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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 March 2014 to 13 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
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:
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:
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: 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)
Deviations:
yes
Remarks:
Target value for relative humidity exceeded (see below)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Hexanoic acid, 3,5,5-trimethyl-, 1,1'-[2-ethyl-2-[[(3,5,5-trimethyl-1-oxohexyl)oxy]methyl]-1,3-propanediyl] ester
Cas Number:
65870-94-2
Molecular formula:
C33H62O6
IUPAC Name:
Hexanoic acid, 3,5,5-trimethyl-, 1,1'-[2-ethyl-2-[[(3,5,5-trimethyl-1-oxohexyl)oxy]methyl]-1,3-propanediyl] ester
Test material form:
liquid
Details on test material:
Description: clear colourless liquid
Expiry date: 29 June 2013
Storage conditions: room temperature, in the dark

Test animals

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 199-226g, females 150-183g
- Housing: Housed in groups of 5 by sex in 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: 10 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

Administration / exposure

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 – 25 mg/mL
Intermediate – 75 mg/mL
High – 250 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 96 to 99% of the nominal concentration.
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 substance 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.
Doses / concentrationsopen allclose all
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:
1000 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. High dose level of 1000 mg/kg bw/day was tolerated well.
- 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.

Examinations

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 treatment period. Observed daily during the treatment-free period.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to dosing on 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 efficiency was calculated retrospectively.

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.
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28 for non-recovery groups 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, triglycerides, 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 for non-recovery groups 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 26. Functional performance tests and assessment of sensory reactivity performed during week 4. Motor activity was performed at least 2 hours after dosing.
- 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, Urinalysis (Volume and Specific Gravity), 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)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No unscheduled deaths on the study. No clinical signs observed.
Mortality:
no mortality observed
Description (incidence):
No unscheduled deaths on the study. No clinical signs observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean body weight gains were slightly lower than control for females at all dose levels during the first week of treatment. Recovery evident thereafter. Slight body weight loss for recovery females during latter half of the treatment-free period.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Slightly lower than controls for treated females during the first week. Similar to controls thereafter.
Water consumption and compound intake (if drinking water study):
no effects observed
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 during the treatment period. Hematological parameters similar to controls at the end of the treatment-free period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reduction or increase in the blood chemical parameters for males and/or females with histopathological findings, but reversible in recovery animals. No difference at the end of the treatment-free period. See details below.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No toxicologically significant effects. Only statistically significant reduction or increase in the urinalytical parameters. Within historical background and generally similar to controls.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Group mean liver weight both absolute and body weight relative significantly higher than control for females and significantly lower for males at 1000 mg/kg bw/day. No differences at the end of the treatment period.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY

There were no deaths on the study. There were no clinical signs for any of the animals throughout the study.

BODY WEIGHT AND WEIGHT GAIN

There was no effect of treatment with the test item at any dose level on male body weight development throughout the treatment period. During the treatment-free period, the mean body weight gains for the recovery males previously given the high dose were also similar to controls.

At all dose levels, the group mean body weight gains for females over the first week of the treatment period were slightly but statistically significantly lower than controls (p<0.05), albeit without a dose-relationship. Thereafter, body weight gains for these females showed an improvement such that the group mean gains were generally similar to controls throughout the remaining dosing period. Overall gains for the treated females at the end of the treatment period were comparable with controls, and therefore any initial effect on body weight development was considered to be of no toxicological importance. During the latter half of the treatment-free period, recovery females showed a slight body weight loss, which was likely due to the stress associated with blood sampling on Day 42 of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE

Food consumption for animals of both sexes given the test item at all doses remained similar to controls throughout the study.

FOOD EFFICIENCY

Food conversion efficiency in treated males was similar to controls throughout the study. For treated females, it was slightly lower than controls over the first week of dosing. Thereafter food conversion efficiency in these females was generally similar to controls with occasional differences reflecting slight differences in body weight gains.

WATER CONSUMPTION AND COMPOUND INTAKE

Gravimetric measurement of water consumption during Week 3 of the study did not reveal any treatment-related differences.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY

At the end of the treatment period, males given the test item at all dose levels showed marginally but statistically significantly lower erythrocyte counts and haemoglobin levels when compared with controls (p<0.05). Males given 1000 mg/kg bw/day also showed statistically significantly lower group mean hematocrit value than controls (p<0.05). There was no dose-relationship and individual values were within historical background data ranges. In the absence of any associated histopathology findings from animals receiving the high dose, these findings were considered unlikely to be of any toxicological significance.

