Registration Dossier

Administrative data

Description of key information

Treatment of rats at dosages up to 750 mg/kg bw/day for twenty-eight consecutive days was well tolerated and the ‘No Observed Adverse Effect Level’ (NOAEL) for toxicity was considered to be 750 mg/kg bw/day.
In a supporting study treatment of rats for 28-days by oral gavage gave a NOAEL of 500 mg/kg bw/day, the highest dose tested.
Effects observed during the treatment period were reversed during a two-week recovery period.

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:
15 June 2012 to 20 February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, labour and Welfare (MHLW) and Ministry of the environment (MOE) guidelines of 21 November 2003 for a twenty-eight day repeat dose oral toxicity study
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: USA Environmental Protection Agency (EPA) Health Effects Test guidelines, OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Wistar Han (TM): HsdRccHan (TM):WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Harlan UK Limited, Blackthorn, Bicester, Oxon, UK- Weight at study initiation: males 195-238g, females 142-168g- Fasting period before study: Overnight- Housing:Suspended solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flake bedding- Diet (e.g. ad libitum): ad libitum- Water (e.g. ad libitum): ad libitum- Acclimation period: 6 daysENVIRONMENTAL CONDITIONS- Temperature: 22+/-3 °C- Humidity (%): 40-75 %- Air changes (per hr): at least 15- Photoperiod (hrs dark / hrs light): 12 hours continuous light/dark.IN-LIFE DATES: NDA
Route of administration:
oral: gavage
Vehicle:
other: Arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test article was diluted in arachis oil.VEHICLE- Justification for use and choice of vehicle (if other than water): Arachis oil was used as the substance formed stable homogenous solutions in this vehicle- Concentration in vehicle: Dose-dependent Low – 30 mg/mlIntermediate – 300 mg/mlHigh – 750 mg/ml- Amount of vehicle (if gavage): 4 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the substance in the test material formulations was determined by chemical analysis and all formulations were within +/- 6% of nominal.
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
The substances was administered daily, for 28 consecutive days, by gavage. Control animals were treated in an identical manner with 4 ml/kg/day of arachis oil.Recovery group animals were maintained for a further fourteen days treatment-free period following termination of treatment.The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.
Remarks:
Doses / Concentrations:0mg/kg/day test substanceBasis:actual ingested
Remarks:
Doses / Concentrations:30 mg/kg/day test substanceBasis:actual ingested
Remarks:
Doses / Concentrations:300 mg/kg/day test substanceBasis:actual ingested
Remarks:
Doses / Concentrations:750 mg/kg/day test substanceBasis:actual ingested
No. of animals per sex per dose:
5 animals of each sex (m/f) were allocated to each dose group. In addition, 5 animals of each sex (m/f) were allocated to each control goup
Control animals:
yes, concurrent vehicle
other: recovery control (concurrent vehicle) [if relevant]
Details on study design:
- Dose selection rationale: Results of Preliminary Range Finding study.- Rationale for animal assignment (if not random): Random- Rationale for selecting satellite groups: Regulatory requirement- Post-exposure recovery period in satellite groups: 14 days, treatment free]
Positive control:
Not Used
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes - Time schedule: Immediately before dosing, up to 30m minutes post-dosing and 1 and 5 hours after dosing during the working week.DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: Immediately before dosing, up to 30m minutes post-dosing and 1 and 5 hours after dosing during the working week.BODY WEIGHT: Yes - Time schedule for examinations: Day 1 and weekly thereafter.FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not applicableFOOD EFFICIENCY:- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No dataFood consumption was recorded for each cage group at weekly intervals throughout the study.WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicableOPHTHALMOSCOPIC EXAMINATION: No HAEMATOLOGY: Yes - Time schedule for collection of blood: Day 28 and day 42 for the recovery groups- Anaesthetic used for blood collection: No - Animals fasted: No- How many animals: all- Parameters examined: Haemoglobin, Erythrocyte count, Haemotocrit, Erythrocyte indices, total leucocyte count, differential leucocyte count, platelet count, reticulocyte count, prothrombin time, activated partial thrombopastin time.CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: Day 28 and day 42 for the recovery groups- Animals fasted: No - How many animals: all- Parameters examined: Urea, glucose, total protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorus, gamma GT, ASAT, ALAT, AP, creatinine, triglycerides, total cholesterol, total bilirubin, bile acidsURINALYSIS: Yes - Time schedule for collection of urine: During week 4 and during week 6 for recovery group animals- Metabolism cages used for collection of urine: Yes- Animals fasted: Yes - Parameters examined: volume, Ketones, Specific Gravity, bilirubin, pH, protein glucose, urobilirubin, reducing substances, blood. NEUROBEHAVIOURAL EXAMINATION: Yes - Time schedule for examinations: Prior to start of treatment, days 7, 14, 21 and 25. Functional performance tests performed during wek 4.- Dose groups that were examined: control, low dose, intermediate dose, high dose- Battery of functions tested: YesBehavioural Assessments: gait and co-ordination, tremors, twitches, convulsions, abnormal behaviour, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin colour, respiration, palpebral closure, urination, defecation, transfer arousal, tail elevationFunctional Performance Tests:- Motor actiivity, forelimb/hindlimb grip strengthSensory Reactivity Tests:- Grasp response, vocalisation, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, blink reflex, startle reflex
Sacrifice and pathology:
