3-{3-[(3,5,5-trimethylhexanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propoxy}-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate

Endpoint summary

Description of key information

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 (OECD 407).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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:

other: Study conducted to GLP in accordance with recognised guideline

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

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

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.

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.

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)

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.

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'

Critical effects observed:

not specified

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.

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Nov. 1992 - Dec. 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance CAS 68424-31-7. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Alpk:APfSD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Zeneca Pharmaceuticals, Alderly Park, Macclesfield, Cheshire, UK
- Age at study initiation: 28 d
- Weight at study initiation: Males: 148.45 g; Females: 122.6 g
- Housing: sexes separately, five per cage, Cages had measurements of 26.5x50.0x20.0 cm and were constructed of stainless steel mesh with one solid side.
- Diet: ad libitum (Based on CT1 diet; Special Diets Services Limited, Witham, Essex, UK
- Water: ad libitum
- Acclimation period: approx. 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 45-65 (71 at one occasion)
- Air changes (per hr): 25-30
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: November 1992 To: December 1992

Route of administration:

oral: feed

Vehicle:

other: ethyl acetate

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: All diet were based on CT1 diet (Special Diets Services Limited, Witham, Essex, UK). They were prepared by grinding the appropriate amount of test substance with 1 kg of milled CT1 diet. This premix was then added to 14 kg of diet and mixed thoroughly with a Pharma Blender Model PMA 100S (T K Filder).

DIET PREPARATION
- Rate of preparation of diet (frequency): 15 kg batches
- Mixing appropriate amounts with (Type of food): CT1 diet (Special Diets Services Limited, Witham, Essex, UK)
- Storage temperature of food: - 20°C, stored at RT for usage up to 14 days

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical stability was determined for diets over a period of 5 weeks following storage at RT or at -20°C.
Samples were extracted by chemical shaking with ethyl acetate. The supernatant was diluted with ethyl acetate to give solutions containing appropriate concentrations of the test substance. Extracts were analysed by gas chromatography using flame ionisation detection. The extract concentration was calculated by reference to data from a standard containing a known concentration.

Duration of treatment / exposure:

daily

Frequency of treatment:

28 d

Remarks:

Doses / Concentrations:
0 ppm, 1000 ppm, 5000 ppm, 12500 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
112 mg/kg/d, 562 mg/kg/d, 1450 mg/kg/d
Basis:
other: actual ingested for males

Remarks:

Doses / Concentrations:
119 mg/kg/d, 586 mg/kg/d, 1613 mg/kg/d
Basis:
other: actual ingested for females

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Dose selection rationale: Based on results of preliminary feeding studies
- Rationale for animal assignment (if not random): The sexes were randomised separately.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked: changes in clinical condition and behaviour and significant changes were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on days 8, 15, 22, 29
- observations included, but were not limited to the assessment of autonomic function (e.g. lachrymation, salivation, piloerection, exophthalmus, urination, defecation, pupillary function, ptosis), description, incidence and severity of any convulsions, tremors, abnormal motor function, alteration in respiration, reactivity to stimuli, changes in the level of arousal, sensorimotor responses

BODY WEIGHT: Yes, measurement in replicate order immediately before feeding and at the same day once a week until termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as mg food/kg body weight: Yes, on a weekly basis
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes: At termination, all rats were bled by cardiac puncture and samples were collected. Parameters determined: Hemoglobin, red cell count, haematocrit, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, platelet count, white blood cell count, neutrophil count, lymphocyte count, monocyte count, eosinophil count, prothrombin time and kaolin-cephalin time

CLINICAL CHEMISTRY: Yes, At termination, all rats were bled by cardiac puncture and samples were collected. Parameters determined: Albumin, total protein, cholesterol, triglycerides, urea, creatinine, glucose, total bilirubin and alkaline phosphatase, plasma gamma-glutamyl transferase, plasma alanine aminotransferase, plasma aspartate aminotransferase, plasma creatine kinase, plasma sodium, plasma potassium, plasma chloride, plasma calcium and plasma phosphorus

