Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Some information in this page has been claimed confidential.

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 June 2015 to 23 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
various deviations from study plan with no impact on integrity of results (see below)
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
yes
Remarks:
various deviations from study plan with no impact on integrity of results (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:
various deviations from study plan with no impact on integrity of results (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:
various deviations from study plan with no impact on integrity of results (see below)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Details on test material:
- Physical state: Pale amber liquid
- Analytical purity: 100 %
- Expiration date of the lot/batch: 08 January 2017
- Storage condition of test material: Approximately 4 °C in the dark (see deviation from study plan, below)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: 188 to 233 g (males) and 147 to 173 g (females)
- Housing: Groups of five animals by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd, Cheshire UK)
- Diet: Free access to a pelleted diet (Rodent 2014C Teklad Global Certified Diet supplied by Harlan Laboratories UK Ltd, Oxon, UK)
- Water: Free access to mains drinking water supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 9 days
- Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd, Cheshire, UK).
- Diet, drinking water, bedding and environmental enrichment items were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Animals (groups 1-4) were randomly allocated to treatment groups using a stratified body weight randomisation procedure and the group mean body weights were then determined to ensure similarity between treatment groups. Cage distribution within the holding rack (groups 1-4) was also randomised.
- An additional dose group (group 5) of five males and five females was incorporated into the study on day 6 of dosing for the remaining dose groups cages for this dose group were placed on a separate holding rack.
- Animals were uniquely identified within the study by an ear punching system.

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

IN-LIFE DATES: From: 19 June 2015 To: 17 July 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
TEST ITEM PREPARATION
- Test item was prepared at the approximate concentrations as a solution in arachis oil BP.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability and homogeneity of test item formulations was determined by Harlan Laboratories Ltd, Shardlow, UK.
- The formulations were shown to be stable for at least 21 days when stored at approximately 4 °C in the dark.
- Prepared formulations were found to be within 93 % to 105 % of the nominal concentration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
- Test item was administered daily (by gavage using a stainless steel cannula attached to a disposable plastic syringe).
- Control animals were treated in an identical manner with 4 mL of arachis oil BP.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
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:
400 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 (0 mg/mL): 5 males and 5 females
- Recovery control (0 mg/mL): 5 males and 5 females
- Low (25 mg/mL): 5 males and 5 females
- Intermediate (75 mg/mL): 5 males and 5 females
- Intermediate II (100 mg/mL): 5 males and 5 females
- High (250 mg/mL): 5 males and 5 females
- Recovery High (250 mg/mL): 5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- The volume of test or control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS
- All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one hour after dosing (see deviations from study plan).
- During the treatment-free period, animals were examined daily.
- All observations were recorded.

FUNCTIONAL OBSERVATIONS
- Prior to the start of treatment, and on days 7, 14, 21 and 25 of dosing, all non-recovery animals were observed for signs of functional/behavioural toxicity.
- Functional performance tests were also performed on all non-recovery animals during week 4 of dosing together with an assessment of sensory reactivity to different stimulii.
- Observations were carried out from approximately two hours after dosing on each occasion.


FUNCTIONAL PERFORMANCE TESTS
- The scoring system and explanation for behavioural assessments and sensory reactivity test are provided in an attached document.

Motor activity
- Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity.
- Non-recovery animals of one sex were tested at each occasion and were randomly allocated to the activity monitors.
- The tests were performed at approximately the same time on each occasion (at least two hours after dosing) and under similar laboratory conditions.
- The evaluation period was one hour for each animals.
- The time in seconds that each animal was active and mobile was recorded for the overall one hour period and also during the final 20 % of the period (considered to be the asymtotic period; Reiter and Macphail, 1979).

Forelimb/hindlimb grip strength
- An automated grip strength meter was used.
- Each non-recovery animal was allowed to grip the proximal metal bar of the meter with its forepaws.
- The animal was pulled by the base of the tail until its grip was broken.
- The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar.
- A record of the force required to break the grip for each animal was made..
- Three consecutive trials were performed for each animal.
- The assessment was developed from the method employed by Meyer et all (1979).
Sacrifice and pathology:
NECROPSY
- On completion of the dosing period, all surviving animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
- All animals were subjected to a full external and internal examination with any abnormalities being recorded.

