Rape oil, reaction products with diethylenetriamine

Endpoint summary

Description of key information

Short-term repeated dose oral toxicity: Treatment at dosages of up to 1000 mg/kg bw/day for 28-days was well tolerated and the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day (OECD 422).

 

Sub-chronic repeated dose toxicity: Daily repeated administration of test item to rats by oral gavage for 90 days at doses of 300, 600 or 1000 mg/kg/day, followed by a 4-week recovery period was well tolerated and did not result in any adverse effects of treatment. There were no treatment-related macroscopic or microscopic changes. Slight disturbances were observed in some hematological and blood chemistry parameters and there was a slight increase in uterus and cervix weights however these were minor in degree, generally recovered fully and were not associated with any microscopic pathology correlates. The no-observed-adverse-effect-level (NOAEL) in this study was therefore considered to be 1000 mg/kg/day and the no-observed-effect-level (NOEL) was considered to be 300 mg/kg/day (OECD 408).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

16 March 2016 to 14 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline required

Principles of method if other than guideline:

The study was performed to provide information for further repeated dose toxicity studies. Test item was administered to the Wistar Han:RccHan:WIST strain rat for a period of fourteen consecutive days at dose levels of 500, 750 and 1000 mg/kg bw/day. A control group was dosed with vehicle alone (Arachis oil BP). The dose levels were chosen based on preliminary toxicity work, performed as part of this study. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item. The results of the study will aid dose level selection for subsequent studies.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Wistar Han

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMAL INFORMATION
- A sufficient number of male and female Wistar Han:RccHan:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK.
- On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for 15 days (to allow time for the preliminary sighting investigations to be completed) during which time their health status was assessed.
- A total of twenty-four animals (twelve males and twelve females) were accepted into the study.
- At the start of treatment the males weighed 340 to 402 g, the females weighed 205 to 222 g, and were approximately 7 to 8 weeks old.
- Animals for the preliminary phase of the study were of the same strain and were taken from available stock within the animal facilities.

ANIMAL CARE AND HUSBANDRY
- The animals were housed in groups of three by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd, Cheshire, UK).
- The animals were allowed free access to food and water.
- A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was used.
- Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd, Cheshire, UK).
- The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility.
- Rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.
- Environmental conditions were continuously monitored by a computerised system, and print-outs of hourly
temperatures and humidities were included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; there were no deviations from these targets.
- Animals for the preliminary phase of the study were housed under similar conditions except that as there was only one male and one female used at each dosage, the animals were individually housed.
- The animals were allocated to dose groups using a randomization procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups. The animals were uniquely identified within the study, by an ear punch system routinely used in the laboratories.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

TEST ITEM PREPARATION AND ANALYSIS
- The test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. Due to the nature of the substance, it was necessary to warm the test item (maximum temperature approximately 70°C) during the preparation of the dosing formulations.
- No analysis was conducted to determine the homogeneity or stability of the test item formulation, however satisfactory homogeneity and four hours stability was demonstrated in the subsequent main study performed at the test facility
- All animals within this preliminary study were dosed within four hours of dose preparation. No analysis was conducted to determine the concentration of the test item formulation. This is an exception with regard to GLP and was reflected in the GLP compliance statement.

DOSE ADMINISTRATION
- In the absence of any toxicity data, preliminary toxicity work was undertaken. Initially a dosage of 500 mg/kg bw/day was investigated using one male and one female. As this appeared to be well tolerated, a further pair of animals were assessed at 1000 mg/kg bw/day.
- Animals were weighed on Days 1 and 4 and clinical signs, including post dosing observations, were recorded daily during the treatment period. For animals at 500 mg/kg bw/day, an additional body weight was recorded on Day 3 to ensure that there was no adverse effects on body weight before treatment of the animals at 1000 mg/kg bw/day commenced (these data were retained with the raw data but are not presented in the report).
- All animals received a macroscopic necropsy examination at termination Day 4.
- Following preliminary work, animals were allocated to treatment groups as shown in the table below.
- The test item was administered daily, for fourteen consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 6 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 on Days 4, 8 and 11. The initial dosage volume for the preliminary phase of the study was 4 mL/kg but this was increased to 6 mL/kg due to the viscosity of the dose formulations at high concentrations. This higher volume was also used for the main phase of the study with formulations also kept warm in a water bath to ensure the continued suitability of the formulations for dosing.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