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on days 8, 15, 22, 29
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (Adrenals, Aorta, Bladder, Bone and Bone marrow (femur), Brain, Caecum, Colon, Cervical lymph node, Cervix, Colon, Duodenum, Epididymis, Eye and harderian gland, Heart, Ileum, Jejunum, Kidney, Liver, Lungs, Mammary gland, Mesenteric lymph node, Nasal passages, Oesophagus, Oral cavity, Ovaries, Pancreas, Parathyroid glad, Pituary gland, Prostate gland, Rectum, Salivary glands, Sciatic nerve, Seminal vesicles, Skin, Spinal chord, Spleen, Sternum, Stomach, Testes, Thymus, Thyroid gland, Trachea, Uterus, Voluntary muscle)

Statistics:

Bodyweights were considered by analysis of covariance on initial body weight, separately for males and females.
Time to tail flick and fore and hindlimb grip strength at weeks 2, 3, 4 and 5 were considered by analysis of variance, separately for both sexes.
Haematological and clinical blood parameters were considered by analysis of variance.
Organ weights were considered by analysis of variance and covariance on final body weight separately for both sexes.

Details on results:

DIET ANALYSIS:
All diets prepared were found to be within 4 % of the target concentration. The homogeneity of the test material in the diet, determined at 1000 and 12500 ppm inclusion levels was within 2 % of the overall mean concentration for both levels. Chemical stability of the test material , assessed at the 1000 and 12500 ppm inclusion levels stored at room temperature or at -20 °C was satisfactory over the period of use.
Dose rates (based on nominal dietary levels) were highest at the start of the study and declined rapidly during the period of rapid growth to week 4.

MORTALITY
There were no mortalities.

FUNCTIONAL OBSERVATION BATTERY
There were no mortalities.
A slightly reduced splay reflex was observed in one female on the 1000 ppm group (on days 29 and 30), one male in the 5000 ppm group (on day 29) and one male in the 12500 ppm group (on day 29). As isolated observations, these were considered to be incidental to treatment.
There were no differences in time to tail flick in either sex which could be attributed to treatment. The statistically significant increase in time to response observed on day 22 for males (5000 ppm) and day 8 for females (1000 ppm) were considered to be incidental to treatment in the absence of similar changes at higher dose levels.
There was no evidence of any treatment related effects on forelimb or hindlimb grip strength.
Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant effects on body weight and all final bodyweights were within 3% of the respective controls, after adjusting for initial weight differences.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption in all treated groups remained similar to, or exceeded that, of the respective control group throughout the study.

HAEMATOLOGY
There were statistical significant reductions in haemoglobin and haematocrit at 12500 ppm in male rats. Statistically significant reductions in haemoglobin and haematocrit were seen in females at 1000 and 5000 ppm and in white blood cell count at 1000 ppm. In the absence of a coherent dose-response relationship, these differences were considered incidental to treatment.
Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

CLINICAL CHEMISTRY
There were minor reductions in plasma cholesterol, triglyceride and total protein levels and plasma alanine transferase activities in males at 12500 ppm compared to controls. Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.
ORGAN WEIGHTS
Kidney weights adjusted for body weight were statistically significant increased in males at 5000 and 12500 ppm. All the females in the treatment groups had slightly raised kidney weights compared to control, but none achieved statistical significance, and there was no evidence of a coherent dose response relationship.
Liver weights adjusted for body weight were statistically significant increased in both sexes at 12500 ppm and in males at 5000 ppm.
Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

PATHOLOGY:
Macroscopic findings:
No treatment-related macroscopic findings were apparent at the end of the study.
Microscopic findings:
Treatment related findings were present in the kidney of male rats from all dose groups. In the 5000 and 12500 ppm dose group these comprised increased tubular hyaline droplet formation and tubular basophilia in all animals, and granular cast formation in four of the 5000 ppm animals and all of the 12500 ppm animals; the latter occurring at the cortico-medullary injection. In the 1000 ppm group, increased renal hyaline droplet formation and/or tubular basophilia were seen, but not granular cast formation.
In the liver, there was minimal hepatocyte hypertrophy in four out of five male rats in the 12500 ppm group.

