Registration Dossier

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Administrative data

Description of key information

No adverse effects relevant to humans were observed after repeated dose administration via the oral route and the NOAEL was reported as 750 mg/kg bw/day (OECD 407).

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:
09 July 2014 to 24 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline.
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
yes
Remarks:
Target value for relative humidity exceeded (see below)
Qualifier:
according to guideline
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:
yes
Remarks:
Target value for relative humidity exceeded (see below)
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
Target value for relative humidity exceeded (see below)
Qualifier:
according to guideline
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:
yes
Remarks:
Target value for relative humidity exceeded (see below)
Qualifier:
according to guideline
Guideline:
other: The Notification and Testing of New Substances: Chemicals and Polymers: CEPA (Canadian Environmental Protection Act) 1999
Deviations:
yes
Remarks:
Target value for relative humidity exceeded (see below)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:Harlan Laboratories U.K Ltd.,Oxon, UK
- Age at study initiation:6 to 8 weeks
- Weight at study initiation:males 195-228g, females 160-190g
- Housing:solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet (e.g. ad libitum): Rodent 2014C Teklad Global Certified Pelleted Diet, Harlan Laboratories U.K Ltd., Oxon, UK
- Water (e.g. ad libitum):ad libitum
- Acclimation period:7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22+/-3 °C
- Humidity (%):50+/-20 %
- Air changes (per hr):at least 15
- Photoperiod (hrs dark / hrs light):12 hours continuous light/dark

IN-LIFE DATES: From: 25 July 2014 To:05 September 2014
Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
Arachis oil BP
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

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:
Low – 100 mg/ml
Intermediate – 300 mg/ml
High – 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 +/- 11% of nominal
Duration of treatment / exposure:
Test duration: 28 days
Control animals were treated in an identical manner with 4 ml/kg/day of Arachis oil BP.
Recovery group animals were maintained for a further 14 days treatment-free period following termination of treatment.
Frequency of treatment:
The substances was administered daily, for 28 consecutive days, by gavage.
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:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
750 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5 animals of each sex (m/f) were allocated to each dose group
Control animals:
yes, concurrent vehicle
other: recovery control (concurrent vehicle)... (see attached file)
Details on study design:
- Dose selection rationale: 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 30 minutes post-dosing and 1 hour after dosing during the working week, weekends and public holidays. Observed daily during the treatment-free period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, up to 30 minutes post-dosing and 1hour after dosing during the working week. During treatment-free period recovery, observed daily. Functional before the first exposure to the test item and once weekly thereafter, 2 hours after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 and weekly thereafter. Also performed prior to terminal kill and on Day 36 and 43 for recovery group animals.

FOOD CONSUMPTION
- Food consumption was recorded for each cage group at weekly intervals throughout the study.
- Food conversion efficicency was calculated retrospectively.

FOOD 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:

WATER CONSUMPTION
- Except during week three where water intake was measured gravimetrically, water intake was observed daily for each cage group by visual inspection of the water bottles for any overt changes.
- A possible intergroup difference was detected during Week 3 and water consumption was therefore recorded for each cage group until study termination.

OPHTHALMOSCOPIC 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: Hemoglobin, Erythrocyte count, Hematocrit, Erythrocyte indices, total leucocyte count, differential leucocyte count, platelet count, reticulocyte count, prothrombin time, activated partial thromboplastin 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, total cholesterol, total bilirubin, bile acids

URINALYSIS: 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, urobilinogen, blood.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to start of treatment, days 7, 14, 21 and 27. Functional performance tests performed during week 4
- Dose groups that were examined: control, low dose, intermediate dose, high dose
- Battery of functions tested: Yes
Behavioural Assessments: gait, tremors, twitches, convulsions, abnormal behaviour, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin colour, respiration, palpebral closure, urination, defecation, transfer arousal, tail elevation.
Functional Performance Tests: Motor activity, forelimb/hindlimb grip strength.
Sensory 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 10% buffered formalin except where stated.
The following tissues: Adrenals, aorta, bone and bone marrow, brain, caecum, colon, duodenum, esophagus, epididymides, eyes, gross lesions, heart, ileum, jejenum, kidneys, liver, lungs, lymph nodes, mammary gland, muscle, ovaries, pancreas, pituitary, prostate, rectum, salivary glands, sciatic nerve, seminal vesicles, skin, spinal cord, spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder, uterus and cervix, vagina