750 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:

Three males and three females

Control animals:

yes, concurrent no treatment

Details on study design:

MAJOR COMPUTERISED SYSTEMS
- Provantis
- Delta Controls - ORCAview

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 after dosing and one hour after dosing.
- Additional observations were also made four hours following dosing (not at weekends or on public holidays).
- All observations were recorded.

BODY WEIGHT
- Individual body weights were recorded on Days 1, 4, 8, 11 and 15.

FOOD CONSUMPTION
- Food consumption was recorded for each cage group for Days 1 to 4, 4 to 8, 8 to 11 and 11 to 15.
- Food conversion efficiency was calculated retrospectively.

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

Sacrifice and pathology:

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

Statistics:

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

Clinical signs:

no effects observed

Description (incidence and severity):

- There were no clinical signs apparent for either sex during the study at 500, 750 or 1000 mg/kg bw/day.

Mortality:

no mortality observed

Description (incidence):

- There were no unscheduled animal deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- There was no obvious effect of treatment on body weight performance for either sex at 500, 750 or 1000 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- Intergroup differences for food consumption did not indicate any obvious effect of treatment for either sex at 500, 750 or 1000 mg/kg bw/day.

Food efficiency:

no effects observed

Description (incidence and severity):

- Intergroup differences for food conversion efficiency did not indicate any obvious effect of treatment for either sex at 500, 750 or 1000 mg/kg bw/day.

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

- For males at 1000 mg/kg bw/day water consumption was higher than control from Day 9. Higher water intake had also been observed for males on occasions prior to Day 9 but water consumption values during this period showed no consistent pattern.
- Intergroup differences for water consumption did not indicate any obvious effect of treatment for either sex at 500 or 750mg/kg bw/day or for females at 1000 mg/kg bw/day.

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

Description (incidence and severity):

- No macroscopic abnormalities were apparent at necropsy for either sex at 500, 750 or 1000 mg/kg bw/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Remarks on result:

not measured/tested

Remarks:

range-finding study intended to inform further repeated dose investigation(s)

Critical effects observed:

no

Conclusions:

Other than an increase in water consumption during the latter part of the treatment period, there was no obvious effect of exposure to test item over fourteen days at dosages up to 1000 mg/kg bw/day. A high dosage of 1000 mg/kg bw/day would therefore be suitable as a high dosage for any further investigation of toxicity and dosages of 0 (Control), 100, 300 and 1000 mg/kg bw/day are recommended.

Executive summary:

OBJECTIVE

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

 

METHODS

The test item was administered by gavage to three groups, each of three male and three female Wistar Han:RccHan:WIST strain rats, for up to fourteen consecutive days, at dose levels of 500, 750 and 1000 mg/kg bw/day. A control group of three males and three females was dosed with vehicle alone (Arachis oil BP). Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. All animals were subjected to gross necropsy examination.

 

RESULTS

No unscheduled animal deaths took place during the study and there were no clinical signs apparent for either sex during the study at 500, 750 or 1000 mg/kg bw/day. No effect of treatment on food consumption or food conversion efficiency was observed for either sex at 500, 750 or 1000 mg/kg bw/day Intergroup differences for food consumption or food conversion efficiency did not indicate any obvious effect of treatment for either sex at 500, 750 or 1000 mg/kg bw/day. At 1000 mg/kg bw/day water consumption for males was higher than control from Day 9. There was no effect of treatment on water consumption for either sex at 500 or 750 mg/kg

bw/day or for females at 1000 mg/kg bw/day. No macroscopic abnormalities were apparent at necropsy for either sex at 500, 750 or 1000 mg/kg bw/day.