The increased kidney weights and microscopic findings of renal tubular basophilia, granular cast formation and increased hyaline droplet formation present in male rats at 5000 and 10000 ppm are clearly treatment related. These findings are consistent with the well characterized light hydrocarbon nephropathy described for male rats, following to a variety of chemicals including light hydrocarbons such as unleaded gasoline and trimethyl pentane. The characteristics include an increased accumulation of hyaline droplets in male rat kidneys, the main constituent of which is alpha 2µ-globulin (Alden et al. Adv. Modern Environ Toxicol 7: 107-120 (1984); Stonard et al. Renal Heterogeneity and Target Cell Toxicity. Bach PH and Lock EA Eds, John Wiley and Sons (1985)). It is widely accepted that this phenomenon is specific to male rat and as such appears to have no relevance for man (Swenberg et al. Toxicol and App. Pharmacol. 97: 35-46 (1989)).

Dose descriptor:

NOAEL

Effect level:

12 500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects observed in female rats

Dose descriptor:

NOAEL

Effect level:

1 613 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects observed in female rats

Dose descriptor:

NOAEL

Effect level:

12 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Treatment related histopathological changes and changes in kidney and liver weight in male rats at 5000 ppm and above are considered species-specific effects which are not relevant for humans and therefore not considered for NOAEL determination.

Dose descriptor:

NOAEL

Effect level:

1 450 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Treatment related histopathological changes and changes in kidney and liver weight in male rats at 5000 ppm and above are considered species-specific effects which are not relevant for humans and therefore not considered for NOAEL determination.

Critical effects observed:

not specified

Reference 3

Endpoint:

repeated dose toxicity: oral

Remarks:

other: range-finding study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

19 February 2014 to 21 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed under GLP conditions

Qualifier:

no guideline required

Principles of method if other than guideline:

The study was designed to provide information for further repeated dose toxicity studies.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Wistar Han

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS AND ANIMAL HUSBANDRY
- A sufficient number of male and female Wistar Han: RccHan: WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Oxon, UK.
- On receipt the animals were examined for signs of ill-health or injury.
- The animals were acclimatized for 7 days during which time their health status was assessed.
- A total of twenty four animals (twelve males and twelve females) were accepted into the study.
- At the start of treatment the males weighed 186 to 204 g, the females weighed 145 to 162 g, and animals were approximately six to eight weeks old.
- Animals were housed in groups of three by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
- The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
- The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK, Barrier Maintained Rodent Facility.
- The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.
- Environmental conditions were continuously monitored by a computerised system, and print-outs of hourly temperatures and humidities were included in the study records.
- The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20 % respectively; short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.
- The animals were allocated to dose groups using a randomisation procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups.
- The animals were uniquely identified within the study, by an ear punch system routinely used in these laboratories.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

TEST ITEM PREPARATION
- The test item was prepared at the appropriate concentrations as a solution in Arachis oil BP.
- The test item was administered within two hours of it being formulated. It is assumed that the formulation was stable for this duration.
- No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and was reflected in the GLP compliance statement.

PROCEDURE
- Animals were allocated to treatment groups as shown in the table below.
- The test material was administered daily, for seven consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg/day of dried Arachis oil BP.
- The volume of test and control item administered to each animal was based on the most recently scheduled body weight and was adjusted on Days 3 and 5.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Seven days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
250 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
500 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:

- Control: 3 male and 3 female
- Low (250 mg/kg/day): 3 males and 3 females
- Intermediate (500 mg/kg/day): 3 males and 3 females
- High (1000 mg/kg/day): 3 males and 3 females

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS
- All animals were examined for overt signs of toxicity, ill health or behavioral change immediately before dosing, up to thirty minutes after dosing and one hour after dosing.
- Animals were also examined four hours after dosing on week days.
- All observations were recorded.

BODY WEIGHT
- Individual body weights were recorded on Days 1, 3, 5 and 8.

FOOD CONSUMPTION
- Food consumption was recorded for each cage group for Days 1 to 3, 3 to 5 and 5 to 8.
- Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION
- Water intake was measured and recorded daily for each cage group

Sacrifice and pathology:

NECROPSY
- On completion of the dosing period, all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination and subjected to an internal and external macroscopic examination.
- No tissues were retained.