Organ weight of the following organs: adrenals, brain, epididymis, heart, kidneys, pituitary, prostate, liver, ovaries, seminal vesicles, spleen, testes, thymus, thyroid/parathyroid, uterus and cervix
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 differences from 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, Absolute Organ Weights, Body Weight-Relative Organ Weights
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed below:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variances were assessed 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 (non-parametric).
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):
There were no unscheduled deaths on the study. Clinical signs were restricted to instances of increased salivation for both sexes from all treatment groups.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were no unscheduled deaths on the study. Clinical signs were restricted to instances of increased salivation for both sexes from all treatment groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males showed a reduction in body weight gain at 750 mg/kg bw/day during the first week of treatment. Recovery evident thereafter. No effects of treatment at 750 mg/kg bw/day for females or at 300 and 100 mg/kg bw/day for both sexes.
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 both sexes during the final two weeks of treatment at 750 mg/kg bw/day
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicological significant effects. Only statistically significant reduction or increase in the hematological parameters.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No toxicologically significant effects. Only statistically significant reduction or increase in the blood chemical parameters.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No toxicologically significant effects. Only statistically significant reduction or increase in the urinalytical parameters.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increase in liver weight both absolute and body weight relative at 750 mg/kg bw/day for animals of either sex, at 300 mg/kg bw/day for females. Increase in kidney weight both absolute and body weight relative for males at 750 mg/kg bw/day.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY: No unscheduled deaths. Instances of increased salivation were evident throughout the treatment period in animals of
either sex from all treatment groups.
Behavorial assessments: There were no treatment-related changes in the behavioral parameters at 100, 300 and 750 mg/kg
bw/day.
Functional Performance tests: There were no toxicologically significant changes in functional performance.
Males treated with 750 and 300 mg/kg bw/day showed a statistically significant reduction in overall activity. In the absence of a true dose related response or any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup difference was considered to be of no toxicological significance.
Sensory reactivity assessments: There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 100, 300 and 750 mg/kg bw/day.

BODY WEIGHT AND WEIGHT GAIN: Males treated with 750 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment. Recovery was evident thereafter. No such effects were detected in females treated with 750 mg/kg bw/day or in animals of either sex treated with 300 or 100 mg/kg bw/day or in recovery animals during the treatment free period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not applicable

FOOD EFFICIENCY: No adverse effect on food consumption or food conversion efficiency was detected in treated
animals when compared to control animals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable. Animals of either sex treated with 750 mg/kg bw/day showed an increase in water consumption during the final two weeks of treatment. No such effects were detected in animals of either sex treated with 300 or 100 mg/kg bw/day.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No toxicologically significant effects were detected in the hematological parameters examined.
Males treated with 750 mg/kg bw/day showed a statistically significant increase in hematocrit (p<0.05) and statistically significant reductions in total leukocyte count (p<0.05) and lymphocyte count (p<0.05). Females from this treatment group showed statistically significant reductions in erythrocyte count (p<0.05), hematocrit (p<0.05) and lymphocyte count (p<0.05). Males treated with 750 and 300 mg/kg bw/day also showed a reduction in mean corpuscular hemoglobin
concentration (p<0.01). Females from all treatment groups showed a statistically significant increase in neutrophil count (p<0.05-0.01). The majority of the individual values were within the normal background range for these parameters and in the absence of any associated histopathological bone marrow changes or a true dose related response the intergroup differences were considered of no toxicological importance. Recovery 750 mg/kg bw/day males showed a statistically significant reduction in neutrophil count (p<0.05) whilst recovery 750 mg/kg bw/day females showed a statically significant reduction in mean corpuscular hemoglobin concentration (p<0.01). All of the individual values were within the normal background range for these parameters and in the absence of a similar effect seen at the end of the treatment period the intergroup differences were considered of no toxicological importance.

CLINICAL CHEMISTRY: No toxicologically significant effects were detected in the blood chemical parameters examined.
Males treated with 100 mg/kg bw/day showed a statistically significant reduction in potassium concentration (p<0.05). All of the individual values were within the normal background range for this parameters and in the absence of a true dose related response the intergroup difference was considered of no toxicological importance. Females from all treatment groups showed a statistically significant increase in phosphorus (p<0.05-0.01), bilirubin (p<0.05) and bile acid
(p<0.05). Females treated with 750 mg/kg bw/day also showed a statistically significant increase in alkaline phosphatase (p<0.05) and sodium concentration (p<0.01). The majority of the individual values were within the normal background range for these parameters and in the absence of true dose related responses the intergroup differences were considered of no toxicological importance. Recovery 750 mg/kg bw/day females showed a statistically significant increase increatinine (p<0.05). In the absence of a similar effect seen at the end of the treatment period the intergroup difference was considered of no toxicological importance.

