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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:
27 April 2016 to 15 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
various with no impact on results or integrity of the study (see below)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Confidential
IUPAC Name:
Confidential
Test material form:
solid
Details on test material:
- Appearance/physical state: Pale yellow waxy solid
- Storage conditions: Room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
ANIMAL INFORMATION
- The animals were acclimatised for nineteen days during which time their health status was assessed.
- Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate oestrous cycling activity were excluded from treatment groups at least five days before the start of treatment.
- A total of one hundred and sixteen animals (fifty eight males and fifty eight females) were accepted into the study.
- At the start of treatment, the males weighed 290 to 344g and were approximately eleven weeks old. The females weighed 203 to 240g, and were approximately fourteen weeks old.

ANIMAL CARE AND HUSBANDRY
- The animals 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 the treatment groups.
- The cage distribution within the holding rack was also randomised.
- The animals were uniquely identified within the study by an ear punching system routinely used in the laboratories.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
TEST ITEM PREPARATION
- For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. Due to the nature of the test item, it was necessary to warm the test item (maximum temperature approximately 70°C) during the preparation of the dosing formulations.
- The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services as part of this study.
- Results show the formulations to be stable for at least four hours. Formulations were therefore prepared daily and dosed within this known stability period.

DOSE ADMINISTRATION
- Animals were allocated to treatment groups as shown in the table below.
- The 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 8 mL/kg of Arachis oil BP.
- The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
SAMPLING
- Samples of each test item formulation were taken on three separate occasions and analysed for concentration of test item at Envigo Research Limited, Shardlow, UK, Analytical Services.
- The method used for analysis of formulations and the results obtained are given in Annex 2 (attached).
- The results indicate that the prepared formulations were within 96-107% of the nominal concentration confirming the accuracy of the formulation procedure.
Duration of treatment / exposure:
42 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 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:
See information on dose administration and allocation of treatment groups in table below.
Control animals:
yes, concurrent vehicle
Details on study design:
NON-RECOVERY DOSE GROUPS
(i) Males and females were housed for a suitable acclimatization period which allowed at least two weeks of pre-treatment vaginal smears to be performed for females enabling the exclusion of females considered not to be showing appropriate oestrous cycling.
(ii) Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). During the pre-pairing period, vaginal smears were performed for females. The first day of dosing was designated as Day 1 of the study.
(iii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
(iv) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
(v) Following evidence of mating (designated as Day 0 post-coitum) the males were returned to their original cages and females were transferred to individual cages.
(vi) On completion of the pairing phase, five selected males per dose group were evaluated for functional/sensory responses to various stimuli during Week 6.
(vii) Pregnant females were allowed to give birth and maintain their offspring until Day 13 post-partum. Litter size, offspring weight and sex, ano-genital distance and visible nipple counts (males only) and clinical signs were also recorded during this period.
(viii) At Day 12 post-partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
(ix) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 43. The male dose groups were killed and examined macroscopically on Day 44 or 45.
(x) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 13 post-partum. At Day 13 post-partum, all surviving offspring were killed and examined macroscopically. All females were killed on Day 14 post-partum and examined macroscopically.
(xi) Where possible, blood samples were taken from two offspring on Day 4 post-partum and one male and one female offspring on Day 13 post-partum. In addition, blood samples were taken from all adult males and females at termination. Blood (plasma) samples from all adult males and blood (serum) samples from Day 13 offspring were analysed for Thyroxine (T4).

RECOVERY DOSE GROUPS
(i) Groups of five male and five female rats were dosed according to dose group continuously up to the point of sacrifice of non-recovery males at which time treatment was discontinued.
(ii) The males and females were maintained without treatment for a further fourteen days.
(iii) Urinalysis was performed for all males during the final week of recovery.
(iv) Blood samples were taken for haematological and blood chemical assessment on Day 56.
(v) After fourteen days of recovery, males and females were killed and examined macroscopically.
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS
- During the treatment period, all animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one hour after dosing (except for females during parturition where applicable). During the treatment-free period, recovery animals were observed daily. All observations were recorded.

BODY WEIGHT
- Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing.
- During the pairing phase, females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post-coitum, and on Days 1, 4 and 7 post-partum.
- Body weights were also recorded at terminal kill (Day 44 or 45 for males and Day 14 post-partum for females).
- Recovery animals were weighed on Day 1 (prior to dosing) and then weekly until termination including the day of termination (Day 15 of the recovery period).