GROSS PATHOLOGY: Yes DETAILS: Full external and internal examination, any macroscopic abnormalities were recorded..HISTOPATHOLOGY: Yes Samples from the following tissues were removed from all animals and preserved in buffered 10% formalin except where stated.The following tissues:-Adrenals, aorta, bone and bone marrow, brain, caecum, colon, duodenum, epididymes, eyes, gross lesions, heart, ileum, jejenum, kidneys, liver, lungs, lymph nodes, mammary gland, muscle, oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salaivary glands, sciatic nerve, seminal vesicles, skin, spinal cord, spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder, uterus and cervix, vagina
Other examinations:
None
Statistics:
Data were processed to give summary incidence or group mean and standard deviation values were appropriate. All data were summarised in tabular form. Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differencesfrom control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight Change, Haematology, Blood Chemistry, Urinalysis (Volume and Specific Gravity), Absolute Organ Weights, Body Weight-Relative Organ Weights Data were analysed using the decision tree fromthe ProvantisTM Tables and Statistics Module as detailed below:Where appropriate, data transformations wereperformed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance wereassessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (nonparametric). Probability values (p) are presented as follows: p<0.01 ** p<0.05 * p0.05 (not significant)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Mortality. There were no unscheduled deaths on the study. Clinical signs were restricted totransient post–dosing salivation on isolated occasions for both sexes at 300 and 750 mg/kg bw/day.
Mortality:
mortality observed, treatment-related
Description (incidence):
Mortality. There were no unscheduled deaths on the study. Clinical signs were restricted totransient post–dosing salivation on isolated occasions for both sexes at 300 and 750 mg/kg bw/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body Weight. At 750 mg/kg bw/day body weight gain was lower than control during the last two weeks of treatment and the first week of the recovery period. There were no clear effects of treatment at 30 or 300 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Increased water consumption was apparent for females throughout treatment and for males for a shorter duration, at 750 mg/kg bw/day. Higher water intake was apparent for females at 300 mg/kg bw/day throughout much of the treatment period.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Erythrocyte count, haemoglobin, haematocrit and mean cell haemoglobin concentration were lower than control at 750 mg/kg bw/day.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Minor effects at 300 and 750 mg/kg bw/day for total protein, blood urea and AAT.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 300 and 750 mg/kg bw/day, dosage related and statistically significant increases in absolute and body weight relative liver and thyroid weights were apparent for both sexes at the end of treatment, compared to control.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
For both sexes at 750 mg/kg bw/day, histopathological examination revealed centrilobular hypertrophy for the liver and follicular cell hypertrophy for the thyroids.
Details on results:
MortalityThere were no unscheduled deaths on the study.Clinical ObservationsNeither the type, incidence, or distribution of clinical signs observed during the study indicated any adverse effect of treatment at dosages up to 750 mg/kg bw/day.At 300 and 750 mg/kg bw/day both sexes showed post dosing salivation on isolated occasions during the study. At both dosages all animals were affected but there was a higher incidence of this finding among animals at 750 mg/kg bw/day compared to that at 300 mg/kg bw/day.No clinical signs were apparent during the study at 30 mg/kg bw/day for either sex.Functional ObservationsBehavioural AssessmentsAssessment of the animals in a standard arena did not reveal any obvious adverse effects of treatment at dosages up to 750 mg/kg bw/day.At 750 mg/kg bw/day, one animal showed moderate autonomic salivation during assessment on Day 14.At 750 mg/kg bw/day, there was a slightly higher incidence of urination for females during assessment in the standard arena for the last two weeks of treatment.Functional Performance TestsAssessment of functional performance using grip strength and measurement of motor activity did not indicate any obvious effects of treatment at dosages up to 750 mg/kg bw/day. During the assessment of motor activity, lower overall activity for males at 300 and 750 mg/kg bw/day attained statistical significance.Sensory Reactivity AssessmentsSensory reactivity to different stimuli (auditory, visual and proprioceptive) appeared unaffected by treatment.Body WeightFor males at 750 mg/kg bw/day body weight gain was slightly lower than control during the last two weeks of treatment, differences attaining statistical significance. Body weight gain remained statistically significantly lower than control during the first week of the recovery period but was similar to control by the end of the second recovery week.For males at 30 and 300 mg/kg bw/day, body weight gain was lower than control during the last week of treatment.There were no adverse effects of treatment on body weight or body weight gain for females at dosages up to 750mg/kg bw/day.Food Consumption and Food Conversion EfficiencyIntergroup differences in food consumption and food conversion efficiency did not indicate any adverse effect of treatment for either sex at dosages up to 750 mg/kg bw/day.Water ConsumptionAt 750 mg/kg bw/day water consumption for females was higher than control throughout the treatment period; water intake was similar to control during the treatment-free recovery period. For males at 750 mg/kg bw/day, water intake tended to be higher than control for the first sixteen days of treatment; thereafter water intake was similar to control.At 300 mg/kg bw/day water consumption for females tended to be higher than control throughout much of the treatment period. Water intake for males at 300 mg/kg bw/day was unaffected by treatment.At 30 mg/kg bw/day water consumption for both sexes was unaffected by treatment.Laboratory InvestigationsHaematologyAt 750 mg/kg bw/day, erythrocyte count, haemoglobin, haematocrit and mean cell haemoglobin concentration for both sexes were