URINALYSIS: No toxicologically significant effects were detected in the urinalytical parameters examined.
Females treated with 750 mg/kg bw/day showed a statistically significant increase (P<0.05) in urine volume. In isolation and in the absence of any histopathological renal changes in females, the intergroup difference was considered not to be of toxicological significance.

NEUROBEHAVIOUR: No

ORGAN WEIGHTS: Non-recovery animals of either sex treated with 750 mg/kg bw/day and females treated with 300 mg/kg bw/day showed an increase in liver weight both absolute and relative to terminal body weight. Non-recovery males treated with 750 mg/kg bw/day also showed an increase in absolute and relative kidney weight. Recovery 750 mg/kg bw/day males showed a statistically significant increase in adrenal weight both absolute and relative to terminal body weight. No such effects were detected in males treated with 300 mg/kg bw/day, in animals of either sex treated with 100 mg/kg bw/day or in recovery females following fourteen days without treatment.

GROSS PATHOLOGY: No treatment-related macroscopic abnormalities were detected.

HISTOPATHOLOGY: NON-NEOPLASTIC
Liver: centrilobular hepatocellular hypertrophy was evident in all males and in one female treated with 750 mg/kg bw/day. Complete recovery was evident following fourteen days without treatment.
Kidneys: hyaline droplets (consistent with Alpha2u-globulin nephropathy) and basophilic tubules were evident in males from all treatment groups. Granular casts were also evident in males treated with 750 and 300 mg/kg bw/day. Partial recovery was evident following fourteen days without treatment.
Adrenal: diffuse hypertrophy of the zona glomerulosa was evident in males from all treatment groups and in females treated with 750 mg/kg bw/day. No recovery was evident following fourteen days without treatment.
Lungs: agregates of alveoplar macrophages were evident in females treated with 750 mg/kg bw/day. Complete recovery was evident following fourteen days without treatment.
Dose descriptor:
NOAEL
Remarks:
750 mg/kg bw/day
Effect level:
mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Remarks:
300 mg/kg bw/day
Effect level:
mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

DEVIATION FROM STUDY PLAN

- The study plan target values for relative humidity controls was 50± 20 % respectively.

- There was no deviation from the target range for temperature but the target range for relative humidity was exceeded on 24 separate days during the study with the maximum relative humidity achieved being 80.15 %.

- While it is accepted that these deviations from the target range for relative humidity were less than ideal, overall it is considered that these deviations had no adverse impact on the scientific purpose of the study.

The oral administration of the test material to rats for a period of twenty-eight consecutive days at dose levels of 100, 300 and 750 mg/kg bw/day resulted in treatment related effects detected in males from all treatment groups and in females treated with 750 mg/kg bw/day. Increased salivation was evident in treated animals throughout the treatment period. Increased water consumption was also evident during the final two weeks of treatment in animals of either sex treated with 750 mg/kg bw/day. Observations of this nature are commonly observed following the oral gavage administration of an unpalatable or slightly irritant test item formulation. The physical condition of males treated with 750 mg/kg bw/day was initially affected, with lower body weight gains evident during the first week of treatment. A full recovery however was evident thereafter. There were no toxicologically significant effects observed during the weekly open field arena observations or in the hematological/blood chemical parameters measured. No macroscopic abnormalities were evident in treated animals however microscopic examination revealed changes in the liver, adrenals, kidneys (males only) and lungs (females only). Histopathological examination of the liver revealed diffuse hepatocellular hypertrophy in animals of either sex treated with 750 mg/kg bw/day. Organ weight data supported this finding with increased absolute and relative liver weights observed in these animals and in females treated with 300 mg/kg bw/day. Complete recovery was evident following fourteen days without treatment. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of any degenerative or inflammatory changes is generally considered to be an adaptive change or considered to reflect a metabolic disturbance that is a result of hepatic induction. Microscopic examination of lung sections revealed alveolar macrophages present at low severity in females treated with 750 mg/kg bw/day. While this is a common incidental finding, in the absence of a similar effect in control animals, the possibility that this was related to treatment cannot be excluded. Complete recovery was evident following fourteen days without treatment therefore it was considered not to represent an adverse effect of treatment. Treatment-related hypertrophy of the adrenal zona glomerulosa was present at extremely low severity in males from all treatment groups and in females treated with 750 mg/kg bw/day. No recovery was evident following fourteen days without treatment and recovery males did show an increase in absolute and relative adrenal weight. This is a physiological change, consistent with increased aldosterone secretion in order to increase fluid retention in the kidneys. In the absence of any degenerative changes, this condition is considered to be adaptive in nature.