THYROID HORMONE ASSESSMENT
- At termination, blood samples were taken from the exsanguination procedure and the serum from each animal was stored frozen at approximately minus 20 °C.
- No treatment-related effects on the pituitary-thyroid axis were identified and the samples were discarded without analysis.

Other examinations:
BODY WEIGHT
- Individual body weights were recorded on day 1 and at weekly intervals thereafter.
- Body weights were also determined prior to terminal kill.

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

WATER CONSUMPTION
- Water intake was observed daily for each cage group (initially by visual inspection of the water bottles for overt changes).
- During week 3, water consumption was measured gravimetrically and, due to possible intergroup differences, water consumption was measured and recorded for each cage group until the termination of the study.
Statistics:
- Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical analysis was achieved at a level of p < 0.05.
- Statistical analysis was performed on the following parameters: grip strength, motor activity, body weight change, hematology, blood chemistry, uring volume, specific gravity, absolute organ weights, body weight-relative organ weights.
- Data were analysed using the decision tree from Provantis tables and statistics module (see details below).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
not treatment-related (see below)
Mortality:
mortality observed, treatment-related
Description (incidence):
not treatment-related (see below)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
not treatment-related (see below)
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
not treatment related (see below)
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
not considered to be adverse (see below)
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
no toxicological significance (see below)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
not considered to be adverse (see below)
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
not treatment related (see below)
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
no toxicological significance (see below)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
not treatment related (see below)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
test item-related changes in kidneys, liver and thyroid (see below)
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY
- There were no treatment-related deaths during the study.
- One male from the 100 mg/kg bw/day dose group was found dead during the blood sampling procedure on day 28 of dosing. There were no clinical signs for this animal prior to death whilst macroscopic findings at necropsy included dark and enlarged liver. Microscopic examination of the selected tissues from this male identified liver congestion, an agonal change, but the reason for death could not be established. As there were no premature decedents in any other dose groups, this death was considered likely to have resulted from the stress associated with the blood sampling procedure and unrelated to treatment with the test item.

CLINICAL OBSERVATIONS
- There were no clinical signs considered to be related to the toxicity of the test item.
- At 1000 mg/kg bw/day, animals of either sex showed sporadic instances of increased post-dose salivation from the first week of dosing.
- Animals treated 300 mg/kg bw/day showed similar clinical signs from the first week of treatment and animals treated with 400 mg/kg bw/day showed similar clinical signs from the second week of treatment. The clinical signs occurred in both groups to a lesser extent than with the high dose animals.
- Although increased post-dose salivation was also recorded from some control males and females on day 16, such observations in test-item treated animals are commonly observed following administration of an unpalatable or slightly irritant test item formulation, and are considered to be of no toxicological significance.
- During the 14 day treatment-free period, there were no clinical signs for any of the recovery animals.

BEHAVIOURAL ASSESSMENTS
- There were no changes in the assessed behavioural patterns considered to be related to treatment with the test item at any dose level.
- At 400 mg/kg bw/day, individual non-recovery animals of either sex were observed with slight or moderate salivation during the behavioural assessment in the final week of dosing. None of the remaining non-recovery animals on the study (including those from the high dose group) showed similar observations and as such this finfing was deemed likely to be incidental.

FUNCTIONAL PERFORMANCE TESTS
- There were no treatment-related changes in functional performance at any dose level.
- Grip strength evaluations during the last week of the treatment period revealed statistically significant intergroup differences in hind limb strength for the non-recovery females from all dose groups (p < 0.05). However, there was no dose relationship and all individual values were within the historical control data ranges.
- There were no clinical signs of neurotoxicity for any of the animals on the study and these observations were considered unlikely to be related to treatment with the test item.

SENSORY REACTIVITY ASSESSMENTS
- There were no treatment-related changes detected at any dose level.

