Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 30 to October 05, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
Range-finding study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From June 23 to July 21, 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Dose range-finding study.
Qualifier:
no guideline required
Principles of method if other than guideline:
The purpose of this study was to provide information as the basis for selection of dose levels for further repeated dose toxicity studies.
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on August 19, 2008/signed on March 04, 2009)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Blackthorn, Bicester, Oxon, UK
- Age at study initiation: 6-8 weeks
- Weight at study initiation: Males: 170-195 g; females: 142-159 g
- Housing: Animals were housed in groups of 3 by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd, Cheshire, UK).
- Diet: Pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories UK Ltd, Oxon, UK), ad libitum
- Water: Mains drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 °C
- Humidity: 55 ± 15 %
- Air changes: 15 changes/h
- Photoperiod: 12 h dark/ 12 h light

IN-LIFE DATES: From: June 23, 2009 To: July 21, 2009.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material was prepared at the appropriate concentrations as a solution in Arachis oil BP. Test material formulations were stable for at least 8 days and therefore prepared weekly and stored at 4 ºC in the dark.

VEHICLE
- Concentration in vehicle: 62.5, 125 and 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate. The test material formulations were sampled and analysed within three days of preparation. The concentration of test material in the formulations was determined by gas chromatography (GC). The results indicate that the prepared formulations were within ± 4 % of the nominal concentration.
Duration of treatment / exposure:
14 days
Frequency of treatment:
Daily
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Details on study design:
No data
Positive control:
None
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill health or behavioural change immediately before dosing, up to 30 minutes after dosing and 1 h after dosing. Additional observations were also made 5 h following dosing whenever possible (not at weekends or on public holidays).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Days 1, 3, 5, 8, 11, 14 and 15.

FOOD CONSUMPTION: Yes
Food consumption was recorded for each cage group for Days 1-3, 3-5, 5-8, 8-11 and 11-15.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was measured and recorded daily for each cage group.
Sacrifice and pathology:
On completion of the dosing period, all surviving animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination and subjected to an internal and external macroscopic examination. Animals that died during the study were also necropsied. No tissues were retained.
Other examinations:
None
Statistics:
None
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Animals of either sex treated with 1000 mg/kg bw/day were killed in extremis following severe clinical signs on Day 3. One female treated with 500 mg/kg bw/day also showed severe clinical signs resulting in termination on Day 3. There were no further unscheduled deaths.
- One male treated with 1000 mg/kg bw/day showed lethargy, moderate/severe ataxia and hunched posture one hour after dosing on Day 2. On Day 3, all males showed lethargy. Hunched posture was evident for two males and one male also displayed ataxia. One female treated with 1000 mg/kg bw/day showed signs of pilo-erection, hypothermia, hunched posture and moderate/severe ataxia, one hour after dosing on Day 2. This female was found comatose after treatment on Day 3 and remaining females from this dose group showed signs of moderate/severe ataxia, exophthalmia and/or lethargy. Due to the severity of these observations, this dose level was considered excessive and the dose group was subsequently terminated on Day 3.
- One female treated with 500 mg/kg bw/day showed mild/moderate ataxia during the 1 h observations on Day 2 and lethargy and moderate/severe ataxia were evident following treatment on Day 3. The observations were considered excessive and this animal was subsequently terminated on Day 3.
- Lethargy and mild ataxia were also observed for one male treated with 500 mg/kg bw/day 1 h after dosing on Day 4; however a complete regression of these signs was evident thereafter. Remaining clinical signs were confined to increased salivation detected soon after dosing between Days 4 and 14, with the observation occasionally observed up to 1 h after dosing.
- Mild ataxia and lethargy were observed one male treated with 250 mg/kg bw/day 1 h after dosing on Day 9. No other clinical signs were observed at 250 mg/kg bw/day dose.
CONCLUSION: Adverse effects at 500 and 1000 mg/kg bw
Mortality:
mortality observed, treatment-related
Description (incidence):
Due to the severity of clinical signs observed, the 1000 mg/kg bw/day dose group was terminated following treatment on Day 3, together with one 500 mg/kg bw/day female. No further unscheduled deaths were observed.
CONCLUSION: Adverse effects at 500 and 1000 mg/kg bw
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Reduced bodyweight gains were observed for animals of either sex treated with 1000 mg/kg bw/day when compared to controls, prior to termination on Day 3.
- Slight reductions in bodyweight gains were also evident on Day 3 for animals of either sex treated with 500 mg/kg bw/day when compared to controls. Values were compared to controls thereafter.
- No adverse effect on bodyweight change was evident for animals of either sex treated with 250 mg/kg bw/day.
CONCLUSION: Adverse effects at 500 and 1000 mg/kg bw
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- A reduction in dietary intake was evident for animals of either sex treated with 1000 mg/kg bw/day when compared to controls prior to termination on Day 3.
- No adverse effects on dietary intake were evident for animals of either sex treated with 500 and 250 mg/kg bw/day when compared to controls.
CONCLUSION: adverse effect in high dose group
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
- Increased water consumption was evident for animals of either sex treated with 1000 mg/kg bw/day on Day 2 and Day 3 when compared to controls.
- A slight increase in water intake was also evident for females treated with 500 mg/kg bw/day during the treatment period when compared to controls.
- No obvious differences in water intake were evident at 250 mg/kg bw/day.
CONCLUSION: adverse effect in high dose groups
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Key result
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Remarks on result:
other: mortality, reduced food consumption and body weight gain was observed at 1000 mg/kg bw/day and treatment related deaths at 500 mg/kg bw/day.
Key result
Critical effects observed:
no