Microscopic examination of the kidneys revealed hyaline droplets in males from all treatment groups. Kidney weights were also elevated in 750 mg/kg bw/day males. Partial recovery was evident following fourteen days without treatment. Hyaline droplets can be directly linked to the accumulation of alpha 2u-globulin, which is unique to the male rat. This finding is not found in immature rats, female rats or humans and therefore is considered to be of no relevance to man.

Therefore, a ‘No Observed Adverse Effect Level’ (NOAEL) for males can be established based on the observed effects excluding those related to alpha 2u-globulin nephropathy.

Conclusions:
The oral administration of the test material to rats for a period of twenty-eight consecutive days at dose levels of 100, 300 and 750 mg/kg bw/day resulted in treatment related microscopic effects detected in males from all treatment groups and in females treated with 750 mg/kg bw/day. The No Observed Effect Level (NOEL) for females was therefore considered to be 300 mg/kg bw/day and was not established for males.
The effect on body weight gain in 750 mg/kg bw/day males was considered to reflect an initial insult to the test item and recovery was evident thereafter therefore it was not considered to represent an adverse effect of treatment.
The microscopic liver changes identified in animals of either sex treated with 750 mg/kg bw/day, which were likely to represent adaptive changes, were considered not to represent “serious damage” to health. The microscopic adrenal changes were considered to be a physiological change and were considered not to represent an adverse effect of treatment. The low level severity of microscopic lung changes were also considered not to represent an adverse effect of treatment. The effects relating to male rat renal changes are species and sex specific and therefore are not relevant to humans.
For these reasons 750 mg/kg bw/day may be regarded as a “No Observed Adverse Effect Level” (NOAEL) for animals of either sex.
Executive summary:

GUIDELINE

The study was designed to investigate the systemic toxicity of the test item and is compatible with the following regulatory guidelines:

- Commission Directive 96/54/EC (Method B7).

- The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labor and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003 for a twenty-eight day repeat 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.

- The OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008).

- USA Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.

- The Notification and Testing of New Substances: Chemicals and Polymers: CEPA (Canadian Environmental Protection Act) 1999.

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).

METHOD

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 100, 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, functional observations, 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 animals were subjected to gross necropsy examination and histopathological examination of selected tissues was performed.

RESULTS

Mortality

There were no unscheduled deaths.

Clinical Observations

Instances of increased salivation were evident throughout the treatment period in animals of either sex from all treatment groups.

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters at 100, 300 and 750 mg/kg bw/day.

Functional Performance Tests

There were no toxicologically significant changes in functional performance.

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 100, 300 and 750 mg/kg bw/day.

Body Weight

Males treated with 750 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment. Recovery was evident thereafter. No such effects were detected in females treated with 750 mg/kg bw/day or in animals of either sex treated with 300 or 100 mg/kg bw/day or in recovery animals during the treatment free period.

Food Consumption

No adverse effect on food consumption was evident in treated animals.

Water Consumption

Animals of either sex treated with 750 mg/kg bw/day showed an increase in water consumption during the final two weeks of treatment. No such effects were detected in animals of either sex treated with 300 or 100 mg/kg bw/day.

Hematology

No toxicologically significant effects were detected in the hematological parameters examined.

Blood Chemistry

No toxicologically significant effects were detected in the blood chemical parameters examined.

Urinalysis

No toxicologically significant effects were detected in the urinalytical parameters examined.

Necropsy

No treatment-related macroscopic abnormalities were detected.