 

CONCLUSION

Other than an increase in water consumption during the latter part of the treatment period, there was no obvious effect of exposure to test item over fourteen days at dosages up to 1000 mg/kg bw/day. A high dosage of 1000 mg/kg bw/day would therefore be suitable as a high dosage for any further investigation of toxicity and dosages of 0 (Control), 100, 300 and 1000 mg/kg bw/day are recommended.

Reference 2

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

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

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.

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

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

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

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

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

Critical effects observed:

no

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.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 June 2018 to 23 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

various (see below) with no impact on results or integrity of the study

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Han Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS
- Number of animals: 65 males and 65 females. Spare animals were removed from the study room after treatment commenced.
- Duration of acclimatisation: At least 12 days before commencement of treatment.
- Age at start of treatment: 55 to 62 days (main study and recovery animals).
- Weight of male rats at start of treatment: 198 to 254 g (main study and recovery animals).
- Weight of female rats at start of treatment: 149 to 190 g (main study and recovery animals).

ALLOCATION AND IDENTIFICATION
- Allocation: Randomly allocated on arrival. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
- Identification of animals: Each animal was assigned a number and identified uniquely within the study by a microchip inserted shortly after arrival.
- Identification of cages: Each cage label was colour-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

ANIMAL REPLACEMENT
- Any individuals rejected during the acclimatisation period were replaced with spare animals of suitable weight from the same batch.
- Replacement before treatment commenced: one male and one female (ocular abnormalities).

ANIMAL CARE AND HUSBANDRY
- Animal facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
- Temperature and relative humidity: Monitored and maintained within the range of 20 to 24ºC and 40 to 70%. Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were not considered to have influenced the health of the animals and/or the outcome of the study.
- Lighting: Artificial lighting (12 hours light: 12 hours dark).
- Electricity supply: Public supply with automatic stand-by generators.

ANIMAL ACCOMMODATION
- Cages: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
- Cage distribution: Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimize possible effects of spatial variations.
- Number of animals per cage: Four or three of the same sex (main study and recovery).
- Bedding: Wood based bedding which was changed at appropriate intervals each week.

ENVIRONMENTAL ENRICHMENT
- Aspen chew block: Provided to each cage throughout the study and replaced when necessary.
- Plastic shelter: Provided to each cage throughout the study and replaced when necessary.

DIET SUPPLY
- Availability: Non-restricted (removed overnight before blood sampling for haematology or blood chemistry).

IDENTITY OF TREATMENT GROUPS
- The study consisted of one control and three treated groups identified as shown in the table below.
- Some serial observations needed to be performed without the knowledge of the treatment group; therefore, the animal numbering system was such that it was not easy to identify a treatment group from the animal number.

Route of administration:

oral: gavage

Details on route of administration:

ADMINISTRATION
- Route: Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
- Treated at: Constant doses in mg/kg.
- Dose volume: 8 mL/kg body weight.
- Individual dose volume: Calculated from the most recently recorded scheduled body weight.
- Control (Group 1): Vehicle at the same volume dose as the treated groups.
- Frequency: Once daily at approximately the same time each day.
- Formulation: Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure. In order to keep the dosing formulations in a liquid state they were placed in a water bath, on a heated magnetic stirrer and heated to 40°C ± 2 °C. A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.