BODY WEIGHT
- No detrimental effects of treatment with the test item were observed on body weight development in animals of either sex.
- During the 28 day treatment period, animals of either sex treated with 100, 300 or 400 mg/kg bw/day showed some instances of slightly reduced weekly body weight gains, occasionally achieving statistical significance ( p < 0.01 or p < 0.05). As there was no dose relationship and group mean body weight gains for animals of both sexes treated with 1000 mg/kg bw/day were comparable with controls throughout dosing, any intergroup differences for the lower dose groups were considered to be due to biological variation.
- Over the first week of the treatment-free period, group mean body weight gains for recovery animals of either sex previously treated with 1000 mg/kg bw/day were lower than controls and achieved statistical significance in males (p < 0.05). Whilst recovery was evident in males over the second week, both controls and previously test item-treated females showed comparable body weight losses during this period. Although overall body weight gain in recovery females previously given 1000 mg/kg bw/day during the treatment-free period was lower than controls, there was no detrimental effect of treatment on female body weight performance during the dosing period and as such any intergroup differences were considered most likely to be due to inter-individual variation.



Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on the in-life results and histopathology findings
Remarks on result:
other: Based on the in-life results and histopathology findings, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day for males and 400 mg/kg bw/day for females.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Based on the in-life results and histopathology findings
Remarks on result:
other: Based on the in-life results and histopathology findings, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day for males and 400 mg/kg bw/day for females.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

FOOD CONSUMPTION

- Throughout the treatment and recovery periods, dietary intake for animals of both sexes receiving the test material was comparable with controls at all dose levels.

- Occasional differences in food conversion efficiency were deemed to be reflective of intergroup differences in body weight gains.

WATER CONSUMPTION

- When compared with controls, gravimetric measurement of water consumption during weeks 3 and 4 of the dosing period identified slight increases in water intake for animals of either sex treatedwith 1000 mg/kg bw/day .

- Both males and females treated with 400 or 300 mg/kg bw/day and females treated with 100 mg/kg bw/day also showed some instances of increased water intake in relation to controls resulting in slightly higher overall water intake. A dose relationship was only apparent for males.

- During the recovery period, water consumption levels for males previously given 1000 mg/kg bw/day were occasionally lower tha controls whilst the corresponding values for females were generally comparable with controls indicating reversibility and any intergroup differences were considered not to be of an adverse nature.

LABORATORY INVESTIGATIONS

- No toxicologically significant effects were detected at any dose level.

- At the end of the dosing period, males receiving the test item at 400 or 1000 mg/kg bw/day and females from all dose groups showed statistically significantly higher reticulocyte counts when compared with the respective controls (p < 0.01 for females treated with 400 or 1000 mg/kg bw/day and p < 0.05 for the remaining instances). Dose dependance was only evident in males from the corresponding dose groups. These differences are associated with atypically low values for most control animals whilst the majority of individual values from the test item-treated animals were within the background data ranges. in the absence of any treatment-related changes in any associated parameters, these intergroup differences were considered to be of no toxicological significance. At the end of the recovery period, the corresponding values in animals of both sexes previously treated with 1000 mg/kg bw/day were comparable with controls.

- At 400 mg/kg bw/day, non-recovery females showed statistically significantly lower group mean hemoglobin level in relation to controls (p < 0.05). As all individual results were within the historical control ranges and the corresponding group mean value for 1000 mg/kg bw/day dose group males was similar to controls, this observation was considered to be incidental.

- At the end of the 14 day treatment-free period, the mean corpuscular hemoglobin concentration and neutrophil counts in males previously administered 1000 mg/kg bw/day were statistically significantly higher than controls (p < 0.05). There were no changes in any associated parameters whilst the corresponding values in females were similar to controls and as such these observations were considered to be of no toxicological significance.

BLLOD CHEMISTRY

- At the end of the treatment period, group mean plasma levels of cholesterol in males treated with 300, 400 or 1000 mg/kg bw/day were statistically significantly higher than controls (p < 0.05 at 300 mg/kg bw/day and p < 0.01 for the remaining dose groups) in a dose related manner. All individual values were within the historical control data ranges and the corresponding group mean value from the recovery males previously treated with 1000 mg/kg bw/day was comparable with controls. Microscopic examination of the liver inidcated minimal centrilobular hypertrophy in most non-recovery males treated with 1000 mg/kg bw/day. Variations in this plasma marker and hepatic changes may well be indicative in fluctuations in metabolic processes associated with test item detoxification and taken together these observations were deemed not to represent an adverse effect of treatment.