Statistics:

EVALUATION OF DATA
- Data were processed to give summary incidence or group mean and standard deviation values where appropriate.
- All data were summarised in tabular form.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

see below

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY
- There were no deaths during the study.

CLINICAL OBSERVATIONS
- There were no clinical signs for any of the animals throughout the dosing period.

BODY WEIGHT
- At 1000 mg/kg bw/day, group mean body weight for males over days 1 to 5 of dosing was slightly lower than controls resulting in the overall gain for these animals being 19 % lower than controls.
- At 500 mg/kg bw/day, the overall gain for males was also 19 % lower than controls. This was mainly due to reduced body weight gain towards the end of the treatment period and was not considered to be treatment-related.
- At 250 mg/kg bw/day, body weight gain in males was similar to controls.
- There was no effect of treatment with the test item on body weight development in females at any dose level.

FOOD CONSUMPTION
- Group mean food intake remained unaffected in animals of both sexes receiving the test item at all dose levels.
- Males given 1000 mg/kg bw/day showed a slight reduction in food conversion efficiency between days 1 and 3, which reflects a slight reduction in body weight gain for these males at the start of the treatment period.
- There were no other inter-group differences in food conversion efficiency considered to be related to treatment with the test item.

WATER CONSUMPTION
- Gravimetric measurement of water consumption did not indicate any intergroup differences in animals of either sex given the test item when compared with controls.

NECROPSY
- No macroscopic abnormalities were detected.

Critical effects observed:

not specified

Conclusions:

Oral administration of test item to rats by gavage at dose levels of 250, 500 and 1000 mg/kg bw/day was well tolerated. Males given the test item at 1000 mg/kg bw/day showed slightly lower body weight gains at the start of dosing, which was associated with slightly lower food conversion efficiency. Based on these results, dose levels of 100, 300 and 1000 mg/kg bw/day were considered to be suitable for a 28 -day repeat dose toxicity study in the rat.

Executive summary:

METHOD

The test item was administered by gavage to three groups of male and female Wistar Han:RccHan:WIST strain rats for seven consecutive days. Dose levels were 250, 500 or 1000 mg/kg bw/day. A control group of three males and three females was dosed with vehicle alone (arachis oil).

Clinical signs (body weight change, dietary intake and water consumption) were monitored during the study. All animals were subjected to gross necropsy examination.

RESULTS

Mortality: There were no deaths during the study.

Clinical observations: No clinical signs were detected for any of the animals during the treatment period.

Body weight: At 1000 mg/kg bw/day, group mean weight gain over days 1 to 5 of dosing for males was slightly lower than controls resulting in a slightly lower overall body weight gain (19 %) for those animals. Body weight development in males given 250 or 500 mg/kg bw/day and females at all dose levels remained unaffected.

Food consumption: During the dosing period, food intake across all test item-treated groups was similar to controls. At 1000 mg/kg bw/day, males showed a slight reduction in food conversion efficiency between days 1 and 3, which reflects a slight initial reduction in body weight gain for these animals. There were no other intergroup differences in food conversion efficiency for animals of both sexes considered to be related to treatment with the test item.

Water consumption: Water intake levels across all groups receiving the test item were similar to controls.

Necropsy: There were no macroscopic findings at necropsy.

CONCLUSION

Oral administration of test item to rats by gavage at dose levels of 250, 500 and 1000 mg/kg bw/day was well tolerated. Males given the test item at 1000 mg/kg bw/day showed slightly lower body weight gains at the start of dosing, which was associated with slightly lower food conversion efficiency. Based on these results, dose levels of 100, 300 and 1000 mg/kg bw/day were considered to be suitable for a 28 -day repeat dose toxicity study in the rat.

Reference 4

Endpoint:

sub-chronic toxicity: oral

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Reference 5

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

To register EC453-460-3 under REACH program, a read-across approach for fulfillment of the endpoints has been adopted. EC453-460-3 is an aliphatic ester and fits the description of polyol esters category in HPV program (High Production Volume, US-EPA). The distinguishing feature of polyol esters category is that the fatty acids were linked to one or more of the multiple hydroxyl groups present in the polyol (alcohol portion of ester). The focus of this correspondence is to support read across to EC613-848-7 (target) which provided key studies to determine hazard profile of EC453-460-3 (target), published information on other structurally analogous polyol esters were served as supporting evidence.