FOOD CONSUMPTION
- During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase.
- For females showing evidence of mating, food consumption was recorded for the periods covering post-coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1-4, 4-7 and 7-14 post-partum. Weekly food consumptions were performed for each cage of recovery group animals throughout the study period.

FOOD EFFICIENCY
- Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase.
- Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION
- Water intake was observed daily by visual inspection of water bottles for any overt changes.

FUNCTIONAL OBSERVATIONS
- At approximately weekly intervals, all non-recovery animals were observed for signs of functional/behavioural toxicity (see Deviations from Study Plan). These observations were performed on mated females on Days 4, 11 and 18 post-coitum and for littering females on Days 4 and 12 post-partum.
- Functional performance tests were also performed on five selected non-recovery males and non-recovery females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

BEHAVIOURAL ASSESSMENT
- Detailed individual clinical observations were performed for each non-recovery animal using a purpose-built arena.
- The parameters observed are listed in the table below.
- The test was developed from the methods used by Irwin (1968) and Moser et al (1988).

FUNCTIONAL PERFORMANCE TESTS
- Motor activity: Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Non-recovery animals 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), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty-minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).
-Forelimb/hindlimb grip strength: An automated 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. The animal was pulled by the base of the tail until its grip was broken. 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 al (1979).

SENSORY REACTIVITY
- Each non-recovery animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli.
- Parameters observed are listed in the table below.
- The assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).

HEMATOLOGY
- Haematological and blood chemical investigations were performed on five males and five females selected from each non-recovery test and control group prior to termination (Day 43 for males and Day 13 post-partum for females).
- In addition, haematological and blood chemical investigations were performed on all recovery group animals after the fourteen-day treatment-free period at termination (Day 56).
- Blood samples were obtained from the lateral tail vein.
- Where necessary repeat samples were taken by cardiac puncture at termination.
- Animals were not fasted prior to sampling.
- Parameters shown in the table below were measured on blood collected into tubes containing potassium EDTA anti-coagulant.
- Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L)

URINALYSIS
- Urinalytical investigations were performed on five non-recovery males from the control and each test group during the final week of treatment and on all recovery males during the final week of the recovery period.
- Urine samples were collected overnight by housing the rats in metabolisms cages.
- Animals were maintained under conditions of normal hydration during collection but without access to food.
- The parameters listed in the table below were measured on collected urine.

BLOOD CHEMISTRY
- Parameters shown in the table below were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant.

ORGAN WEIGHTS
- Organs listed in the table below were dissected free from fat and weighed before fixation from five selected adult males and five selected adult females from each non-recovery dose group and from all recovery group animals.
- Selected tissues were weighed from all remaining animals (see table, below).
- Additionally, for one male and one female offspring (where possible), the weight of the thyroid was recorded.

HISTOPATHOLOGY
- Except where stated, samples of tissues listed in the table below were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin.
- Selected tissues were preserved from all remaining animals (see table, below).
- Tissues were dispatched to the Test Site (Envigo CRS Limited, Eye, Suffolk) for processing.
- The tissues from five selected control and 1000 mg/kg bw/day dose group animals and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination.
- The tissues from remaining control and 1000 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed.
- In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.



Sacrifice and pathology:
NECROPSY
- Adult non-recovery males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult non-recovery females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 14 post-partum. Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent. Any females which failed to achieve pregnancy or produce a litter were killed around the same time as the littering females.
- For all non-recovery females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski, 1964).
- Recovery group animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 57.
- All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

PATHOLOGY
- Microscopic examination was conducted by the Study Pathologist.
- A peer review of the findings observed was conducted by Envigo CRS Limited) at the histopathology peer review test site.
Statistics:
See below