Vehicle:

arachis oil

Details on oral exposure:

FORMULATION OF TEST ITEM
- Method of preparation: Due to the nature of the test item, it was necessary to warm the test item, to a maximum temperature of 70 °C, during the preparation of the dosing formulations (see table below). The required amount of test item was weighed and placed into a suitable container. The required amount of arachis oil vehicle was warmed to 70 °C. Starting with the lowest concentration, approximately 50% of the final volume of vehicle was added to the test item and heated to 70 °C on a magnetic stirrer. The formulation was stirred at 70 °C until uniformly mixed. Further warmed vehicle was added until the formulation was at the required volume. Further concentrations were prepared, in ascending concentration order, following the above method.
- Frequency of preparation: Weekly
- Storage of formulation: Formulations from 10 to 250 mg/mL were stable for up to 15 days at ambient temperature and 15 days refrigerated.
- Test item accounting: Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SAMPLING
- Achieved concentration: Samples of each formulation prepared for administration in Weeks 1, 4 and 13 of treatment were analysed for achieved concentration of the test item.

FORMULATION ANALYSIS
- Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analysed to assess the stability and homogeneity of the test item in the liquid matrix.

Duration of treatment / exposure:

- All main study and recovery animals were treated for 90 consecutive days, main study animals were killed on Day 91
- Cessation of treatment for the recovery phase animals started the day after the final treatment (Day 91 of the treatment phase was Day 1 of the recovery phase). Recovery phase animals were killed on recovery Day 29.

Frequency of treatment:

Daily

Dose / conc.:

0 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)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

- Control (arachis oil): 10 males plus 10 females (main study) and 10 males plus 10 females (recovery phase)
- Test item (300 mg/kg bw/day): 10 males plus 10 females (main study) and 0 males plus 0 females (recovery phase)
- Test item (600 mg/kg bw/day): 10 males plus 10 females (main study) and 0 males plus 0 females (recovery phase)
- Test item (1000 mg/kg bw/day): 10 males plus 10 females (main study) and 10 males plus 10 females (recovery phase)

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

SERIAL OBSERVATIONS
- Clinical and behavioural observations: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatisation and recovery periods, observations of the animals and their cages were recorded at least once per day.
- Signs associated with dosing: Daily during the treatment period, detailed observations were recorded daily at the following times in relation to dose administration:
(i) Daily during week 1 of treatment: Pre-dose observation 1 to 2 hours after completion of dosing all groups. As late as possible in the working day.
(ii) Daily from Week 2 of treatment up to recovery phase termination: Pre-dose observation 1 to 2 hours after completion of dosing all groups.
- Detailed physical examination and arena observations: Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities. After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behaviour. Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.
- Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all animals (before dosing) during Week 12 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing. The measurements, reflexes and responses shown in the table below were recorded. At any point during the observations, additional comments were made as free text where considered appropriate.
- Motor activity: During Week 12 of treatment (before dosing), the motor activity of each animal was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo. Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one-hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.
- Body weight: The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy.
- Food consumption: The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.
- Opthalmic examination: The eyes of the animals were examined by means of a binocular indirect ophthalmoscope as follows: pretreatment for all animals; Week 12 for all animals of Groups 1 and 4 (main study and recovery phase). Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.
- Haematology, peripheral blood: Blood samples were collected after overnight withdrawal of food and prior to dosing (where appropriate) at the following occasions: Week 13 (all main study animals); Recovery Week 4 (all recovery animals). Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for characteristics shown in the table below using a Bayer Advia 120 analyser. Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of: Prothrombin time (PT) - using IL PT Fibrinogen reagent. Activated partial thromboplastin time (APTT) - using IL APTT reagent.
- Blood chemistry: Blood samples were collected after overnight withdrawal of food and prior to dosing (where appropriate) at the following occasions: Week 13 (all main study animals); Recovery Week 4 (all recovery animals). Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer as shown in the table below. Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analysed albumin concentration.