statistically significantly lower than control. For treated males, all individual values were within the historical control range, however for treated females two individual erythrocyte counts and four individual values for haematocrit were below this historical range. For females at this dosage, mean cell haemoglobin was also statistically significantly lower than control; one individual control and one treated value exceeded the historical control range.For males at 300 mg/kg bw/day, lower erythrocyte count for males attained statistical significance but all individual values were within the historical control range. For females at 30 and 300 mg/kg bw/day, lower mean cell haemoglobin concentration attained statistical significance compared with control but, again, all individual values were within the historical control range.At 750 mg/kg bw/day, total leucocyte count for females was statistically significantly higher than control with the majority of individual values exceeding the historical control range. There was no statistically significant increase for any particular white cell type.At 300 and 750 mg/kg bw/day, activated partial thromboplastin time for females was shorter than control; although differences attained statistical significance there was no dosage-relationship. Only one individual value at 300 mg/kg bw/day was below the historical control range while one individual control values exceeded this historical range.No statistically significant differences from control were apparent for haematological parameters for males at 30 mg/kg bw/day or for either sex at 750 mg/kg bw/day following the treatment-free recovery period.Blood ChemistryFor males at 300 and 750 mg/kg bw/day, mean total protein was lower than control with differences attaining statistical significance. All individual values at 750 mg/kg bw/day and the majority at 300 mg/kg bw/day were below the historical control range compare to only two control values.For males at 300 and 750 mg/kg bw/day, increased albumin/globulin ratio also attained statistical significance when compared to control; although no dosage-relationship was apparent all individual values exceeded the historical control. Additionally for males at 750 mg/kg bw/day, there was a statistically significant decrease in blood urea although all individual values were within the historical range.For females at 300 and 750 mg/kg bw/day an increase in alanine aminotransferase levels attained statistical significance when compared to control. There was no dosage relationship but the majority of individual values for treated animals exceeded the historical control range compare to only one control value.Additionally for females at 750 mg/kg bw/day there was a statistically significant decrease in blood sodium levels, three individual values were below the historical control range compare to only one control value.For males at 300 and 750 mg/kg bw/day, decreased total cholesterol attained statistical significance when compared to control; no dosage-relationship was apparent and all individual values were within the historical control range.For females at 750 mg/kg bw/day following the two week treatment-free recovery period, higher total cholesterol and calcium levels attained statistical significance when compared to control. For total cholesterol one individual value for treated animals exceeded the historical control range, while three values exceeded this historical range for calcium levels.No statistically significant differences from control were apparent for blood chemistry parameters for either sex at 30 mg/kg bw/day or for males at 750 mg/kg bw/day following the treatment-free recovery period.UrinalysisAt 750 mg/kg bw/day the presence of ketones was detected in the urine of both sexes. Following the two week recovery period only one female showed ketones in the urine. One female at 300 mg/kg bw/day showed ketones in the urine at the end of treatment. Ketones were not detected for males at this dosage or for either sex at 30 mg/kg bw/day.PathologyNecropsyMacroscopic findings observed during necropsy of the animals at termination was restricted to increased pelvic space in the right kidney for one male receiving 750 mg/kg bw/day.Organ WeightsAt 300 and 750 mg/kg bw/day, dosage related and statistically significant increases, compared to control, in absolute and body weight relative liver weights were apparent for both sexes at the end of treatment. Body weight relative values are generally considered to be the better indicator of hepatic effect and all individual values at these dosages exceeded the historical control range; while one female control body weight relative liver weight also exceeded this historical range thus were to a much lesser extent than observed for the treated animals. For females at 750 mg/kg bw/day, liver weights remained statistically significantly higher than control at the end of the two week treatment-free recovery period, with only three individual body weight relative values exceeded the historical control range.At 300 and 750 mg/kg bw/day, absolute and body weight relative thyroid weights for non-recovery animals of both sexes were higher than control; differences attaining statistical significance and showing a dosage-relationship. Thyroid weights were also statistically significantly higher than control for males at 30 mg/kg bw/day but these values showed no dosages relationship when compared with the thyroid weights at 300 mg/kg bw/day.At 750 mg/kg bw/day, absolute and body weight relative kidney weights for non-recovery males were higher than control; differences attaining statistical significance. Although kidney weights were also higher than control for females at this dosage, differences did not attain statistical significance.For males at 750 mg/kg bw/day, absolute and body weight relative prostate and seminal vesicle weights were statistically significantly lower than control at the end of the two week treatment-free recovery period.HistopathologyAt 750 mg/kg bw/day histopathological examination of tissues revealed centrilobular hypertrophy for the liver and follicular cell hypertrophy for the thyroids of both sexes.
Dose descriptor:
NOAEL
Effect level:
> 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects were observed
Critical effects observed:
not specified