- Other statistically significant intergoup differences in non-recovery animals included increased albumin concentrations in males given 400 mg/kg bw/day (p < 0.05) and females given 100 mg/kg bw/day (p < 0.05) and increased albumin/globulin ratio and chloride levels in females from the 400 mg/kg bw/day dose group (p < 0.05). The corresponding values in males and females from the 1000 mg/kg bw/day group were similar to controls and these intergroup differences were considered to be incidental.

- At the end of the recovery period, group mean plasma levels of phosphorus in males and albumin/globulin ratio in females previously given 1000 mg/kg bw/day were statistically significantly lower than controls (p < 0.05 and p < 0.01 respectively). With no changes in any associated parameters and the corresponding values in the opposite sex being similar to controls, these observations were considered to be of no toxicological significance.

URINALYSIS

- Towards the end of the dosing period, urine volume and specific gravity in non-recovery males from all test item-treated dose groups were slightly higher than controls, but there was no dose relationship and statistical analysis did not reveal any significance. The corresponding values in non-recovery females from all dose groups were comparable with controls. Towards the end of the recovery period, there were again no statistically significant intergroup differences for males or females previously treated with 1000 mg/kg bw/day in relation to the respective controls.

- Towards the end of the recovery period, urine samples from 2/5 males and 3/5 females previously treated with 1000 mg/kg bw/day tested positive for the presence of protein. As this was only seen in one non-recovery male and female each from the 1000 mg/kg bw/day dose group with one non-recovery control male also showing a similar result, these observations were considered to be incidental. Ketones were also detected in urine from one non-recovery and one recovery male each treated with 1000 mg/kg bw/day whilst one non-recovery female from this dose group tested positive for the presence of erythrocytes in urine. Due to the isolated nature of these incidents, they were considered unlikely to be related to treatment with the test item.

NECROPSY

- At terminal necropsy, there were no observations considered to be related to treatment with the test item.

- One male given 400 mg/kg bw/day showed small prostate and seminal vesicles (with coagulating gland). As there were no similar observations for any other males on the study, this observation was considered to be unrelated to treatment with the test item.

ORGAN WEIGHTS

- At the end of the dosing period, group mean absolute and body weight-related kidney weights in males given 400 or 1000 mg/kg bw/day and liver weights in animals of either sex from these dose groups as well as males receiving 300 mg/kg bw/day were statistically significantly higher than controls (p < 0.01 in all instances). Dose relationships were evident with intergroup differences being particularly marked for the 1000 mg/kg bw/day animals. Most individual values from the animals given 1000 mg/kg bw/day and some individual values from the 400 mg/kg bw/day animals were above the background data ranges. At the end of the 14 day recovery period, only males previously given 1000 mg/kg bw/day showed statistically significantly higher group mean absolute and body weight-related liver and kidney weights in relation to controls (p < 0.01 for liver, p < 0.05 for kidney). It is worth noting, however, that the magnitude of these intergroup differences in recovery males was considerably lower than for non-recovery animals; the majority of the individual values from recovery animals were also within the background data ranges.

- At 400 or 1000 mg/kg bw/day, group mean spleen weights in males were statistically significantly higher than controls at the end of the treatment period (p < 0.01 and p < 0.05 respectively). There was no dose relationship and most individual values from the test item-treated males were within the historical control data ranges. The corresponding values from the recovery males previously given 1000 mg/kg bw/day were also similar to controls. In the absence of any histopathology correlates, these findings were considered to be of no toxicological significance.

- At the end of the treatment-free period, group mean absolute and body weight-related brain weights in females previously treated with 1000 mg/kg bw/day were statistically significantly lower than controls (p < 0.05). The differences were minimal and with group mean absolute and body weight-related brain weights for non-recovery animals of either sex and for recovery males being similar to controls, this observation was considered to be of no toxicological significane.