None

Conclusions:
Based on the results of this dose range-finding study, dose levels of 30, 300 and 600 mg/kg bw/day were selected for subsequent toxicity studies.
Executive summary:

In a 14-day repeated dose toxicity range-finding study, groups of Wistar Han™:HsdRccHan™:WIST strain rats (3/sex/dose) were administered daily with test material at dose levels of 0 (vehicle control), 250, 500 and 1000 mg/kg bw/day in arachis oil BP by oral gavage. Animals were then observed for mortality, clinical condition, bodyweight, food consumption, water consumption and were all macroscopically necropsied after sacrifice.

- Mortality: Due to the severity of clinical signs observed, the 1000 mg/kg bw/day dose group was terminated following treatment on Day 3, together with one 500 mg/kg bw/day female. No further unscheduled deaths were observed.

- Clinical observations: Lethargy, moderate/severe ataxia, hunched posture, pilo-erection, and hypothermia were observed for animals of either sex treated with 1000 mg/kg bw/day and one female at this dose level was found comatosed. This dose level was considered excessive and all animals from this dose group were terminated following dosing on Day 3. Similar clinical signs were evident for one female treated with 500 mg/kg bw/day, which was also terminated on Day 3. Signs of lethargy and mild ataxia were observed for one male treated with 500 mg/kg bw/day and one male treated with 250 mg/kg bw/day. These signs were isolated and complete regression was evident thereafter.

- Bodyweight: Reduced bodyweight gains were observed in animals of either sex treated with 1000 mg/kg bw/day when compared to controls, prior to termination on Day 3. No adverse effect on bodyweight change was evident for animals of either sex treated with 500 or 250 mg/kg bw/day.

- Food consumption: Reduced dietary intake was evident for animals of either sex treated with 1000 mg/kg bw/day when compared to controls prior to termination on Day 3. No adverse effects on dietary intake were evident for animals of either sex treated with 500 and 250 mg/kg bw/day when compared to controls.

- Water consumption: Increased water consumption was evident for animals of either sex treated with 1000 mg/kg bw/day on Day 2 and Day 3 when compared to controls. No adverse effects on water intake were evident at 500 or 250 mg/kg bw/day.