Treatment at dosages up to 750 mg/kg bw/day was generally well tolerated with no findings observed that were considered to represent an adverse effect of treatment. Increased water intake and transient post dosing salivation at 300 and 750 mg/kg bw/day suggested unpalatable/slightly irritant of test item formulation but, the absence of any microscopic gastric or renal changes, indicated that this was of no toxicological importance. Lower body weight gain was observed for males for the last two weeks of treatment and persisted during the first week of recovery, however by termination body weight gain was similar to control and the differences in body weight change were regarded as insufficient to represent an adverse effect of treatment. A number of haematology and blood chemistry parameters attained statistical significance but, while some these may have been influenced by adaptive histopathological liver changes, there were no histopathological correlates that indicated an adverse effect of treatment. Similarly the presence of ketones in the urine for both sexes at 750 mg/kg bw/day was considered most likely to be related to alter liver metabolism. In view of the low incidence of this finding after the recovery period and the absence of any adverse histopathological correlates, this finding was considered not to represent an adverse effect of treatment. Absolute and body weight relative liver and thyroid weights were increased, compared to control for both sexes at 300 and 750 mg/kg bw/day, with an increase in thyroid weights also being apparent for males at 30 mg/kg bw/day. Microscopic examination of the liver revealed centrilobular hypertrophy for both sexes at 750 mg/kg bw/day. This hepatocellular hypertrophy was not accompanied by increased degenerative or inflammatory changes and, therefore, the finding was deemed to represent an adaptive change during hepatic clearance ofthe test item. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of any associated inflammatory or degenerative changes, is generally considered not to represent an adverse health effect. Associated with this adaptive liver change, microscopic examinationsof the thyroid revealed follicular hypertrophy in both sexes at 750 mg/kg bw/day. This finding is considered to be a secondary response to the hepatocellular hypertrophy. It is well documented (Greaves (2000)) that an increased metabolism by the liver of the thyroid hormones (thyroxine and triiodothyronine) causes an increased release of TRH (thyrotropin releasing hormone) from the hypothalamus and TSH (thyroid-stimulating hormone) from the pituitary and consequent activation of the thyroid epithelium (Hypothalamic-Pituitary-Thyroid Axis). The thyroid of rats is highly sensitive to this phenomenon which is particularly species specific with no safety relevance for humans and is generally regarded as adaptive in nature. Differences for a small number of other organ weights achieved statistical significance when compared to control but, in the absence of any supporting histopathological changes, were considered not to represent an adverse effect of treatment