Reason / purpose for cross-reference:

read-across source

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.

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'

Critical effects observed:

not specified

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.

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.

Reference 6

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

To register EC453-460-3 under REACH program, a read-across approach for fulfillment of the endpoints has been adopted. EC453-460-3 is an aliphatic ester and fits the description of polyol esters category in HPV program (High Production Volume, US-EPA). The distinguishing feature of polyol esters category is that the fatty acids were linked to one or more of the multiple hydroxyl groups present in the polyol (alcohol portion of ester). The focus of this correspondence is to support read across to EC613-848-7 (target) which provided key studies to determine hazard profile of EC453-460-3 (target), published information on other structurally analogous polyol esters were served as supporting evidence.

Reason / purpose for cross-reference:

read-across source

Details on results:

DIET ANALYSIS:
All diets prepared were found to be within 4 % of the target concentration. The homogeneity of the test material in the diet, determined at 1000 and 12500 ppm inclusion levels was within 2 % of the overall mean concentration for both levels. Chemical stability of the test material , assessed at the 1000 and 12500 ppm inclusion levels stored at room temperature or at -20 °C was satisfactory over the period of use.
Dose rates (based on nominal dietary levels) were highest at the start of the study and declined rapidly during the period of rapid growth to week 4.

MORTALITY
There were no mortalities.

FUNCTIONAL OBSERVATION BATTERY
There were no mortalities.
A slightly reduced splay reflex was observed in one female on the 1000 ppm group (on days 29 and 30), one male in the 5000 ppm group (on day 29) and one male in the 12500 ppm group (on day 29). As isolated observations, these were considered to be incidental to treatment.
There were no differences in time to tail flick in either sex which could be attributed to treatment. The statistically significant increase in time to response observed on day 22 for males (5000 ppm) and day 8 for females (1000 ppm) were considered to be incidental to treatment in the absence of similar changes at higher dose levels.
There was no evidence of any treatment related effects on forelimb or hindlimb grip strength.
Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant effects on body weight and all final bodyweights were within 3% of the respective controls, after adjusting for initial weight differences.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption in all treated groups remained similar to, or exceeded that, of the respective control group throughout the study.

HAEMATOLOGY
There were statistical significant reductions in haemoglobin and haematocrit at 12500 ppm in male rats. Statistically significant reductions in haemoglobin and haematocrit were seen in females at 1000 and 5000 ppm and in white blood cell count at 1000 ppm. In the absence of a coherent dose-response relationship, these differences were considered incidental to treatment.
Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

CLINICAL CHEMISTRY
There were minor reductions in plasma cholesterol, triglyceride and total protein levels and plasma alanine transferase activities in males at 12500 ppm compared to controls. Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.
ORGAN WEIGHTS
Kidney weights adjusted for body weight were statistically significant increased in males at 5000 and 12500 ppm. All the females in the treatment groups had slightly raised kidney weights compared to control, but none achieved statistical significance, and there was no evidence of a coherent dose response relationship.
Liver weights adjusted for body weight were statistically significant increased in both sexes at 12500 ppm and in males at 5000 ppm.
Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

PATHOLOGY:
Macroscopic findings:
No treatment-related macroscopic findings were apparent at the end of the study.
Microscopic findings:
Treatment related findings were present in the kidney of male rats from all dose groups. In the 5000 and 12500 ppm dose group these comprised increased tubular hyaline droplet formation and tubular basophilia in all animals, and granular cast formation in four of the 5000 ppm animals and all of the 12500 ppm animals; the latter occurring at the cortico-medullary injection. In the 1000 ppm group, increased renal hyaline droplet formation and/or tubular basophilia were seen, but not granular cast formation.
In the liver, there was minimal hepatocyte hypertrophy in four out of five male rats in the 12500 ppm group.