- Necropsy: No macroscopic abnormalities were detected for both interim death and terminal kill animals.

Based on the results of this dose range-finding study, dose levels of 30, 300 and 600 mg/kg bw/day were selected for subsequent toxicity studies.  

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
Adopted 03 October 2008
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
OECD GLP (inspected on November 05 to 09 and 26 to 30, 2007/ signed on November 12, 2008)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Remarks:
paste / solid block
Details on test material:
- Physical state: solid, pale yellow to yellow block, or pasty
- Stability under test conditions: no data
- Storage condition of test material: in the dark, preferably at about 4-10°C and under nitrogen.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Oxon, UK.
- Age at study initiation: 6-8 weeks
- Weight at study initiation: Males: 208-237 g, females: 149-171 g
- Housing: Animals were housed in groups of 5/sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
- Diet: Pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories UK Limited, Oxon, UK), ad libitum
- Water: Mains drinking water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 ºC
- Humidity: 55 ± 15 %
- Air changes: 15 changes/ h
- Photoperiod: 12 h dark/ 12 h light

IN-LIFE DATES: From: July 30, 2009 To: October 05, 2009.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material was prepared at the appropriate concentrations as a solution in Arachis oil BP. Test material formulations were stable for at least 8 days and therefore prepared weekly during the treatment period and stored at 4 ºC in the dark, under nitrogen.

VEHICLE
- Concentration in vehicle: 7.5, 75 and 150 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The concentration of test material in the formulations was determined by gas chromatography (GC) using an external standard technique. Test material formulations were sampled and analysed within three days of preparation for verification of concentration.
- The results indicate that the prepared formulations were within ± 9 % of the nominal concentration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were chosen based on the results from previous toxicity work (Study Number 0161-0687).
- Rationale for animal assignment (if not random): Animals were randomly allocated to treatment groups using a stratified bodyweight randomisation procedure and the group mean bodyweights were then determined to ensure similarity between the treatment groups.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS / CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed immediately before dosing, up to 30 minutes post dosing and 1 and 5 h after dosing during the working week. At weekends, animals were observed immediately before and after dosing and 1 h after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 and at weekly intervals thereafter. Bodyweights were also recorded prior to terminal kill. Due to an increase in dose level for the high dose group, bodyweights were also recorded on Day 3.

FOOD CONSUMPTION: Yes
- Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY: Yes
- Food efficiency (group mean bodyweight gain / group mean food consumption) was calculated.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological investigation was performed on all animals from each test and control group at the end of the study (Day 28).
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 5 animals/sex/dose
- Parameters examined: Haemoglobin, erythrocyte count, haematocrit, erythrocyte indices (mean corpuscular haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration), total leucocyte count, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), platelet count, reticulocyte count, prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Clinical chemistry investigation was performed on all animals from each test and control group at the end of the study (Day 28).
- Animals fasted: No
- How many animals: 5 animals/sex/dose
- Parameters examined: Urea, glucose, total protein, albumin, albumin/Globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, total bilirubin, bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and on Days 7, 14, 21 and 27 for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately 2 h after dosing on each occasion.
- Dose groups that were examined: 5 animals/sex/dose
- Battery of functions tested:
a) Behavioural assessments: Gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behaviour, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin colour, respiration, palpebral closure, urination, defecation, transfer arousal and tail elevation.
b) Functional performance tests: Forelimb/hindlimb grip strength, motor activity.
c) Sensory reactivity: Sensory reactivity to auditory, visual and proprioceptive stimuli.