.
Conclusions:
Treatment of rats at dosages up to750 mg/kg bw/day for twenty-eight consecutive days was well tolerated. The ‘No Observed Adverse Effect Level’ (NOAEL) for toxicity was considered to be 750 mg/kg bw/day
Executive summary:

Test Guidance

The study was designed to investigate the systemic toxicity of the test item and designed to be compatible with the following regulatory guidelines: i) Commission Directive 96/54/EC (Method B7). ii) The Japanese Ministry ofEconomy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003 for a twenty-eight dayrepeat dose oral toxicity study as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973 of Ministry of International Trade and Industry (MITI) amended 2004. iii) The OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008). iv) USA Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000. This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods.

The test item was administered by gavage to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 30, 300 and 750 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (750 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, body weight change, food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all nonrecovery group animals at the end of the treatmentperiod and for all recovery group animals at the end of the treatment-free period.

Results.

Mortality: There were no unscheduled deaths on the study.

Clinical Observations: Clinical signs were restricted to transient post–dosing salivation on isolated occasions for both sexes at 300 and 750 mg/kg bw/day.

Behavioural Assessment: There were no adverse effects of treatment at 30, 300 or 750 mg/kg bw/day.

Functional Performance Tests: There were no adverse effects of treatment at 30, 300 or 750 mg/kg bw/day.

Sensory Reactivity Assessments: There were no effects of treatment at 30, 300 or 750 mg/kg bw/day.

Body Weight: At 750 mg/kg bw/day body weight gain was lower than control during the last two weeks of treatment and the first week of the recovery period. There were no clear effects of treatment at 30 or 300 mg/kg bw/day.

Food Consumption: Food consumption and food conversion efficiency were unaffected by treatment at 30, 300 or 750 mg/kg bw/day.

Water Consumption: Increased water consumption was apparent for females throughout treatment and for males for a shorter duration, at 750 mg/kg bw/day. Higher water intake was apparent for females at 300 mg/kg bw/day throughout much of the treatment period. For males at 300 mg/kg bw/day and both sexes at 30 mg/kg bw/day water consumption was unaffected by treatment.

Haematology: Erythrocyte count, haemoglobin, haematocrit and mean cell haemoglobin concentration for both sexes and mean cell haemoglobin for females were statistically significantly lower than control at 750 mg/kg bw/day. Additional lower erythrocyte count for males at 300 mg/kg bw/day and lower mean cell haemoglobin concentration for females at 30 and 300 mg/kg bw/day, attained statistical significance compared with control. At 750 mg/kg bw/day, total leucocyte count for females was statistically significantly higher than control. No other statistically significant differences from control were considered to indicate a treatment related effect.

Blood Chemistry: At 300 and 750 mg/kg bw/day, mean total protein for males was statistically significantly lower than control and increased albumin/globulin ratio also attained statistical significance. Additionally for males at 750 mg/kg bw/day, there was a statistically significant decrease in blood urea. For females at 300 and 750 mg/kg bw/day there was a non-dosage related statistically significant increase in alanine aminotransferase levels compared to control. For males at 300 and 750 mg/kg bw/day there was a non-dosage related decrease in total cholesterol that attained statistical significance when compared to control. No other statistically significant differences from control were considered to indicate a treatment related effect.

Urinalysis: There were no effects of treatmentat 30, 300 or 750 mg/kg bw/day.

Necropsy: Macroscopic necropsy findings did not indicate any effect of treatment at 30, 300 or 750 mg/kg bw/day.

Organ Weights: At 300 and 750 mg/kg bw/day, dosage related and statistically significant increases in absolute and body weight relative liver and thyroid weights were apparent for both sexes at the end of treatment, compared to control. Thyroid weights were also statistically significantly higher than control for males at 30 mg/kg bw/day. At 750 mg/kg bw/day, absolute and body weight relative kidney weights for non recovery males were statistically significantly higher than control. No other statistically significant differences from control were considered to indicate a treatment related effect.

Histopathology: For both sexes at 750 mg/kg bw/day, histopathological examination revealed centrilobular hypertrophy for the liver and follicular cell hypertrophy for the thyroids.

Conclusion.

