw/day. Prothrombin time was also increased for one female receiving 750 mg/kg bw/day. Following two weeks of recovery, these changes had resolved. The biochemical examination of the blood plasma obtained after 28 days of treatment revealed high urea, blood urea nitrogen concentration and alanine amino transferase activity, low glucose concentration, high cholesterol and triglyceride concentrations, high bile acid, low sodium and chloride and slightly low albumin with an associated low albumin/globulin ratio at the high dose. All changes had fully or partially resolved on completion of the recovery period. Urinalysis investigations after 28 days of treatment revealed low pH, high total protein and creatinine for females, and high specific gravity for males receiving 750 mg/kg bw/day. None of these changes were apparent on completion of the recovery period. There were no treatment-related macroscopic abnormalities detected in treatment or recovery groups. Analysis of organ weights, when compared with the controls, high liver weights for animals which received 300 or 750 mg/kg bw/day. In males, just the body weight-adjusted weights were high, whilst in females, both the absolute and body weight adjusted weights were increased. In addition, body weight-adjusted kidney weights were high for males which received 300 or 750 mg/kg bw/day. Absolute and body weight-adjusted ovary weights for females which received 750 mg/kg/day were low when compared with the controls. After two weeks of recovery, all findings showed partial or full recovery. There were no significant microscopic or histopathological findings in treatment or recovery groups. There were no test item related effects seen in 30 mg/kg bw/day.

The oral (gavage) administration of the test item to males/females at dose levels of 30, 300 or 750 mg/kg bw/day resulted in transient clinical signs, increased motor activity and treatment-related effects on body weight, water consumption, clotting factors, clinical chemistry and urine parameters and liver and kidney weights. These findings showed at least partial recovery following two weeks off treatment. There were no pathological changes. Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 750 mg/kg/day for males/females since findings are either adaptive or have no relevance to human health.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study is GLP compliant and of a high quality (Klimisch 1)

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:

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 30, 300 or 1000 mg/kg/day. During the definitive test following day 1 and unexpected mortality, the high dose was reduced to 750 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 (750 mg/kg bw/day; reduced from 1000 mg/kg bw/day on day 2) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. There were no test item related effects seen in 30 mg/kg bw/day. There was unscheduled mortality at 1000 mg/kg bw/day due to poor clinical condition which led to a reduction of dose level to 750 mg/kg bw/day on day 2. Transient clinical signs related to dose including swaying and elevated gait, body tremors, uncoordinated behaviour, piloerection and reduced body temperature at 1000 mg/kg bw/day on Day 1, and to a lesser extent on Days 2 and 3 following a reduction in dose level to 750 mg/kg bw/day. These signs were not evident from Day 4. salivation and associated chin rubbing were observed during the first five days of treatment for animals receiving 750 mg/kg bw/day; chin rubbing was also observed for two females receiving 300 mg/kg bw/day. These clinical signs are considered to be typically related to the taste of the test-article and not of toxicological importance. Treatment related motor activity effects in males were considered of doubtful toxicological significance. Sensory reactivity responses and grip strength were unaffected by treatment with no significant difference from controls. Overall body weight gain for males receiving 750 mg/kg bw/day was lower than that of the controls, predominantly due to low gains in the last two weeks of treatment. Recovery was evident following cessation of treatment. Food consumption and water consumption was high during the treatment period for males and females receiving 750 mg/kg bw/day. There was evidence of recovery following cessation of treatment. Haematological examination after 28 days of treatment revealed, when compared with the controls, increased group mean prothrombin and activated partial thromboplastin times for males receiving 300 or 750 mg/kg bw/day. Prothrombin time was also increased for one female receiving 750 mg/kg bw/day. Following two weeks of recovery, these changes had resolved. The biochemical examination of the blood plasma obtained after 28 days of treatment revealed high urea, blood urea nitrogen concentration and alanine amino transferase activity, low glucose concentration, high cholesterol and triglyceride concentrations, high bile acid, low sodium and chloride and slightly low albumin with an associated low albumin/globulin ratio at the high dose. All changes had fully or partially resolved on completion of the recovery period. Urinalysis investigations after 28 days of treatment revealed low pH, high total protein and creatinine for females, and high specific gravity for males receiving 750 mg/kg bw/day. None of these changes were apparent on completion of the recovery period. There were no treatment-related macroscopic abnormalities detected in treatment or recovery groups. Analysis of organ weights, when compared with the controls, high liver weights for animals which received 300 or 750 mg/kg bw/day. In males, just the body weight-adjusted weights were high, whilst in females, both the absolute and body weight adjusted weights were increased. In addition, body weight-adjusted kidney weights were high for males which received 300 or 750 mg/kg bw/day. Absolute and body weight-adjusted ovary weights for females which received 750 mg/kg/day were low when compared with the controls. After two weeks of recovery, all findings showed partial or full recovery. There were no significant microscopic or histopathological findings in treatment or recovery groups. The oral (gavage) administration of the test item to males/females at dose levels of 30, 300 or 750 mg/kg bw/day resulted in transient clinical signs, increased motor activity and treatment-related effects on body weight, water consumption, clotting factors, clinical chemistry and urine parameters and liver and kidney weights. These findings showed at least partial recovery following two weeks off treatment. There were no pathological changes. Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 750 mg/kg/day for males/females since findings are either adaptive or have no relevance to human health.

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).