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no clinical signs observed that were considered to be related to treatment.
- At 100 mg/kg bw/day, three animals showed noisy respiration at some stage of the study, but in the absence of similar findings at higher dosages, this finding was considered incidental and of no toxicological significance.
Mortality:
no mortality observed
Description (incidence):
- There were no unscheduled animal deaths during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- There was no obvious effect of treatment on body weight and body weight gain of nonrecovery males throughout the study at 100, 300 or 1000 mg/kg bw/day. Occasional differences in body weight gain were observed for males at all dosages during the study but in the absence of any statistically significant difference from control for overall weight gain these were considered to reflect normal biological variation.
- There was no obvious effect of treatment on body weight and body weight gain of nonrecovery females during the pre-pairing, gestation or lactation phase of the study at 100, 300 or 1000 mg/kg bw/day.
- There was no obvious effect of treatment on body weight and body weight gain of nonrecovery females during the pre-pairing, gestation or lactation phase of the study at 100, 300 or 1000 mg/kg bw/day.
- There was no obvious effect of treatment on body weight and body weight gain of recovery animals throughout the study at 1000 mg/kg bw/day. Some differences in mean body weights for both sexes attained statistical significance compared with control during the study but this was considered to reflect initial differences in body weight and normal biological variation in body weight performance and was unrelated to treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- There was no obvious effect of treatment on food consumption of non-recovery males throughout the pre-pairing and post pairing phases of the study at 100, 300 or 1000 mg/kg bw/day.
- There was no obvious effect of treatment on food consumption of non-recovery females during the pre-pairing, gestation or lactation phase of the study at 100, 300 or 1000 mg/kg bw/day.
- There was no obvious effect of treatment on food consumption of recovery animals throughout the study at 1000 mg/kg bw/day.
Food efficiency:
no effects observed
Description (incidence and severity):
- There was no obvious effect of treatment on food conversion efficiency of non-recovery males throughout the pre-pairing and post pairing phases of the study at 100, 300 or 1000 mg/kg bw/day.
- There was no obvious effect of treatment on food conversion efficiency of non-recovery females during the pre-pairing phase of the study at 100, 300 or 1000 mg/kg bw/day.
- There was no obvious effect of treatment on food conversion efficiency of recovery animals throughout the study at 1000 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
- Visual assessment of water bottle residues did not indicate any effect of treatment for non-recovery males throughout the pre-pairing and post pairing phases of the study at 100, 300 or 1000 mg/kg bw/day.
- Visual assessment of water bottle residues did not indicate any effect of treatment for non-recovery females during the pre-pairing phase of the study at 100, 300 or 1000 mg/kg bw/day.
- Visual assessment of water bottle residues did not indicate any effect of treatment for recovery animals throughout the study at 1000 mg/kg bw/day.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no toxicologically significant effects detected in the hematology parameters measured.
- At 1000 mg/kg bw/day, non-recovery males showed a statistically significant higher reticulocyte count compared to concurrent control; however all individual values were within the historical control range. As there was no supporting statistically significant differences in other erythrocyte parameters apparent or supporting histopathological changes observed, this finding was considered to be incidental and of no toxicological significance.
- At all dosages, non-recovery females showed a statistically significant lower white blood cell count, primarily due to statistically significant lower numbers of lymphocytes, compared to control. However, all individual values for treated animals were within the respective historical control ranges while one control value for lymphocytes was higher than the corresponding historical control data. In the absence of any supporting histopathological change this finding was considered incidental and of no toxicological significance.
- At all dosages, non-recovery females showed a statistically significant higher platelet count compared to control, however, three individual control values were lower than the historical control range compared to 1, 2 and 0 individual values at 100, 300 or 1000 mg/kg bw/day respectively. It is considered that the differences represent low control values, particularly as no supporting histopathological change was apparent for treated animals, and were unrelated to treatment with the test item.
- At 1000 mg/kg bw/day, recovery males showed statistically significant higher haemoglobin, mean corpuscular haemoglobin and mean corpuscular volume, compared to control. For the 1000 mg/kg bw/day animals, only one individual value for mean corpuscular haemoglobin and one individual value for mean corpuscular volume exceeded the respective historical control range; for the control group one individual value for mean corpuscular haemoglobin was below the historical control range. In the absence of any effect on these parameters during the treatment period, these findings were considered to be incidental and unrelated to previous exposure to the test item.
- At 1000 mg/kg bw/day, recovery females showed a statistically significant lower haematocrit, compared to control; all values for these treated females were within the historical control range, while one control value exceeded this historical control range. In the absence of an effect on this parameter during the treatment period, this finding was considered to be incidental and unrelated to previous exposure to the test item.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no toxicologically significant effects detected in the blood chemical parameters measured.
- No statistically significant differences from control were apparent for blood chemistry parameters for non-recovery males at all dosages.
- For non-recovery females at all dosages, statistically significant higher mean blood urea were apparent compared to control, with the majority of individual values for treated animals exceeding the historical control range compared to only one control value. However, the group mean values showed no dosage relationship and, in the absence of any supporting histopathological change, this finding was considered likely to be incidental and to be of no toxicological significance.
- For non-recovery females at all dosages, statistically significant higher mean total protein and albumin levels were apparent compared to control, although there was no accompanying effect on albumin/globulin ratio. Group mean values showed no dosage relationship and all individual values for treated animals was within the respective historical control range. In the absence of any supporting histopathological change, this finding was considered to be incidental and of no toxicological significance.