At 300 or 1000 mg/kg bw/day, females showed slightly lower group mean activated partial thromboplastin times than controls, but statistical significance was only achieved for the high dose animals (p<0.01). With the exception of Female 38 from the high dose group with a value slightly lower than the historical background data ranges, the remaining values were within these ranges and this shortening of APTT was considered unlikely to be of any toxicological significance.

At the end of the treatment-free period, hematology parameters from animals of either sex previously given the high dose were similar to the respective controls.

CLINICAL CHEMISTRY

At all dose levels, group mean plasma urea concentration for males were statistically significantly higher than controls (p<0.05 for the low and intermediate dose levels, p<0.01 for the high dose level) at the end of the dosing period. A dose-relationship was apparent between the intermediate and high dose levels, but the majority of individual values were within the historical background data ranges. Since plasma levels of creatinine were similar across all male groups including controls and there were no associated histopathology findings, the increase in urea levels was considered unlikely to be of any toxicological importance. Variations in this and other plasma markers and hepatic changes may well be indicative in fluctuations in metabolic processes associated with test item detoxification.

Group mean plasma triglycerides and bilirubin levels for males at all dose levels were statistically significantly lower than controls (p<0.05) whereas A/G ratios were statistically significantly higher than controls (p<0.05). There was no dose-relation for these parameters and the majority of individual values were within the historical background data ranges. Males given the high dose also showed statistically significantly lower plasma levels of gammaglutamyltranspeptidase and alkaline phosphatase when compared with controls (p<0.05), however, the majority of individual values for these animals were within the historical background data ranges. The histopathological examination of male livers showed centrilobular hypertrophy which was considered likely to be of an adaptive nature and was reversible in recovery males and hence these findings were considered unlikely to be of toxicological significance.

At the end of the treatment period, group mean plasma levels of glucose in treated females were statistically significantly higher than controls (p<0.05), whereas plasma concentrations of potassium were statistically significantly lower than controls (p<0.05). There was no dose relationship for these parameters and the majority of individual values were within the historical background data ranges. Histopathology of female livers revealed fatty vacuolation in the periportal region in three out of five females given the high dose, however considering the minimal nature of this finding and its reversibility in the recovery females, it was unlikely to be of an adverse nature and these clinical pathology findings in females were considered not to be of toxicological importance.

At the end of the treatment-free period, there were no differences in measured parameters from animals of either sex previously given the high dose when compared with the respective controls.

URINALYSIS

A high degree of individual variation was apparent for urine volume and specific gravity, however any inter-group differences between animals of either sex receiving the test material and controls towards the end of the treatment period were not statistically significant or dose-related.

Other measured parameters for the treated animals at all dose levels were also generally similar to controls.

At the end of the treatment-free period, group mean urine volume in males previously given the test item at 1000 mg/kg bw/day was statistically significantly lower than controls (p<0.01) whereas the specific gravity was statistically significantly higher than controls (p<0.05). Three out of five of these males also showed cloudy urine whilst only one control was noted with this.

The corresponding parameters in treated females at the end of the recovery period were generally similar to controls. The majority of individual values were within the historical background ranges.

NEUROBEHAVIOUR

Behavorial assessments: There were no treatment-related changes in the behavioral parameters at any dose level.

Functional Performance tests: There were no treatment-related changes in functional performance at any dose level.

Overall activity in females given the test item at 100 mg/kg bw/day was statistically significantly lower than controls (p<0.05); however, there was no dose-relationship and the corresponding values in females receiving 300 or 1000 mg/kg bw/day were similar to controls and, therefore, this finding was considered unrelated to treatment with the test item.

Sensory reactivity assessments: Sensory reactivity scores across all treated groups were similar to controls.

ORGAN WEIGHTS

At the end of the treatment period, group mean absolute and body weight-related liver weights in females given the test item at 1000 mg/kg bw/day were statistically significantly higher than controls (p<0.05) with the majority of individual values for these animals above the historical background data ranges. The corresponding weight from the treated recovery females was similar to controls.