IMMUNOLOGY: No

OTHER:
- Organ weights: Adrenals, brain, epididymides, heart, kidneys, prostate and seminal vesicles (with coagulating glands and fluid), spleen, testes, thymus, liver, thyroid/parathyroid, ovaries, uterus, cervix and pituitary (post-fixation) were removed from animals that were killed at the end of the study and were dissected free from fat and weighed before fixation.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
On completion of the dosing period, all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. Animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY: Yes (see Table 7.5.1/1 )
Samples of the tissues mentioned in Table 7.5.1/1 were removed from all animals and preserved in buffered 10 % formalin for microscopic analysis.
Other examinations:
- At the end of the study, plasma from each animal was stored frozen at approximately -20 °C for thyroid hormone assessment. No treatment related effects on the pituitary-thyroid axis were identified, therefore these samples were discarded.
- A vaginal smear was taken from all females and the stage of oestrus was recorded at the end of the study.
Statistics:
- Mean values and standard deviations of data were calculated.
- Where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module.
- For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means were assessed using ANOVA or ANCOVA and Bartlett’s test.
- Williams test was used for parametric data and the Shirley test for nonparametric data.
- If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (nonparametric) test to determine significant differences from the control group.
- If required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
- Probability values (P) are presented as follows:
P < 0.01 **
P < 0.05 *
p ≥ 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Hunched posture, ataxia and tiptoe gait were observed in one female treated with highest dose level on the first day of dosing. A regression in symptoms was evident on Day 2 and the dose level was increased to 600 mg/kg bw/day on Day 3 of treatment. On Day 4, the previously observed clinical signs were evident for females until Day 18, and prostration was noted for one female on Days 15, 16 and 17. Similar clinical signs were evident for high dose males following dosing from Day 4, although these were incidental and only affected one male in each case.
- Increased salivation was detected soon after dosing and on occasion, prior to dosing, up to 1 h after dosing and up to 5 h after dosing for animals of either sex treated with 600 mg/kg bw/day from Day 6 for females and from Day 8 for males. These findings were not considered to represent systemic toxicity.
- Hunched posture, abnormal gait and increased salivation were also evident in the 300 mg/kg bw/day dose group, although the incidences were not of the extent observed at the highest dose level.
- No clinical observations were detected at 30 mg/kg bw/day.
CONCUSION: Adverse effects at 600 mg/kg bw in females (ataxia, prostration and lethargy) / reversible after 18 days
Mortality:
no mortality observed
Description (incidence):
No unscheduled death occurred.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- A slight reduction in bodyweight gains was evident for animals of either sex treated with 600 mg/kg bw/day when compared to controls, with the effect extending to the male 300 mg/kg bw/day dose group, although this was not considered to represent an adverse effect.
- No adverse effects on bodyweight change were evident for females treated with 300 mg/kg bw/day or for animals of either sex treated with 30 mg/kg bw/day.
CONCUSION: Non-adverse effects
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
CLINICAL CHEMISTRY
- Male: Males treated with 300 mg/kg bw/day showed increases in alanine aminotransferase and alkaline phosphatase levels in comparison to controls. No significant effects were detected for animals of either sex treated with the highest or
lowest dose levels. These findings were not considered to represent an adverse effect.
- Males treated with 600 mg/kg/day showed an increase in albumin/globulin ratio when compared to controls. The significance achieved was minimal (P<0.05) and in isolation, these findings were most probably attributed to one or two lower than expected control values, and were not considered to represent true effects of treatment.
- Females treated with 600 mg/kg/day showed a reduction in urea levels when compared to their concurrent controls. The significance achieved was minimal (P<0.05) and in isolation, these findings were most probably attributed to one or two lower than expected control values, and were not considered to represent true effects of treatment.