- Any other intergroup differences attaining statistical significance were considered to be unrelated to treatment with the test item.

HISTOPATHOLOGY

- Changes considered to be associated with the oral (gavage) administration of test item were present in the kidneys of males and the liver and thyroid gland of both sexes. No morphological 'No Observed Effect Level' was established for findings in the kidneys. Test item-related changes in the liver were confined to animals given 1000 mg/kg bw/day with thyroid changes seen in animals treated with 300, 400 or 1000 mg/kg bw/day.

- Kidneys: When compared with controls, the severity and incidence of increased intra-epithelial hyaline droplets and basophilic tubules was greater in all non-recovery male test item-treated groups. Granular casts were also observed at the corticomedullary junction in males receiving the test item at dose levels of 300 mg/kg bw/day or above. These findings were consistent an etiological diagnosis of alpha-2u-globulin nephropathy. In the recovery males, the incidence and severity of increased intra-epithelial hyaline droplets was comparable between controls and males previously receiving 1000 mg/kg bow/day, which 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 previously treated males than in controls although at a lower severity than in the non-recovery males. 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.

- Liver: At the end of the dosing period, minimal or mild centrilobular hepatocellular hypertrophy was observed in 4/5 males and all the females given 1000 mg/kg bw/day. It was not seen in females or in males from the other treated groups. In addition, minimal or mild periportal fat vacuolation was seen in 4/5 females given 1000 mg/kg bw/day. This finding was not observed in any other animal on the study. A mild focus of necrosis was present in a female given 1000 mg/kg bw/day. This finding was similar in morphology to that occasionally seen in control rats and was considered to have arisen spontaneously and not to be associated with administration of the test item. At the end of the 14 day recovery period, no centrilobular hepatocellular hypertrophy was observed in the liver of any animal. This finding was therefore considered to have reversed completely. Mild periportal fat vacuolation was observed in 1/5 recovery females previously given 1000 mg/kg bw/day and the single incidence of this finding indicated partial reversal of the effect.

- Thyroid gland: At the end of the treatment period, the incidence and/or severity of minimal or mild diffuse hypertrophy of the follicular epithelium was greater in animals of either sex given 300, 400 or 1000 mg/kg bw/day than in controls. The minor differences, compared to controls, in the incidences of minimal change, seen in both sexes given 100 mg/kg bw/day, were attributed to individual variation and not to the test item. Following the treatment free period, the incidence and severity of diffuse hypertrophy of the follicular epithelium in the thyroid glands remained greater in both sexes which had previously received 1000 mg/kg bw/day than in controls. However, the magnitude of difference was small and there was no morphological reason to suggest that tis finding would not reverse completely given sufficient time.

Applicant's summary and conclusion

Conclusions:
The oral (gavage) administration of test item to male and female Wistar Han:RccHan:WIST strain rats resulted in periportal fat vacuolation in females at a dose level of 1000 mg/kg bw/day and at a mild level was considered to be adverse. Based on the in-life results and histopathology findings, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day for males and 400 mg/kg bw/day for females.
Executive summary:

GUIDELINES

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

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

- The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labor and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003 for a twenty-eight day repeat dose oral toxicity study as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973 of Ministry of International Trade and Industry (MITI) amended 2004.

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

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

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

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

METHODS

Test item was administered by gavage to four groups, each of five male and five female Wistar Han:RccHan:WIST strain rats, for 28 consecutive days, at dose levels of 100, 300, 400 or 1000 mg/kg bw/day. A control group was dosed with vehicle alone (arachis oil BP). It should be noted that initially the study design involved four dose groups and animals for group 5 (used for administration of the test item at a dose level of 400 mg/kg bw/day) were incorporated into the study on day 6 of dosing for the remaining dose groups. Since group 5 animals were of similar specifications to the remaining animals on the study and were dosed for 28 day, this was considered not to have affected the scientific outcome of the study. Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg bw/day) or vehicle alone for 28 consecutive days and then maintained without treatment for a further 14 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 towards the end of the treatment period and for all recovery group animals towards the end of the treatment-free period. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

RESULTS

Mortality: There were no treatment-related deaths during the course of the study.