- For non-recovery females at all dosages, statistically significant higher creatinine levels were apparent compared to control, but these mean values showed no dosage relationship. Only one individual value at 300 mg/kg bw/day exceeded the historical control range and, in the absence of any supporting histopathological change, this finding was considered to be incidental and of no toxicological significance.
- For non-recovery females at all dosages, statistically significant lower mean aspartate aminotransferase activity was apparent compared to control but these mean values showed no consistent dosage relationship. All individual values for treated animals were within the historical control range and the mean control value appeared to be adversely influenced by one particularly high individual control value. A decrease in this enzyme activity is considered unlikely to indicate an adverse effect of treatment and this finding was considered to be incidental and unrelated to treatment.
- For non-recovery females at all dosages, statistically significant higher mean chloride levels were apparent compared to control but mean values showed no consistent dosage relationship. With the exception of one individual value at 300 mg/kg bw/day, all individual values for treated animals were within the historical control and this finding was considered to be incidental and unrelated to treatment.
- For non-recovery females at all dosages, statistically significant lower mean inorganic phosphorus levels were apparent compared to control but mean values showed no consistent dosage relationship. All individual values for treated animals were within the historical control range while three individual control values exceeded this historical control range. The differences observed for mean inorganic phosphorus levels were therefore considered to reflect atypical high control values and were unrelated to treatment
- For recovery males at 1000 mg/kg bw/day, higher albumin, sodium, chloride and calcium level and lower triglyceride level attained statistical significance compared to control. In the absence of any effects on these parameters during the treatment period, these findings were considered to be incidental and unrelated to previous treatment
- No statistically significant differences from control were apparent for blood chemistry parameters for recovery females at 1000 mg/kg bw/day.
Urinalysis findings:
no effects observed
Description (incidence and severity):
- Intergroup differences in urinalysis parameters for non-recovery animals at the end of the treatment period did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.
- Intergroup differences in urinalysis parameters for recovery animals at the end of the recovery period did not indicate any effect of treatment for either sex at 1000 mg/kg bw/day.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- Assessment of the animals in a standard arena did not reveal any obvious effects of treatment for either sex at 100, 300 or 1000 mg/kg bw/day during the treatment period or for either sex at 1000 mg/kg bw/day during the recovery period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- Intergroup differences in offspring absolute and body weight relative thyroid weights did not indicate any obvious effect of maternal treatment for either sex at 100, 300 or 1000 mg/kg bw/day.
- For non-recovery males at 1000 mg/kg bw/day, mean absolute and body weight relative liver weights at the end of treatment period were statistically significantly higher than control. For treated animals, only one body weight relative value at 1000 mg/kg bw/day exceeded the historical control range while one absolute and one body weight relative control value was below the respective historical control range. In the absence of any supporting effects on blood chemistry parameters or evidence of histopathological change for these non-recovery animals, this finding was considered to be incidental and of no toxicological significance.
- For non-recovery males at all dosages, mean absolute and body weight relative kidney weights at the end of treatment period were statistically significantly higher than control, but these mean values showed no consistent dosage relationship. For treated animals, only one body weight relative value at 1000 mg/kg bw/day exceeded the historical control range while one absolute and one body weight relative control value was below the respective historical control range. In the absence of any supporting effects on blood chemistry parameters or evidence of histopathological change for these non-recovery animals, this finding was considered to be incidental and of no toxicological significance.
- For non-recovery females at 1000 mg/kg bw/day, mean absolute and body weight relative brain at the end of treatment period were statistically significantly lower than control. For the treated females 2/5 absolute and 3/5 body weight relative brain weights were lower than the historical control range, however 2/5 body weight relative brain weights were also lower than the historical control range. In the absence of any evidence of histopathological change, this finding was considered to be incidental and of no toxicological significance.
- For recovery males at 1000 mg/kg bw/day, mean absolute and body weight relative pituitary weights at the end of the recovery period were statistically significantly higher than control. Individual values for all these treated animals were within the historical control range, while one absolute control was below the historical control range. In the absence of any supporting findings for the pituitary at the end of treatment period, this finding was considered to be incidental and unrelated to treatment. No statistically significant differences in organ weights were apparent at the end of the recovery period for females that had received 1000 mg/kg bw/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
- Adults: Macroscopic necropsy findings observed at the end of the treatment period at 100, 300 or 1000 mg/kg bw/day or at the end of the recovery period at 1000 mg/kg bw/day did not indicate any obvious effect of treatment for either sex.
- Offspring: Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any obvious effect of maternal treatment on offspring development at 100, 300 or 1000 mg/kg bw/day.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
FUNCTIONAL PERFORMANCE TESTS
- Intergroup differences in grip strength measurements during assessment of animals did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day during the treatment period or for either sex at 1000 mg/kg bw/day during the recovery period.
- At 1000 mg/kg bw/day, non-recovery females showed statistically significantly higher overall motor activity compared to control. No similar findings were apparent for nonrecovery males at this dosage or for either sex at 100 or 300 mg/kg bw/day. In the absence of any supporting findings in other behavioural/functional assessment or clinical signs during the study suggestive of neurotoxicity, this finding was considered to be incidental and of no toxicological significance.