Treatment at dosages up to 750 mg/kg bw/day was generally well tolerated with no findings observed that were considered to represent an adverse effect of treatment. Increased water intake and transient post dosing salivation at 300 and 750 mg/kg bw/day suggested unpalatable/slightly irritant of test item formulation but, the absence of any microscopic gastric or renal changes, indicated that this was of no toxicological importance. Lower body weight gain was observed for males for the last two weeks of treatment and persisted during the first week of recovery, however by termination body weight gain was similar to control and the differences in body weight change were regarded as insufficient to represent an adverse effect of treatment. A number of haematology and blood chemistry parameters attained statistical significance but, while some these may have been influenced by adaptive histopathological liver changes, there were no histopathological correlates that indicated an adverse effect of treatment. Similarly the presence of ketones in the urine for both sexes at 750 mg/kg bw/day was considered most likely to be related to alter liver metabolism. In view of the low incidence of this finding after the recovery period and the absence of any adverse histopathological correlates, this finding was considered not to represent an adverse effect of treatment. Absolute and body weight relative liver and thyroid weights were increased, compared to control for both sexes at 300 and 750 mg/kg bw/day, with an increase in thyroid weights also being apparent for males at 30 mg/kg bw/day. Microscopic examination of the liver revealed centrilobular hypertrophy for both sexes at 750 mg/kg bw/day. This hepatocellular hypertrophy was not accompanied by increased degenerative or inflammatory changes and, therefore, the finding was deemed to represent an adaptive change during hepatic clearance ofthe test item. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of any associated inflammatory or degenerative changes, is generally considered not to represent an adverse health effect. Associated with this adaptive liver change, microscopic examinationsof the thyroid revealed follicular hypertrophy in both sexes at 750 mg/kg bw/day. This finding is considered to be a secondary response to the hepatocellular hypertrophy. It is well documented (Greaves (2000)) that an increased metabolism by the liver of the thyroid hormones (thyroxine and triiodothyronine) causes an increased release of TRH (thyrotropin releasing hormone) from the hypothalamus and TSH (thyroid-stimulating hormone) from the pituitary and consequent activation of the thyroid epithelium (Hypothalamic-Pituitary-Thyroid Axis). The thyroid of rats is highly sensitive to this phenomenon which is particularly species specific with no safety relevance for humans and is generally regarded as adaptive in nature. Differences for a small number of other organ weights achieved statistical significance when compared to control but, in the absence of any supporting histopathological changes, were considered not to represent an adverse effect of treatment.

Treatment of rats at dosages up to750 mg/kg bw/day for twenty-eight consecutive days was well tolerated and the ‘No Observed Adverse Effect Level’ (NOAEL) for toxicity was considered to be 750 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch grade 1, GLP study conducted according to OECD407 test guideline.Klimisch grade 2, GLP study conducted according to OECD407 test guideline.

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

Additional information

Key 28 -day repeated oral dose toxicity study

Test Guidance

The study was designed to investigate the systemic toxicity of the test item and designed to be compatible with the following regulatory guidelines: i) Commission Directive 96/54/EC (Method B7). ii) The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003 for a twenty-eight dayrepeat dose oral toxicity study as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973 of Ministry of International Trade and Industry (MITI) amended 2004. iii) The OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008). iv) USA Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000. This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods.

The test item was administered by gavage to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 30, 300 and 750 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (750 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, body weight change, food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all nonrecovery group animals at the end of the treatmentperiod and for all recovery group animals at the end of the treatment-free period.

Results.

Mortality: There were no unscheduled deaths on the study.

Clinical Observations: Clinical signs were restricted to transient post–dosing salivation on isolated occasions for both sexes at 300 and 750 mg/kg bw/day.

Behavioural Assessment: There were no adverse effects of treatment at 30, 300 or 750 mg/kg bw/day.

Functional Performance Tests: There were no adverse effects of treatment at 30, 300 or 750 mg/kg bw/day.

Sensory Reactivity Assessments: There were no effects of treatment at 30, 300 or 750 mg/kg bw/day.

Body Weight: At 750 mg/kg bw/day body weight gain was lower than control during the last two weeks of treatment and the first week of the recovery period. There were no clear effects of treatment at 30 or 300 mg/kg bw/day.

Food Consumption: Food consumption and food conversion efficiency were unaffected by treatment at 30, 300 or 750 mg/kg bw/day.

Water Consumption: Increased water consumption was apparent for females throughout treatment and for males for a shorter duration, at 750 mg/kg bw/day. Higher water intake was apparent for females at 300 mg/kg bw/day throughout much of the treatment period. For males at 300 mg/kg bw/day and both sexes at 30 mg/kg bw/day water consumption was unaffected by treatment.