At the end of the recovery period, absolute and body weight-related thyroid/parathyroid weights in males were statistically significantly lower than controls (p<0.05) but in females these weights were statistically significantly higher than controls (p<0.05); it is worth noting that were no differences for these organ weights at the end of the treatment period.

GROSS PATHOLOGY

The incidence and distribution of macroscopic necropsy findings in treated animals did not indicate any adverse systemic effect of treatment. The following isolated findings were noted:
- At the end of the treatment period, one male given the test item at 100 mg/kg bw/day showed small testes and epididymides whilst another male receiving the test item at 1000 mg/kg bw/day showed small testes.
- At the end of the treatment-free period, one female previously given the high dose was noted with pale/enlarged adrenals and fluid-filled right horn of the uterus.

HISTOPATHOLOGY: NON-NEOPLASTIC
The incidence and distribution of microscopic findings in treated animals did not indicate any adverse systemic effect of treatment. The following microscopic findings considered to be associated with the administration of the test item were observed in non-recovery animals:

In comparison with controls, the severity and incidence of intra-epithelial hyaline droplets and basophilic tubules in the kidneys was greater in all the male treated groups. In addition, granular casts were observed at the corticomedullary junction in these males. These findings were consistent with an etiological diagnosis of alpha-2u-globulin nephropathy.

Minimal centrilobular hepatocellular hypertrophy was observed in all the males given 1000 mg/kg bw/day. It was not seen in females or in males from the other treated groups. Minimal periportal fat vacuolation was seen in three out of five females given 1000 mg/kg bw/day. It was not seen in females from the other treated groups.

The incidence of minimal or mild diffuse hypertrophy of the follicular epithelium in the thyroid was greater in animals of either sex given 1000 mg/kg bw/day than in controls. The single incidences of minimal change seen in a female given 100 mg/kg bw/day and a male and a female given 300 mg/kg bw/day were attributed to individual variation and not to the test item.

In animals allowed a fourteen-day post-dosing recovery period, no centrilobular hepatocellular hypertrophy or periportal fat vacuolation was observed in the liver of any animal and the incidence and severity of diffuse hypertrophy of the follicular epithelium in the thyroid glands was comparable between controls and treated animals. These two findings were, therefore, considered to have reversed completely.

In the kidneys, the incidence and severity of increased intra-epithelial hyaline droplets was comparable between controls and recovery males previously given the test item at 1000 mg/kg bw/day. This was considered to indicate that the primary cause of the renal pathology had reversed. However, the incidence and severity of basophilic tubules and granular cast formation at the corticomedullary junction remained greater in treated animals than in controls although at a lower severity than in main study animals. In the absence of other more severe kidney findings such as fibrous scarring, it was thought likely that given sufficient recovery time these too would resolve.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

DEVIATION FROM STUDY PLAN

There was one deviation (unplanned change) from the study plan.

Three instances of high relative humidity were noted for this study, there were two episodes of high relative humidity lasting for up to 2 hours on the same day, one episode lasting for up to 4 hours and another period which lasted for up to six hours.

For these periods, the relative humidity recorded ranged between 70.69 and 75.37% RH. Although the episodes of high relative humidity observed on the study were not ideal, they were only marginally above the higher end of the target range and occurred towards the end of the treatment-free period. Since there were no clinical signs for any of the animals throughout the study, overall it was considered that they had no effect on the scientific integrity of the study.

 

DISCUSSION 

The oral (gavage) administration of the test material to rats, at dose levels of 100, 300 and 1000 mg/kg bw/day was well tolerated. Whilst there was no effect of treatment with the test item on body weight development or associated food intake in males, a slight reduction in body weight gain over the first week of dosing was evident in females at all dose levels, albeit without a dose relationship or any effect on food intake. Since the overall body weight gains in these females during the treatment period were similar to controls, any initial effect on body weight gain was considered to be of no toxicological significance.

Hematological investigations did not reveal any adverse effect of treatment for either sex. Some statistically significant inter-group differences were evident in males and females at the end of the treatment period, however the majority of corresponding individual values were within the background data ranges. As there were no associated histopathology findings and the corresponding values in treated recovery animals were comparable with controls, these differences were considered to be of no toxicological importance.