Sacrifice and pathology:

TERMINAL INVESTIGATIONS
- Method of kill: Carbon dioxide asphyxiation with subsequent exsanguination.
- Necropsy: All main study and recovery animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. The retained tissues were checked before disposal of the carcass. The schedule was main study animals were killed following 13 weeks of treatment and recovery animals were killed following 13 weeks of treatment and 4 weeks of recovery. The sequence allowed satisfactory inter-group comparison. The organs weighed, tissue samples fixed and sections examined microscopically were as detailed in the table below (animal ID retained).
- Bone marrow: Bone marrow smears were prepared immediately following death, on completion of the scheduled treatment or recovery periods:
(i) Fixation: Smears were air dried and subsequently fixed in methanol.
(ii) Analysis: No examinations were performed, however, the smears were retained for possible future examination.
(iii) Retention: The smears were transferred to archives and will be retained for the same period as the study raw data.
- Organ weights: For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for main study and recovery animals killed at scheduled intervals.
- Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
(i) Epididymides: In modified Davidson’s fluid.
(ii) Testes: In modified Davidson’s fluid.
(iii) Eyes: In Davidson’s fluid.
(iv) Bone marrow smears: see above.
- Histology:
(i) Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
(ii) Full list: Main study animals of Groups 1 and 4.
(iii) Abnormalities only: All main study animals of Groups 2 and 3 and all recovery phase animals killed at a scheduled interval.
(iv) Routine staining: Sections were stained with hematoxylin and eosin.
- Light microscopy: Tissues preserved for examination were examined as shown in the table below. Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

Statistics:

See below

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no clinical signs observed that were considered related to treatment.
- The routine weekly arena observations also did not indicate any changes in the appearance or general behaviour of the animals that were considered related to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- There was no effect of treatment on body weight or body weight gain during the 90-day treatment period or the 4-week recovery period.
- Occasional differences in body weight gain were observed at all doses but these differences were minor and considered to reflect normal biological variation.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- There was no effect of treatment on food consumption during the 90 day treatment period or the 4-week recovery period.
- All differences in food consumption were considered to reflect normal biological variation or were similar to values observed during the pretreatment phase (slightly high consumption for Group 4 females).
- It was observed that food consumption during the recovery phase was noticeably higher than during the treatment phase however the increase was similar for controls and Group 4 and is considered likely associated with the cessation of treatment for these animals.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- The ophthalmic examinations performed on animals in Group 1 (control) and Group 4 (1000 mg/kg/day) during Week 12 of treatment did not reveal any findings that were considered to be related to treatment.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- Investigations performed during Week 13 revealed slightly lower than control reticulocyte counts for males receiving 1000 mg/kg/day (0.89X control) whereas reticulocyte counts for females at 600 or 1000 mg/kg/day were slightly higher than control (1.2X and 1.1X control respectively).
- Examination of the white blood cell data revealed a slightly higher than control neutrophil count (1.4X of control) and a slightly lower than control group mean lymphocyte count (0.82X control) for males given 1000 mg/kg/day, resulting in a slightly lower than control total white blood cell count (0.90X control). However, examination of the individual data revealed significant overlap with the controls and values for all treated groups of females were considered similar to controls.
- These differences were not apparent in the recovery phase.
- All other differences from controls were minor, due to higher than expected control values (recovery APTT times for control males) or due to individual animals (e.g. high monocyte count for female No. 124) and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Investigations performed during Week 13 revealed slightly increased alanine amino-transferase activities for males and females at 600 or 1000 mg/kg/day (between 1.2X and 1.5X control) with the difference for males and females at 1000 mg/kg/day and females at 600 mg/kg/day attaining a level of statistical significance.
- These differences were not apparent in the recovery phase.
- All other differences from controls, including some attaining a level of statistical significance, were considered minor, due to higher than expected control values (recovery potassium concentration for control females) or values were within the range for control animals and were therefore attributed to normal biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