- Males treated with 300 mg/kg/day showed a reduction in blood sodium levels when compared to controls. Three males showed values lower than the normally expected ranges and one value was higher than expected. Furthermore, two control values were also higher than the normally expected ranges for this parameter. In the absence of an electrolyte imbalance, this finding was considered to have arisen incidentally and was not considered to be related to treatment.
CONCLUSION: Non-adverse effect
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
- Female: Weekly open field arena observations confirmed the clinical signs of increased salivation for one female treated with 300 mg/kg bw/day and one female treated with 600 mg/kg bw/day during Weeks 3 and 4. One female treated with 600 mg/kg bw/day also showed tiptoe gait during Week 4. These findings were not considered to represent systemic toxicity.
- No treatment-related effects were detected for males from all treatment groups or for animals of either sex treated with 30 mg/kg bw/day.
- Overall activity was reduced for animals of either sex treated with 600 mg/kg bw/day and for males treated with 300 mg/kg bw/day when compared to controls. In the absence of a similar effect observed during the asymptotic period, these reductions were considered to be attributed to a slight decline in physical health and unrelated to neurotoxicity. No such effects were evident for females treated with 300 mg/kg bw/day or for animals of either sex treated with 30 mg/kg bw/day.
- No treatment-related effects were detected in grip strength for treated animals when compared to controls.
- No treatment-related effects were detected on sensory reactivity.
CONCLUSION: non-adverse effects /reversible
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Increases in absolute and relative liver weights were evident for animals of either sex treated with 600 mg/kg bw/day when compared to controls, with the effect extending into the male 300 mg/kg bw/day dose group. Hepatocyte enlargement is commonly seen in the rodent liver following the ingestion of xenobiotics and, in the absence of associated degenerative or inflammatory changes, is generally considered to be adaptive in nature.
- Females treated with 600 mg/kg/day showed an increase in absolute and relative adrenal weights when compared to controls (P<0.05), with one absolute weight value higher than the normally expected range. Females treated with 300 and 30 mg/kg/day also showed statistically significant increases in absolute and relative adrenal weights, however, a convincing dose-related response was not observed. In the absence of any histopathological adrenal changes, the increases in adrenal weights in females from all the treatment levels were not considered to be of any toxicological importance.
- Kidney weights, both absolute and relative to terminal bodyweight were higher for females treated with 600 mg/kg/day when compared to controls (P<0.05). Review of the individual data revealed two higher than expected values for absolute kidney weight in the treated group, although one value from the control group was also higher than expected. All individual values for relative kidney weights were within the normally expected ranges, and in the absence of any histopathological correlates, these increases were not considered to represent a toxicologically significant effect of treatment.
- Males treated with 30 mg/kg/day showed lower than expected kidney weights, both absolute and bodyweight relative. In the absence of a dose-related response, this finding was considered to have arisen incidentally.
- Absolute and relative thymus weights were higher for females treated with 300 or 30 mg/kg/day when compared to controls. The significance in each case was minimal and in the absence of an histopathological correlates, these effects were not considered to be of any toxicological significance.
CONCLUSION: Non-adverse effect
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic findings were confined to the presence of enlarged cervical lymph nodes for one female treated with 300 mg/kg bw/day. In isolation, this finding was considered to be unrelated to treatment.
CONCLUSION: non-adverse effect
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Centrilobular hepatocyte enlargement in high dose group. These findings were considered to be adaptive effects
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Centrilobular hepatocyte enlargement was seen in relation to treatment for animals of either sex treated with 600 mg/kg bw/day, and for males treated with 300 mg/kg bw/day. Hepatocyte enlargement is commonly seen in the rodent liver following
the ingestion of xenobiotics and, in the absence of associated degenerative or inflammatory changes, is generally considered to be adaptive in nature.
CONCLUSION: non-adverse effects
Other effects:
no effects observed
Description (incidence and severity):
No differences in stage of oestrus were detected for females from treated groups when compared to controls.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects were detected for animals of either sex treated with 30 mg/kg bw/day.