At the end of the dosing period, statistically significant differences in plasma levels of urea, triglycerides, bilirubin and A/G ratios in males at all dose levels, gamma-glutamyltranspeptidase and alkaline phosphatase in males from the high dose group and glucose and potassium in females at all dose levels were evident. The majority of individual values were within the background data ranges whilst the corresponding values from treated recovery animals were similar to controls. Histopathological examinations of the tissues from animals did not indicate any adverse findings and as such these differences were considered to be of no toxicological significance.

At the end of the treatment period, increased liver weights were apparent in females receiving the test item at 1000 mg/kg bw/day and microscopic evaluation of the liver from these animals revealed minimal periportal fat vacuolation in three animals. It remains debatable whether this minor change was spontaneous in nature as suggested by the degree of change or treatment related as it was confined to the females from the high dose group. Considering the second option as more likely, this change probably signaled a slight perturbation in hepatic lipid metabolism; however, as this finding was not seen in recovery animals and was therefore completely reversible (liver weights in treated recovery females were also similar to controls), it was considered to be non-adverse. Minimal centrilobular hypertrophy of hepatocytes was present in the liver of all the males given 1000 mg/kg bw/day. It was not observed in animals of either sex from the other treated groups or recovery animals. In the absence of any other evidence of liver pathology, this finding was considered to be an adaptive change resulting from enzyme induction and therefore non-adverse.

In the thyroid glands of both sexes receiving the test item at 1000 mg/kg bw/day, there was a greater incidence of minimal or mild diffuse hypertrophy of the follicular epithelium than in controls. The underlying mechanism for this treatment-related change was not apparent but the most likely explanation for its development is a perturbation of thyroid hormone synthesis, transport or metabolism probably associated with liver enzyme induction. At the end of the treatment-free period, inter-group differences in thyroid/parathyroid weights were noted for treated animals of either sex when compared with controls; however, the incidence and severity of diffuse hypertrophy of the follicular epithelium in these animals was comparable indicating reversibility. Since the rat thyroid gland has been shown to be markedly more sensitive than humans in its response to xenobiotics (Chandra, S.et al, 2013), it seems reasonable to conclude that the minor difference seen in this study would not carry significant risk to humans and therefore this finding was considered to be non-adverse.

In the kidneys of males from all the treated groups, there was an increase in the amount of intraepithelial hyaline droplets, an increase in the incidence and severity of foci of basophilic tubules and granular cast formation at the corticomedullary junction. These findings were considered indicative of alpha-2u-globulin nephropathy and, since this condition is species specific, of no relevance to human exposure and was therefore considered to be non-adverse. After the fourteen-day period of recovery, the incidence and severity of intra-epithelial hyaline droplets was comparable between controls and treated animals indicating reversibility of the primary cause of the condition. Granular casts and foci of basophilic tubules were still present in recovery animals given the highest dosage but were not seen in controls. While these two findings would be expected to take longer to resolve than the increased accumulation of hyaline droplets, in the absence of other more severe kidney findings, it was thought likely that given sufficient recovery time these too would resolve.

 

Overall, it is considered that a dosage of 1000 mg/kg bw/day could be assigned a No Observed Adverse Effect Level (NOAEL) for systemic toxicity because all treatment-related findings were either reversible, non-adverse and/or of no relevance to human exposure.

Applicant's summary and conclusion

Conclusions:
The oral (gavage) administration of the test material to male and female Wistar Han™:RccHan™: WIST strain rats, at dose levels of 100, 300 and 1000 mg/kg bw/day was well tolerated. Based on the in-life results and histopathology findings, a dose level of 1000 mg/kg bw/day is considered to be a No Observed Adverse Effect Level (NOAEL) for systemic toxicity.
Executive summary:

Introduction

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.

 

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 100, 300 and 1000 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Recovery groups, each of five males and five females, were treated with the high dose (1000 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. 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 deaths on the study.

Clinical Observations

There were no clinical signs for any of the animals throughout the study.

Behavioral Assessment

Behavioral assessment scores across the treated animals of either sex remained similar to the respective controls.

Functional Performance Tests

There were no treatment-related changes in functional performance at any dose level.

Sensory Reactivity Assessments

Sensory reactivity scores remained unaffected by treatment with the test item.

Body Weight

Body weight development in males remained unaffected by treatment with the test material at all dose

levels. Females receiving the test item at all dose levels showed slightly lower group mean body weight gains over the first week of dosing. As there was no dose-relationship and body weight gains for these females were generally comparable with control from the second week of the treatment period, this finding was considered to be of no toxicological significance.