SENSORY REACTIVITY ASSESSMENTS
- Intergroup differences observed in the scores for sensory reactivity for animals did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day during the treatment period or for either sex at 1000 mg/kg bw/day during the recovery period.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- Microscopic examination of tissues from animals at the end of the treatment period did not reveal any obvious effect of treatment for either sex at 1000 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

Key result
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: absence of test item-related effects at any dosage level

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Treatment at dosages of up to 1000 mg/kg bw/day was well tolerated and the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day. The No Observed Effect Level (NOEL) for reproduction, including the survival, growth and development of the offspring was also considered to be 1000 mg/kg bw/day.
Executive summary:

GUIDELINE

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development), to evaluate some endocrine disruptor relevant endpoints and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 28 July 2015). It also assesses the ability of the animals to recover from any toxicity following the withdrawal of treatment.  The 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 twelve male and twelve female Wistar Han:RccHan:WIST strain rats, for approximately six weeks for males and approximately seven to eight weeks for females (including a two-week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for forty-three consecutive days and then maintained without treatment for a further fourteen days.

 

Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of ano-genital distance (both sexes) and visible nipple count (male offspring only).

 

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 12 post-partum. Urinalysis was performed on five non-recovery males per dose group during the final week of treatment and five non-recovery males and females from each dose group were selected for haematology and blood chemistry assessments prior to termination. Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post-partum, for thyroid hormone analysis; plasma samples from all adult males and serum samples from Day 13 offspring were analysed for Thyroxine (T4).

 

Adult non-recovery males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post-partum, respectively. Any female which failed to achieve pregnancy or which did not produce a litter was terminated around the same time as the littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed. Following forty-two days of treatment, recovery group animals were maintained without treatment for a further fourteen days. Urinalysis was performed on all recovery group males during the final week of the treatment period. In addition, haematological and blood chemical assessments were performed on all recovery group animals at the end of the treatment-free period. These animals were then subjected to a gross necropsy.

 

RESULTS

Mortality: There were no unscheduled animal deaths during the study.

 

Clinical observations: There were no clinical signs observed that were considered to be related to treatment.

 

Behavioural assessment: Behavioural assessments did not indicate any obvious effect of treatment at 100, 300 or 1000 mg/kg bw/day.

 

Functional/performance tests: Functional performance tests did not indicate any obvious adverse effect of treatment at 100, 300 or 1000 mg/kg bw/day.

 

Sensory reactivity assessments: Sensory reactivity assessments did not indicate any obvious effect of treatment at 100, 300 or 1000 mg/kg bw/day.

 

Body weight: There was no obvious effect of treatment on body weight and body weight gain for males during the pre- and post-pairing phases of the study and for females during the pre-pairing, gestation and lactation phases of the study at 100, 300 or 1000 mg/kg bw/day. Additionally, there was no obvious effect of treatment on body weight and body weight gain for either sex at 1000 mg/kg bw/day during a two-week recovery period.