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (actual dose received)
System:
central nervous system
Organ:
other: not applicable
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, NOAEL and NOEL were considered to be 300 and 30 mg/kg bw/day, respectively.
Executive summary:

In a repeated dose toxicity study performed in accordance with OECD test guideline No. 407 and in compliance with GLP, test material solution in arachis oil was administered by gavage to three groups of Wistar Han™:HsdRccHan™:WIST strain rats (5/sex/dose) at dose levels of 30, 300 and 600 mg/kg bw/day for 28 consecutive days. Control rats were given the vehicle alone. The highest dose group was initially treated at a lower dose level (300 mg/kg bw/day) for the first two days of the treatment to allow animals in this dose group to acclimatise to the test material at a lower dosage before exposure to the full dosage of 600 mg/kg bw/day. This action was undertaken to reduce the risk of treatment-related deaths at the highest dose level following the effects observed in a previous study undertaken with this test material. Clinical signs, functional observations, bodyweight development, dietary intake and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Although no deaths were evident in this study, clinical signs were evident for one female from the highest dose group on the first day of treatment. Regression was evident on Day 2 and the dose level was increased to 600 mg/kg bw/day on Day 3. Clinical signs appeared in one male and two females on Day 4 (the second day of treatment at 600 mg/kg bw/day) and incidental clinical signs were evident during the treatment period. Incidents of hunched posture, and abnormal gait were also evident for two females treated with the intermediate dose on the first day of treatment however complete regression was evident thereafter. The clinical signs observed during the daily clinical observations were confirmed during the weekly arena observations. Furthermore, motor activity assessments revealed lower overall motor activity for animals of either sex treated with 600 mg/kg bw/day, extending into the male 300 mg/kg/day dose group. No significant reductions were evident during the final 20% of the assessment period, known as the asymptotic period, which would suggest that the reduced activity observed was most probably attributed to a slight decline in the physical health of the animals, and did not represent a neurotoxic effect of treatment. Remaining clinical signs consisted of increased salivation following dose, which was observed in both the high and intermediate dose groups. This observation is commonly observed following administration of a slightly unpalatable or irritant test material formulation. There were no toxicologically significant differences in dietary or water intake for treated animals when compared to control values and histopathological examinations did not reveal any effects of the gastro-intestinal tract which may be suggestive of irritancy. In the absence of any changes to suggest neurotoxicity the increased salivation detected in this study was not considered to represent an adverse effect of treatment and is most likely to be attributed to the poor taste of the test substance. A slight reduction in overall bodyweight change was observed for animals of either sex treated with the highest dose level in comparison to controls, with the effect extending into the intermediate male dose group. No adverse effects on dietary intake or food conversion efficiency were detected, therefore, the slightly reduced bodyweight gain was not considered to represent an adverse effect. Blood chemical analysis did not reveal any significant differences in the highest dose level, however, significantly elevated alanine aminotransferase levels were observed for males treated with 300 mg/kg bw/day, together with an increase in alkaline phosphate. Furthermore, liver weights were elevated for animals of either sex treated with 600 mg/kg bw/day with the effect extending into the intermediate male dose group. Finally, histopathological examination revealed centrilobular hepatocyte enlargement in the highest dose group, with the effect extending into the male 300 mg/kg bw/day dose group. The increases in enzyme activity, elevated liver weights and microscopic hepatic changes are commonly observed following the administration of a xenobiotics, resulting in induction of metabolising enzymes. In the absence of any inflammatory or degeneration changes, these findings were not considered to represent an adverse effect of treatment.

Due to the nature of the clinical signs observed at the highest dose level, consisting of ataxia, prostration and lethargy, together with the remaining treatment-related effects, a ‘No Observed Adverse Effect Level’ (NOAEL) could not be established at the highest dose level. The treatment-related effects detected at 300 mg/kg bw/day were not considered to represent an adverse health effect, therefore a NOAEL was established at 300 mg/kg bw/day. No treatment-related effects were detected at 30 mg/kg/day, therefore a No Observed Effect Level (NOEL) was established at 30 mg/kg bw/day.

Based on the results of this study, test material is not classified for damage to organs through 28 days oral repeated exposure according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for sub-acute oral toxicity endpoint.