Clinical observations: There were no clinical signs at any dose level considered to be related to the toxicity of the test item.

Behavioural 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: Evaluation did not identify any effect of treatment with the test item.

Body weight: There was no detrimental effect on body weight development as a result of treatment with test item. Any intergroup differences were considered likely to be due to biological variation.

Food consumption: There was no effect of treatment on dietary intake or food conversion efficiency at any dose level.

Water consumption: There was considered to be no adverse effect of treatment with the test item on water consumption.

Hematology: No toxicologically significant effects were detected at any dose level in animals of either sex.

Blood chemistry: No toxicologically significant effects were detected at any dose level in animals of either sex.

Urinalysis: No treatment-related effects were detected in the urinalysis parameters examined for animals of either sex at dose levels up to 1000 mg/kg bw/day.

Necropsy: Macroscopic examination did not reveal any treatment-related findings.

Organ weights: Group mean absolute and body weight-related kidney weights in non-recovery males who received 400 or 1000 mg/kg bw/day and liver weights in non-recovery animals of either sex from these dose groups as well as non-recovery males who received 300 mg/kg bw/day were statistically significantly higher than controls (p < 0.01). The differences were particularly marked for 1000 mg/kg bw/day animals and dose-relationships were apparent in all cases. At the end of the 14 day recovery period, only males previously given 1000 mg/kg bw/day showed statistically significantly higher group mean absolute and body weight-related liver and kidney in relation to controls (p < 0.05). The magnitude of the intergroup differences in the recovery males was considerably lower than non-recovery males and, taking into consideration the associated histopathology findings from the relevant tissues, it was considered likely that these changes would have been fully reversible over time. Other intergroup differences attaining statistical significance were considered to be incidental or of no toxicological relevance.

Histopathology: Treatment-related histopathology findings were as shown below:

(a) Kidneys: When compared with controls, the severity and incidence of increased intra-epithelial hyaline droplets and basophilic tubules was greater in all non-recovery male test item-treated groups. Granular casts were also observed at the corticomedullary junction in males receiving the test item at dose levels of 300 mg/kg bw/day or above. These findings were consistent an etiological diagnosis of alpha-2u-globulin nephropathy. In the recovery males, the incidence and severity of increased intra-epithelial hyaline droplets was comparable between controls and males previously receiving 1000 mg/kg bow/day, which 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 previously treated males than in controls although at a lower severity than in the non-recovery males. 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. Since alpha-2u-globulin nephropathy is a rodent specific phenomenon, of no relevance to human exposure, these finding were considered to be non-adverse.

(b) Liver: At the end of the dosing period, minimal or mild centrilobular hepatocellular hypertrophy was observed in most animals of either sex treated with 1000 mg/kg bw/day and was considered to be of an adaptive nature; the finding was shown to be completely reversible after a treatment-free period of 14 days. Most non-recovery females from the 1000 mg/kg bw/day dose group also showed minimal or mild periportal fat vacuolation with the latter considered to be adverse. One recovery female previously treated with 1000 mg/kg bw/day showed mild periportal fat vacuolation at the end of the treatment-free period indicating partial reversibility for this observation.

(c) Thyroid glands: The incidence and/or severity of minimal or mild diffuse hypertrophy of the follicular epithelium was greater in non-recovery animals of either sex given 300, 400 or 1000 mg/kg bw/day than in controls. After the treatment-free period, the incidence and severity of diffuse hypertrophy of the follicular epithelium in the thyroid glands remained greater in both sexes which had previously received 1000 mg/kg bw/day than in controls. 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 the finding was considered to be non-adverse.

CONCLUSION

The oral (gavage) administration of test item to male and female Wistar Han:RccHan:WIST strain rats resulted in periportal fat vacuolation in females at a dose level of 1000 mg/kg bw/day and at a mild level was considered to be adverse. Based on the in-life results and histopathology findings, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day for males and 400 mg/kg bw/day for females.