Haematology: Erythrocyte count, haemoglobin, haematocrit and mean cell haemoglobin concentration for both sexes and mean cell haemoglobin for females were statistically significantly lower than control at 750 mg/kg bw/day. Additional lower erythrocyte count for males at 300 mg/kg bw/day and lower mean cell haemoglobin concentration for females at 30 and 300 mg/kg bw/day, attained statistical significance compared with control. At 750 mg/kg bw/day, total leucocyte count for females was statistically significantly higher than control. No other statistically significant differences from control were considered to indicate a treatment related effect.

Blood Chemistry: At 300 and 750 mg/kg bw/day, mean total protein for males was statistically significantly lower than control and increased albumin/globulin ratio also attained statistical significance. Additionally for males at 750 mg/kg bw/day, there was a statistically significant decrease in blood urea. For females at 300 and 750 mg/kg bw/day there was a non-dosage related statistically significant increase in alanine aminotransferase levels compared to control. For males at 300 and 750 mg/kg bw/day there was a non-dosage related decrease in total cholesterol that attained statistical significance when compared to control. No other statistically significant differences from control were considered to indicate a treatment related effect.

Urinalysis: There were no effects of treatmentat 30, 300 or 750 mg/kg bw/day.

Necropsy: Macroscopic necropsy findings did not indicate any effect of treatment at 30, 300 or 750 mg/kg bw/day.

Organ Weights: At 300 and 750 mg/kg bw/day, dosage related and statistically significant increases in absolute and body weight relative liver and thyroid weights were apparent for both sexes at the end of treatment, compared to control. Thyroid weights were also statistically significantly higher than control for males at 30 mg/kg bw/day. At 750 mg/kg bw/day, absolute and body weight relative kidney weights for non recovery males were statistically significantly higher than control. No other statistically significant differences from control were considered to indicate a treatment related effect.

Histopathology: For both sexes at 750 mg/kg bw/day, histopathological examination revealed centrilobular hypertrophy for the liver and follicular cell hypertrophy for the thyroids.

Conclusion.

Treatment at dosages up to 750 mg/kg bw/day was generally well tolerated with no findings observed that were considered to represent an adverse effect of treatment. Increased water intake and transient post dosing salivation at 300 and 750 mg/kg bw/day suggested unpalatable/slightly irritant of test item formulation but, the absence of any microscopic gastric or renal changes, indicated that this was of no toxicological importance. Lower body weight gain was observed for males for the last two weeks of treatment and persisted during the first week of recovery, however by termination body weight gain was similar to control and the differences in body weight change were regarded as insufficient to represent an adverse effect of treatment. A number of haematology and blood chemistry parameters attained statistical significance but, while some these may have been influenced by adaptive histopathological liver changes, there were no histopathological correlates that indicated an adverse effect of treatment. Similarly the presence of ketones in the urine for both sexes at 750 mg/kg bw/day was considered most likely to be related to alter liver metabolism. In view of the low incidence of this finding after the recovery period and the absence of any adverse histopathological correlates, this finding was considered not to represent an adverse effect of treatment. Absolute and body weight relative liver and thyroid weights were increased, compared to control for both sexes at 300 and 750 mg/kg bw/day, with an increase in thyroid weights also being apparent for males at 30 mg/kg bw/day. Microscopic examination of the liver revealed centrilobular hypertrophy for both sexes at 750 mg/kg bw/day. This hepatocellular hypertrophy was not accompanied by increased degenerative or inflammatory changes and, therefore, the finding was deemed to represent an adaptive change during hepatic clearance ofthe test item. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of any associated inflammatory or degenerative changes, is generally considered not to represent an adverse health effect. Associated with this adaptive liver change, microscopic examinationsof the thyroid revealed follicular hypertrophy in both sexes at 750 mg/kg bw/day. This finding is considered to be a secondary response to the hepatocellular hypertrophy. It is well documented (Greaves (2000)) that an increased metabolism by the liver of the thyroid hormones (thyroxine and triiodothyronine) causes an increased release of TRH (thyrotropin releasing hormone) from the hypothalamus and TSH (thyroid-stimulating hormone) from the pituitary and consequent activation of the thyroid epithelium (Hypothalamic-Pituitary-Thyroid Axis). The thyroid of rats is highly sensitive to this phenomenon which is particularly species specific with no safety relevance for humans and is generally regarded as adaptive in nature. Differences for a small number of other organ weights achieved statistical significance when compared to control but, in the absence of any supporting histopathological changes, were considered not to represent an adverse effect of treatment.