The increased kidney weights and microscopic findings of renal tubular basophilia, granular cast formation and increased hyaline droplet formation present in male rats at 5000 and 10000 ppm are clearly treatment related. These findings are consistent with the well characterized light hydrocarbon nephropathy described for male rats, following to a variety of chemicals including light hydrocarbons such as unleaded gasoline and trimethyl pentane. The characteristics include an increased accumulation of hyaline droplets in male rat kidneys, the main constituent of which is alpha 2µ-globulin (Alden et al. Adv. Modern Environ Toxicol 7: 107-120 (1984); Stonard et al. Renal Heterogeneity and Target Cell Toxicity. Bach PH and Lock EA Eds, John Wiley and Sons (1985)). It is widely accepted that this phenomenon is specific to male rat and as such appears to have no relevance for man (Swenberg et al. Toxicol and App. Pharmacol. 97: 35-46 (1989)).

Dose descriptor:

NOAEL

Effect level:

12 500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects observed in female rats

Dose descriptor:

NOAEL

Effect level:

1 613 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects observed in female rats

Dose descriptor:

NOAEL

Effect level:

12 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Treatment related histopathological changes and changes in kidney and liver weight in male rats at 5000 ppm and above are considered species-specific effects which are not relevant for humans and therefore not considered for NOAEL determination.

Dose descriptor:

NOAEL

Effect level:

1 450 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Treatment related histopathological changes and changes in kidney and liver weight in male rats at 5000 ppm and above are considered species-specific effects which are not relevant for humans and therefore not considered for NOAEL determination.

Critical effects observed:

not specified

Reference 7

Endpoint:

repeated dose toxicity: oral

Remarks:

other: range-finding study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

To register EC453-460-3 under REACH program, a read-across approach for fulfillment of the endpoints has been adopted. EC453-460-3 is an aliphatic ester and fits the description of polyol esters category in HPV program (High Production Volume, US-EPA). The distinguishing feature of polyol esters category is that the fatty acids were linked to one or more of the multiple hydroxyl groups present in the polyol (alcohol portion of ester). The focus of this correspondence is to support read across to EC613-848-7 (target) which provided key studies to determine hazard profile of EC453-460-3 (target), published information on other structurally analogous polyol esters were served as supporting evidence.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

see below

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY
- There were no deaths during the study.

CLINICAL OBSERVATIONS
- There were no clinical signs for any of the animals throughout the dosing period.

BODY WEIGHT
- At 1000 mg/kg bw/day, group mean body weight for males over days 1 to 5 of dosing was slightly lower than controls resulting in the overall gain for these animals being 19 % lower than controls.
- At 500 mg/kg bw/day, the overall gain for males was also 19 % lower than controls. This was mainly due to reduced body weight gain towards the end of the treatment period and was not considered to be treatment-related.
- At 250 mg/kg bw/day, body weight gain in males was similar to controls.
- There was no effect of treatment with the test item on body weight development in females at any dose level.

FOOD CONSUMPTION
- Group mean food intake remained unaffected in animals of both sexes receiving the test item at all dose levels.
- Males given 1000 mg/kg bw/day showed a slight reduction in food conversion efficiency between days 1 and 3, which reflects a slight reduction in body weight gain for these males at the start of the treatment period.
- There were no other inter-group differences in food conversion efficiency considered to be related to treatment with the test item.

WATER CONSUMPTION
- Gravimetric measurement of water consumption did not indicate any intergroup differences in animals of either sex given the test item when compared with controls.

NECROPSY
- No macroscopic abnormalities were detected.

Critical effects observed:

not specified

Conclusions:

Oral administration of test item to rats by gavage at dose levels of 250, 500 and 1000 mg/kg bw/day was well tolerated. Males given the test item at 1000 mg/kg bw/day showed slightly lower body weight gains at the start of dosing, which was associated with slightly lower food conversion efficiency. Based on these results, dose levels of 100, 300 and 1000 mg/kg bw/day were considered to be suitable for a 28 -day repeat dose toxicity study in the rat.

Executive summary:

METHOD

The test item was administered by gavage to three groups of male and female Wistar Han:RccHan:WIST strain rats for seven consecutive days. Dose levels were 250, 500 or 1000 mg/kg bw/day. A control group of three males and three females was dosed with vehicle alone (arachis oil).

Clinical signs (body weight change, dietary intake and water consumption) were monitored during the study. All animals were subjected to gross necropsy examination.

RESULTS

Mortality: There were no deaths during the study.