Organ Weights

Non-recovery animals of either sex treated with 750 mg/kg bw/day and females treated with 300 mg/kg bw/day showed an increase in liver weight both absolute and relative to terminal body weight. Non-recovery males treated with 750 mg/kg bw/day also showed an increase in absolute and relative kidney weight. No such effects were detected in males treated with 300 mg/kg bw/day or animals of either sex treated with 100 mg/kg bw/day.

Histopathology

The following microscopic abnormalities were detected:

Liver: centrilobular hepatocellular hypertrophy was evident in all males and in one female treated with 750 mg/kg bw/day. Complete recovery was evident following fourteen days without treatment.

Kidneys: hyaline droplets (consistent with Alpha 2u-globulin nephropathy) and basophilic tubules were evident in males from all treatment groups. Granular casts were also evident in males treated with 750 and 300 mg/kg bw/day. Partial recovery was evident following fourteen days without treatment.

Adrenal: diffuse hypertrophy of the zona glomerulosa was evident in males from all treatment groups and in females treated with 750 mg/kg bw/day. No recovery was evident following fourteen days without treatment.

Lungs: agregates of alveoplar macrophages were evident in females treated with 750 mg/kg bw/day. Complete recovery was evident following fourteen days without treatment.

CONCLUSION

Oral administration of the test material to rats for a period of twenty-eight consecutive days at dose levels of 100, 300 and 750 mg/kg bw/day resulted in treatment related microscopic effects detected in males from all treatment groups and in females treated with 750 mg/kg bw/day. The No Observed Effect Level (NOEL) for females was therefore considered to be 300 mg/kg bw/day and was not established for males.

The effect on body weight gain in 750 mg/kg bw/day males was considered to reflect an initial insult to the test item and recovery was evident thereafter therefore it was not considered to represent an adverse effect of treatment.

The microscopic liver changes identified in animals of either sex treated with 750 mg/kg bw/day, which were likely to represent adaptive changes, were considered not to represent “serious damage” to health. The microscopic adrenal changes were considered to be a physiological change and were considered not to represent an adverse effect of treatment. The low level severity of microscopic lung changes were also considered not to represent an adverse effect of treatment. The effects relating to male rat renal changes are species and sex specific and therefore are not relevant to humans. For these reasons 750 mg/kg bw/day may be regarded as a “No Observed Adverse Effect Level” (NOAEL) for animals of either sex.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

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

Oral

Oral administration of the test material to rats for a period of twenty-eight consecutive days at dose levels of 100, 300 and 750 mg/kg bw/day resulted in treatment related microscopic effects detected in males from all treatment groups and in females treated with 750 mg/kg bw/day. The No Observed Effect Level (NOEL) for females was therefore considered to be 300 mg/kg bw/day and was not established for males.

The effect on body weight gain in 750 mg/kg bw/day males was considered to reflect an initial insult to the test item and recovery was evident thereafter therefore it was not considered to represent an adverse effect of treatment.

The microscopic liver changes identified in animals of either sex treated with 750 mg/kg bw/day, which were likely to represent adaptive changes, were considered not to represent “serious damage” to health. The microscopic adrenal changes were considered to be a physiological change and were considered not to represent an adverse effect of treatment. The low level severity of microscopic lung changes were also considered not to represent an adverse effect of treatment. The effects relating to male rat renal changes are species and sex specific and therefore are not relevant to humans. For these reasons 750 mg/kg bw/day may be regarded as a “No Observed Adverse Effect Level” (NOAEL) for animals of either sex.

Inhalation

According to REACH, Annex VIII, Section 8.6.1, short-term repeated dose toxicity should be investigated using one species and the most likely route of human exposure. The substance is a liquid with a vapour pressure of 0.013 Pa at 25°C and, based on evaluation of the life cycle of the substance, it is expected that inhalation exposure will be low and that the most likely route of exposure for workers and consumers is the dermal route. A repeated dose toxicity study via the inhalation route is, therefore, not appropriate.

Dermal

According to REACH, Annex VIII, Section 8.6.1, short-term repeated dose toxicity should be investigated using one species and the most likely route of human exposure. However, no evidence of systemic toxicity was demonstrated during an acute study via the dermal route and, in order to minimise the number of vertebrate animals tested, oral dosing by gavage was considered most appropriate for a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) in rats.

Justification for classification or non-classification

No classification is required for repeat dose toxicity under the terms of Regulation (EC) No 1272/2008 because there were no adverse effects relevant to humans at up to and including the highest dose level of 750 mg/kg bw/day.

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