Treatment of rats at dosages up to750 mg/kg bw/day for twenty-eight consecutive days was well tolerated and the ‘No Observed Adverse Effect Level’ (NOAEL) for toxicity was considered to be 750 mg/kg bw/day.

Supporting 28 -Day Repeated Oral Dose toxicity Study

Test Guidance

OECD Guideline No. 407

Method

This study was designed to assess the toxicity of the test material when administered orally, via gastric intubation to 45 Sprague-Dawley CD rats at dose levels of 50, 158 and 500 mg/kg/day at a dose volume of 2.0 ml/kg/day. The test substance was administered seven days/week over a four week period. Control animals (10/sex) received the vehicle, corn oil, at the same dose volume as adminstered to the treated animals. Body weight and food consumption measurements were performed on all animals pretest and weekly during the treatment period. Physical observations were conducted on all animals prior to each administration of the test material. Hematology and clinical chemistry parameters were evaluated for all animals at study termination and prior to recovery sacrifice. After approximately four weeks of treatment, half of the control and high-dose animals (5/sex/group), half of the mid-dose females (5), all of the low-dose animals (5/sex) and all of the mid-dose males (5) were sacrificed, selected organs were weighed and organ/body weight ratios were calculated. Complete gross postmortem examinations were performed on all animals and histopathological evaluation of selected tissues were performed on all animals. After a two-week recovery period, all surviving animals were sacrificed, selected organs were weighed and organ/body weight ratios were calculated. Complete gross postmortem examinations were performed on all animals and histopathological evaluation of selected tissues were performed on all animals.

Results

One mid-dose female was found dead on day 4. No cause of death was apparent from gorss post mortem examinations. Because this was the only animal which died and no mortality was seen in the high-dose group, this death does not appear to be related to test material administration.

Evaluation of physical observations, body weight and food consumption data, clinical laboratory studies, urinalysis data and gross postmortem observations revealed no adverse effects of the test material administration.

Mean haemoglobin and haematocrit values for high-dose males and females were slightly lower than mean control values at study termination, but recovery was apparent during the two-week recovery period. Evaluation of total and differential leukocyte values and coagulation studies revelaed no evidence of an effect of test material adminstration.

Mean kidney weights and kidney/body weight ratios for treated males were higher than control values at study termination. differences were dose-related (minimal at the low dose, slight at the mid-dose and moderate at the high-dose). Similar differences were not apparent in females, nor were any differences seen between kidney weights of control and high dose males held for a two-week recovery period. In the absence of any renal pathology or any evidence of impaired renal function based on clinical laboratory and urinalysis values, the differences seen at the termination of the dosing period appear to represent a reversible metabolic response to test material administration.

Liver weights and liver/body weight ratios for mid- and high-dose males and females were higher than mean control values at study termination; differences at the high-dose level were pronounced (approximately 40 to 50% higher than control values). Values for high-dose animals at termination of the recovery period were only slightly (less than 20%) higher than concurrent control values. Microscopic examination revealed hypertrophy of the centrilobular hepatocytes in the treated animals (1 of 5 low-dose, 2 of 5 mid-dose and 5 of 5 high-dose animals) killed at termination of test material administration. Similar changes were not evident in control and high-dose females held for a two-week recovery period, and microscopic changes were not seen in livers of treated males. The absence of any indication of hepatic toxicity in clinical laboratory studies and the recovery seen during two-week treatment-free period suggest that the changes seen represent a reversible metabolic response to test material administration.

Conclusion

Treatment of rats at dosages up to 500 mg/kg bw/day for twenty eight consecutive days was well tolerated and the ‘No Observed Adverse Effect Level’ (NOAEL) for toxicity was considered to be 500 mg/kg bw/day


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Key study

Justification for classification or non-classification

In accordance with the CLP Regulation, No 1272/2008, the test material is not classified for repeat dose toxicity via the oral route. The NOAEL from the key study is 750 mg/kg bw/day, the highest dose tolerated by rats (palatability/irritation). Although effects potentially related to treatment with the test material were observed in animals killed at the end of the dosing period, the same effects were not found in high-dose animals terminated after a two-week treatment free recovery period. In addition certain findings were considered to be due to specific sensitivities of the rat and to have no relevance to humans. The test material is therefore not considered to have an adverse effect that would require classification for repeat oral exposure.