Clinical observations: No clinical signs were detected for any of the animals during the treatment period.

Body weight: At 1000 mg/kg bw/day, group mean weight gain over days 1 to 5 of dosing for males was slightly lower than controls resulting in a slightly lower overall body weight gain (19 %) for those animals. Body weight development in males given 250 or 500 mg/kg bw/day and females at all dose levels remained unaffected.

Food consumption: During the dosing period, food intake across all test item-treated groups was similar to controls. At 1000 mg/kg bw/day, males showed a slight reduction in food conversion efficiency between days 1 and 3, which reflects a slight initial reduction in body weight gain for these animals. There were no other intergroup differences in food conversion efficiency for animals of both sexes considered to be related to treatment with the test item.

Water consumption: Water intake levels across all groups receiving the test item were similar to controls.

Necropsy: There were no macroscopic findings at necropsy.

CONCLUSION

Oral administration of test item to rats by gavage at dose levels of 250, 500 and 1000 mg/kg bw/day was well tolerated. Males given the test item at 1000 mg/kg bw/day showed slightly lower body weight gains at the start of dosing, which was associated with slightly lower food conversion efficiency. Based on these results, dose levels of 100, 300 and 1000 mg/kg bw/day were considered to be suitable for a 28 -day repeat dose toxicity study in the rat.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

Key study

The key study was designed to investigate the systemic toxicity of an analogue of the registered substance 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.

- 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).

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.

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: 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: Scores remained unaffected by treatment with the test item.

Body Weight: Development of body weight 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.

The histopathology findings were as follows:

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.

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.

Supporting study

In a supporting study using an analogue substance, repeated dietary administration of the test material to rats, up to and including a dose level of 1450 mg/kg bw/day for male rats and 1613 mg/kg bw/d for female rats, did not produce any evidence of overt toxicity. There were no clinical signs indicative of neurological dysfunction or neuropathological changes in the brain related to treatment with the test material at any dose level.

In male rats, an increased incidence of renal hyaline droplet formation and tubular basophilia was present at all dose levels, with granular formation and increased kidney weights also present at 5000 and 12500 ppm. This phenomenon is believed to be specific to male rats as such and is not considered to be of relevance to man.

Inhalation

The test substance has been shown to have a low vapour pressure (9.08 x 10E-03 Pa at 25 °C) and high onset boiling point range (from approximately 277 °C at 101 kPa). As a result, the potential for generation of inhalable forms of the substance is low and exposure of humans via the respiratory route is predicted to be negligible under normal use conditions. Furthermore, the Log Pow value of > 10.0 does not favour absorption directly across the respiratory tract epithelium by passive diffusion (Log10 Pow > 4) and the substance will not be readily soluble in blood because it is poorly water soluble (< 1 x 10E-06 g/L at 20 °C). Thus experimental evidence is in agreement with ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7c: Endpoint specific guidance (Version 2.0; November 2014), and investigation of repeated dose toxicity via the inhalation route is scientifically invalid.

Dermal

Experimental data shows that analogues of the liquid substance have low oral and dermal toxicity under acute conditions (LD50 > 2000 mg/kg) and repeated exposure of the skin is not expected under normal condition of use. In addition, the test material has been determined to have a low vapour pressure (9.08 x 10E-03 Pa at 25 °C) and high onset boiling point range (from approximately 277 °C at 101 kPa), indicating that the potential for dermal absorption after exposure to vapour is low. Furthermore, the substance has a high average molecular weight, is poorly soluble (< 1 x 10E-06 g/L at 20 °C) and has a Log10 Kow value of > 10.0. Consequently, and in accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7c: Endpoint specific guidance (Version 2.0; November 2014), the substance is considered insufficiently soluble to partition from the stratum corneum into the epidermis and the majority of UVCB constituents are likely to be too large to favour dermal absorption (molecular weight > 100 g/moL and Log10 Pow > 4). Investigation of repeated dose toxicity over a period of 28 days via the dermal route is therefore contraindicated.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP guideline study on read-across substance

Justification for classification or non-classification

No classification for repeat dose toxicity is required as the key study on an analogue of the registered substance reports no adverse effects relevant to humans at the maximum recommended dose level of 1000 mg/kg bw/day.