Food Consumption

Throughout the treatment and treatment-free periods, food consumption for animals of either sex given the test item was comparable with controls. Food conversion efficiency in treated males was also similar to controls throughout the study, however for treated females it was slightly lower than controls over the first week of dosing reflecting slight differences in body weight gains.

Water Consumption

Gravimetric measurement of water consumption during Week 3 of the study did not reveal any treatment-related differences.

Hematology

At the end of the treatment period, males given the test material at all dose levels showed a marginal decrease in erythrocyte counts, hemoglobin levels and hematocrit; the latter was seen in males given 1000 mg/kg bw/day only. Females receiving the test item at 300 or 1000 mg/kg bw/day showed slightly lower group mean activated partial thromboplastin times than controls. These findings were considered unlikely to be of any toxicological significance. At the end of the treatment-free period, hematology parameters for animals of either sex previously given the high dose were similar to the respective controls.

Blood Chemistry

At the end of the treatment period, blood chemistry investigations revealed higher plasma levels of urea and A/G ratios in males given the test material at all dose levels when compared with controls.

Plasma concentrations of triglycerides and bilirubin in these males were lower than controls whilst males given the test item at 1000 mg/kg bw/day also showed lower plasma levels of gamma-glutamyltranspeptidase and alkaline phosphatase. Plasma levels of glucose and potassium in treated females at all dose levels were respectively higher and lower than controls.

These findings were considered unlikely to be of any toxicological significance. At the end of the treatment-free period, blood chemistry parameters for animals of either sex previously given the high dose were similar to controls.

Urinalysis

No toxicologically significant effects were detected in the urinalysis parameters examined at dose levels up to 1000 mg/kg bw/day.

Necropsy

Macroscopic examination did not reveal any treatment-related findings.

Organ Weights

At the end of the treatment period, liver weights for females given the test item at 1000 mg/kg bw/day were slightly higher than controls. At the end of the treatment-free period, thyroid/parathyroid weights in treated males were lower than controls (p<0.05) but in females the corresponding weights were higher than controls. These findings were considered to be of no toxicological importance.

Histopathology

The histopathology findings were as following:

Liver: At the end of the treatment period, minimal centrilobular hypertrophy was evident in males treated with 1000 mg/kg bw/day, which was considered to be of an adaptive nature. Three out of five females from this dose group showed evidence of minimal fatty vacuolation in the periportal region which may indicate a slight perturbation in hepatic lipid metabolism. Since these findings were not seen in animals from the lower dose groups and the microscopic examination of the liver from animals allocated to the treatment-free period showed reversibility, they were considered to be non-adverse.

Thyroid: At the end of the treatment period, diffuse hypertrophy of the follicular epithelium was evident in animals of either sex receiving the test item at 1000 mg/kg bw/day. This finding may indicate perturbation of thyroid hormone synthesis, transport or metabolism probably associated with liver enzyme induction. The incidence and severity of this finding in previously treated animals allocated to the recovery period was similar to controls indicating reversibility. Since the rat thyroid gland is considered to be markedly more sensitive than humans in its response to xenobiotics, the minor difference seen in this study would not carry significant risk to humans, and this finding was therefore considered to be non-adverse.

Kidney: In comparison with controls, the severity and incidence of intra-epithelial hyaline droplets and basophilic tubules in the kidneys was greater in all the male treated groups. In addition, granular casts were observed at the corticomedullary junction in these males. These findings were considered indicative of alpha-2u-globulin nephropathy. Microscopic examination of the kidneys from the recovery animals showed reversibility for intra-epithelial hyaline droplets, the primary cause for this condition. The granular casts and foci of basophilic tubules were still seen in animals previously given the high dose, however in the absence of other more severe kidney findings, it was considered likely that given sufficient recovery time these too would resolve. Since alpha-2u-globulin nephropathy is species specific, of no relevance to human exposure, this finding was considered to be non-adverse.

 

Conclusion

The oral (gavage) administration of the test material to male and female Wistar Han™:RccHan™:WIST strain rats, at dose levels of 100, 300 and 1000 mg/kg bw/day was well tolerated. Based on the in-life results and histopathology findings, a dose level of 1000 mg/kg bw/day is considered to be a No Observed Adverse Effect Level (NOAEL) for systemic toxicity.