 

Food consumption: There was no obvious effect of treatment on food consumption for males during the pre- and post-pairing phases of the study and for females during the pre-pairing, gestation and lactation phases of the study at 100, 300 or 1000 mg/kg bw/day. Additionally, there was no obvious effect of treatment on food intake for either sex at 1000 mg/kg bw/day during the treatment period or a two-week recovery period.

 

Food conversion efficiency: There was no obvious effect of treatment on food conversion efficiency for males during the pre- and post-pairing phases of the study and for females during the pre-pairing phase of the study at 100, 300 or 1000 mg/kg bw/day. Additionally, there was no obvious effect of treatment on food conversion efficiency for either sex at 1000 mg/kg bw/day during the treatment period or a two-week recovery period.

 

Water consumption: Visual assessment of water consumption did not indicate any obvious effect of treatment on water intake for males during the pre- and post-pairing phases of the study and for females during the pre-pairing, gestation and lactation phases of the study at 100, 300 or 1000 mg/kg bw/day. Additionally, there was no obvious effect of treatment on water consumption for either sex at 1000 mg/kg bw/day during the treatment period or a two-week recovery period.

 

Oestrous cycle: Assessment of oestrous cycles during the pre-pairing phase of the study did not indicate any obvious effect of treatment at 100, 300 or 1000 mg/kg bw/day.

 

Mating: Mating performance was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.

Fertility: Fertility was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.

 

Gestation lengths: Gestation length was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.

Offspring litter size, sex ratio and viability: There was considered to be no effect of maternal treatment on the numbers of implantations, post-implantation loss, litter size and sex ratio at birth/Day 1 and subsequent offspring survival to Day 13 of age at 100, 300 or 1000 mg/kg bw/day.

 

Offspring growth and development: There was no obvious effect of maternal treatment on offspring body weight on Day 1 and subsequent body weight gain to Day 13 at 100, 300 or 1000 mg/kg bw/day. Evaluation of ano-genital distance for offspring of either sex on Day 1 post-partum and visible nipple count for male offspring on Day 13 post-partum did not reveal any obvious effect of treatment. The low incidence of clinical signs apparent for the offspring during the study did not indicate any obvious effect of maternal treatment.

Haematology: There was no obvious adverse effect of treatment on haematology parameters at the end of the treatment period at 100, 300 or 1000 mg/kg bw/day or at the end of the recovery period at 1000 mg/kg bw/day.

 

Blood chemistry: There was no obvious adverse effect of treatment on blood chemistry parameters at the end of the treatment period at 100, 300 or 1000 mg/kg bw/day or at the end of the recovery period at 1000 mg/kg bw/day.

 

Urinalysis: There was no obvious adverse effect of treatment on urinalysis parameters at the end of the treatment period at 100, 300 or 1000 mg/kg bw/day or at the end of the recovery period at 1000 mg/kg bw/day.

 

Necropsy: Macroscopic necropsy findings observed for decedent and terminal kill offspring at Day 13 of age, and adult males and females at the end of the treatment period at 100, 300 and 1000 mg/kg bw/day did not indicate any obvious effect of treatment. Macroscopic necropsy findings observed at the end of the recovery period at 1000 mg/kg bw/day did not indicate any obvious effect of treatment for either sex.

 

Organ weights: Intergroup differences in offspring absolute and body weight relative thyroid weights did not indicate any obvious effect of maternal treatment for either sex at 100, 300 or 1000 mg/kg bw/day. Intergroup differences in organ weights for adult animals at the end of the treatment period at 100, 300 or 1000 mg/kg bw/day and at the end of the recovery period at 1000 mg/kg bw/day did not indicate any obvious adverse effect of treatment.

 

Histopathology: Microscopic examination of tissues from animals at the end of the treatment period did not reveal any obvious effect of treatment for either sex at 1000 mg/kg bw/day.

 

Thyroid hormone analysis: Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption.

 

CONCLUSION

Treatment at dosages of up to 1000 mg/kg bw/day was well tolerated and the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day. The No Observed Effect Level (NOEL) for reproduction, including the survival, growth and development of the offspring was also considered to be 1000 mg/kg bw/day.