SENSORY REACTIVITY AND GRIP STRENGTH
- Sensory reactivity observations and grip strength values were considered to be unaffected by treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Examination of the data obtained from animals killed on Day 91 (Week 13) revealed a slightly increased group mean adjusted and body weight relative uterus and cervix weights were observed for females at 600 or 1000 mg/kg/day (between 1.2 and 1.4X control) however, no statistical significance was achieved and there were no correlating macroscopic or microscopic changes.
- Examination of the data obtained following 4 weeks of recovery revealed the uterus and cervix weight for females previously given 1000 mg/kg/day was considered similar to controls.
- All differences from controls, including some that attained statistical significance, were minor, confined to one sex or lacked dose-relationship. They were therefore attributed to normal biological variation.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Animals killed after 13 weeks of treatment: The macroscopic examination performed after 13 weeks of treatment revealed no test item-related lesions. The incidence and distribution of all findings were considered to be unrelated to treatment.
- Animals killed after 4 weeks of recovery: The macroscopic examination performed after 4 weeks of recovery revealed no test item-related lesions. All the findings were considered to be unrelated to treatment.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

MOTOR ACTIVITY
- High beam (rearing activity) and low beam (cage floor activity) motor activity scores for males and females were considered to be unaffected by treatment.
- During Week 12 of treatment, the majority of group mean high beam and low beam activity scores for all treated males and to a lesser extent all treated females were slightly low compared with Controls, however, these differences did not attain statistical significance. Low beam total scores in all treated males and females show a dose-relationship, but no dose-relationship was evident in the high beam total scores. The majority of high beam and low beam individual 6-minute scores and total scores were also within the historical control data (HCD) range. Due to the overall pattern of activity throughout the 1-hour recording period and that no dose-relationship is evident in the high beam scores (high beam scores are generally more sensitive to any effects of treatment than low beam scores), these differences were considered not to be associated with treatment.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- Animals killed after 13 weeks of treatment: The microscopic examination performed after 13 weeks of treatment revealed no test item-related lesions. The incidence and distribution of all findings were considered to be unrelated to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

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: lack of treatment-related effects at highest dose level tested

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

FORMULATION ANALYSIS

- The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection, linearity of detector response, repeatability, method accuracy and precision.

- The homogeneity and stability was confirmed for test item in arachis oil formulations at nominal concentrations of 10 mg/mL and 250 mg/mL at ambient temperature storage (15 to 25 °C) for 15 days and refrigerated storage (2 to 8 °C) for up to 15 days. This analysis was performed during distribution between the bottles and magnetic stirring for 4 hours.

- The mean concentrations of test item in test formulations analyzed for the study were within ± 5 % (-4.8 to +2.9 %) of nominal concentrations, confirming accurate formulation. The difference from mean remained within 2 % (± 1.47 %), confirming precise analysis.

Conclusions:

Daily repeated administration of test item to rats by oral gavage for 90 days at doses of 300, 600 or 1000 mg/kg/day, followed by a 4-week recovery period was well tolerated and did not result in any adverse effects of treatment. There were no treatment-related macroscopic or microscopic changes. Slight disturbances were observed in some hematological and blood chemistry parameters and there was a slight increase in uterus and cervix weights however these were minor in degree, generally recovered fully and were not associated with any microscopic pathology correlates. The No Observed Adverse Effect Level (NOAEL) in this study was therefore considered to be 1000 mg/kg/day and the No Observed Effect Level (NOEL) was considered to be 300 mg/kg/day.

Executive summary:

GUIDELINE

The study designed to meet the requirements of the Organization for Economic Co-operation and Development, Testing of Chemicals Guideline No. 408 (revised 1998). The study was conducted in accordance with the requirements of current, internationally recognized Good Laboratory Practice Standards and was conducted in accordance with the applicable sections of the United Kingdom Animals (Scientific Procedures) Act 1986, Amendment Regulations 2012 (the Act).

 

METHODS

The purpose of the study was to assess the systemic toxic potential of the test item, in a 13-week oral gavage study in Han Wistar rats. Recovery from any effects was evaluated during a 4-week recovery period. The study design was as summarised below:

- Group 1 (test item; 0 mg/kg bw/day): 10 males plus 10 females (main study) and 10 males plus 10 females (recovery phase).

- Group 2 (test item; 300 mg/kg bw/day): 10 males plus 10 females (main study) and 0 males plus 0 females (recovery phase).

- Group 3 (test item; 600 mg/kg bw/day): 10 males plus 10 females (main study) and 0 males plus 0 females (recovery phase).

- Group 4 (test item; 1000 mg/kg bw/day): 10 males plus 10 females (main study) and 10 males plus 10 females (recovery phase).

Animals received the control, arachis oil or the test item, by oral administration for 13 weeks. Recovery animals were similarly treated for 13 weeks followed by a 4 week off dose period. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, ophthalmoscopy, haematology (peripheral blood), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

 

RESULTS

There were no clinical signs or behavioural changes considered related to treatment with the test item and no treatment-related effects on body weight or food consumption. Sensory reactivity responses, grip strength and motor activity were unaffected by treatment. There were no treatment-related ophthalmic changes. Reticulocyte counts were slightly low for males at 1000 mg/kg/day and slightly high for females at 600 or 1000 mg/kg/day. Slightly low lymphocyte and high neutrophil counts were observed for males at 1000 mg/kg/day. Slightly increased alanine amino-transferase activities were observed for males and females at 600 or 1000 mg/kg/day. Full recovery was apparent. Increased group mean uterus and cervix weights were observed in Week 13 for females at 600 or 1000 mg/kg/day with full recovery apparent. There were no microscopic correlates for this change. Macroscopic and microscopic examination did not reveal any treatment-related changes. The incidence and distribution of all findings were considered to be unrelated to treatment.

 

CONCLUSION

Daily repeated administration of test item to rats by oral gavage for 90 days at doses of 300, 600 or 1000 mg/kg/day, followed by a 4-week recovery period was well tolerated and did not result in any adverse effects of treatment. There were no treatment-related macroscopic or microscopic changes. Slight disturbances were observed in some hematological and blood chemistry parameters and there was a slight increase in uterus and cervix weights however these were minor in degree, generally recovered fully and were not associated with any microscopic pathology correlates. The No Observed Adverse Effect Level (NOAEL) in this study was therefore considered to be 1000 mg/kg/day and the No Observed Effect Level (NOEL) was considered to be 300 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

ORAL ROUTE

28-day repeated dose oral toxicity

The key 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).

 

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 highdose (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 groupanimals at the end of the treatment-free period. These animals were then subjected to a gross necropsy.

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 noobvious 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.

 

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.

90-day repeated dose oral toxicity

The study designed to meet the requirements of the Organization for Economic Co-operation and Development, Testing of Chemicals Guideline No. 408 (revised 1998). The study was conducted in accordance with the requirements of current, internationally recognized Good Laboratory Practice Standards and was conducted in accordance with the applicable sections of the United Kingdom Animals (Scientific Procedures) Act 1986, Amendment Regulations 2012 (the Act).

 

The purpose of the study was to assess the systemic toxic potential of the test item, in a 13-week oral gavage study in Han Wistar rats. Recovery from any effects was evaluated during a 4-week recovery period. The study design was as summarised below:

- Group 1 (test item; 0 mg/kg bw/day): 10 males plus 10 females (main study) and 10 males plus 10 females (recovery phase).

- Group 2 (test item; 300 mg/kg bw/day): 10 males plus 10 females (main study) and 0 males plus 0 females (recovery phase).

- Group 3 (test item; 600 mg/kg bw/day): 10 males plus 10 females (main study) and 0 males plus 0 females (recovery phase).

- Group 4 (test item; 1000 mg/kg bw/day): 10 males plus 10 females (main study) and 10 males plus 10 females (recovery phase).

Animals received the control, arachis oil or the test item, by oral administration for 13 weeks. Recovery animals were similarly treated for 13 weeks followed by a 4 week off dose period. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, ophthalmoscopy, hematology (peripheral blood), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

 

There were no clinical signs or behavioural changes considered related to treatment with the test item and no treatment-related effects on body weight or food consumption. Sensory reactivity responses, grip strength and motor activity were unaffected by treatment. There were no treatment-related ophthalmic changes. Reticulocyte counts were slightly low for males at 1000 mg/kg/day and slightly high for females at 600 or 1000 mg/kg/day. Slightly low lymphocyte and high neutrophil counts were observed for males at 1000 mg/kg/day. Slightly increased alanine amino-transferase activities were observed for males and females at 600 or 1000 mg/kg/day. Full recovery was apparent. Increased group mean uterus and cervix weights were observed in Week 13 for females at 600 or 1000 mg/kg/day with full recovery apparent. There were no microscopic correlates for this change. Macroscopic and microscopic examination did not reveal any treatment-related changes. The incidence and distribution of all findings were considered to be unrelated to treatment.

Daily repeated administration of test item to rats by oral gavage for 90 days at doses of 300, 600 or 1000 mg/kg/day, followed by a 4-week recovery period was well tolerated and did not result in any adverse effects of treatment. There were no treatment-related macroscopic or microscopic changes. Slight disturbances were observed in some hematological and blood chemistry parameters and there was a slight increase in uterus and cervix weights however these were minor in degree, generally recovered fully and were not associated with any microscopic pathology correlates. The no-observed-adverse-effect-level (NOAEL) in this study was therefore considered to be 1000 mg/kg/day and the no-observed-effect-level (NOEL) was considered to be 300 mg/kg/day.

INHALATION ROUTE

28-day repeated dose inhalation toxicity

According to REACH, Annex VIII, Section 8.6.1, short-term repeated dose toxicity should be investigated using one species and the most likely route of human exposure. The substance is a waxy solid with determined vapour pressures of 0.0345 Pa at 25 °C, 0.0985 Pa at 55 °C and 0.891 Pa at 145 °C and a high onset boiling point (decomposition from approximately 171 °C at 101 kPa). Based on evaluation of the life cycle of the substance, it is therefore expected that inhalation exposure will be low and that the most likely route of exposure for workers and consumers is the dermal route. A short-term repeated dose toxicity study via the inhalation route is, therefore, not appropriate.

 

90-day repeated dose inhalation toxicity

According to REACH, Annex IX, Section 8.6.2, sub-chronic repeated dose toxicity should be investigated using one species and the most likely route of human exposure. The substance is a waxy solid with determined vapour pressures of 0.0345 Pa at 25 °C, 0.0985 Pa at 55 °C and 0.891 Pa at 145 °C and a high onset boiling point (decomposition from approximately 171 °C at 101 kPa). Based on evaluation of the life cycle of the substance, it is therefore expected that inhalation exposure will be low and that the most likely route of exposure for workers and consumers is the dermal route. A sub-chronic repeated dose toxicity study via the inhalation route is, therefore, not appropriate.

 

DERMAL ROUTE

28-day repeated dose dermal toxicity

According to REACH, Annex VIII, Section 8.6.1, short-term repeated dose toxicity should be investigated using one species and the most likely route of human exposure. However, no evidence of systemic toxicity was demonstrated during an acute study via the dermal route and, in order to minimise the number of vertebrate animals tested, oral dosing by gavage was considered most appropriate for a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) in rats.

 

90-day repeated dose dermal toxicity

According to REACH, Annex IX, Section 8.6.2, sub-chronic repeated dose toxicity should be investigated using one species and the most likely route of human exposure. However, no evidence of systemic toxicity was demonstrated during an acute study via the dermal route and, in order to minimise the number of vertebrate animals tested, oral dosing by gavage was considered most appropriate for investigation of sub-chronic systemic toxicity over 90-days in accordance with OECD 408.

Justification for classification or non-classification

No treatment-related effects were reported in a 28-day investigation at dose levels of 100, 300 and 1000 mg/kg bw (OECD 422) or 300, 600 and 1000 mg/kg bw in a 90-day investigation (OECD 408). Classification for Specific Target Organ Toxicity (STOT RE category 1 or 2) is therefore not required under the terms of Regulation (EC) No 1272/2008 and subsequent amendments.