Rape oil, reaction products with diethylenetriamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Treatment at dosages of up to 1000 mg/kg bw/day was well tolerated and the No Observed Effect Level (NOEL) for reproduction, including the survival, growth and development of the offspring was considered to be 1000 mg/kg bw/day (OECD 422).

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

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
- On receipt the animals were examined for signs of ill-health or injury.
- 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
- During the pairing phase, non-recovery animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- The animals were allowed free access to food and water. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. 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 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 are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Deviations from the target ranges did occur on the study but were considered not to have affected the purpose or scientific integrity of the study (see Deviations from Study Plan).
- 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 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.

Details on mating procedure:

MATING
- Non-recovery animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days.
- Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded.
- The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages.
- Mated females were housed individually during the period of gestation and lactation.

PREGNANCY AND PARTURITION
- Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition.
- Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays.
- The following was recorded for each female:
(I) Date of pairing
(ii) Date of mating
(iii) Date and time of observed start of parturition
(iv) Date and time of observed completion of parturition

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

Parental animals: Observations and examinations:

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.

THYROID HORMONE ANALYSIS
- Where possible, blood samples were taken into EDTA, centrifuged, and the plasma from each blood sample stored frozen at -70 ºC ±10 °C for possible evaluation of thyroid hormones from the following:
(a) Two offspring from each litter at Day 4 post-partum (offspring samples from the same litter were pooled when animals were of the same sex).
(b) One male and one female offspring from each litter at Day 13 post-partum.
(c) All adult males at termination.
(d) All adult females at termination.
- Where possible, additional blood samples were taken, allowed to clot, centrifuged, and the serum from each blood sample stored frozen at lower than -60 °C for possible evaluation of thyroid hormones from the following:
(a) Two offspring (one male and one female where possible) at Day 13 post-partum.
(b) All adult males at termination.
(c) All adult females at termination.
- Analysis of samples was conducted at the Test Sites under the supervision of relevant Principal Investigators for the study phase. Initially only the plasma from adult non-recovery males and serum from Day 13 offspring were analysed for Thyroxine (T4).
- Analysis of plasma samples was conducted at the Test Site (Envigo CRS Limited, Huntingdon).
- Analysis of serum samples was conducted at the Test Site (Envigo CRS Limited, Huntingdon) for this study phase.

Oestrous cyclicity (parental animals):

ESTROUS CYCLE ASSESSMENT
- Vaginal smears were taken daily throughout the two-week pre-pairing treatment period and the stage of the oestrous cycle was recorded for each day.

Sperm parameters (parental animals):

SPERM
- Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
- Since there were no indications of treatment-related changes, examinations were not extended to include animals from the low, intermediate or recovery groups.

Litter observations:

LITTER DATA
- On completion of parturition (Day 0 post-partum), the number of live and dead offspring was recorded.
- Offspring were individually identified within each litter by tattoo on Day 1 post-partum.
- The following was recorded for each litter:
(i) Number of offspring born
(ii) Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post-partum
(iii) Sex of offspring on Days 1, 4, 7 and 13 post-partum
(iv) Clinical condition of offspring from birth to Day 13 post-partum
(v) Individual offspring weights on Days 1, 4, 7 and 13 post-partum (litter weights were calculated retrospectively from this data).

PHYSICAL DEVELOPMENT
- All live offspring were assessed for ano-genital distance on Day 1 post-partum.
- Additionally, visible nipple counts were performed for all male offspring on Day 13 post-partum.

Postmortem examinations (parental animals):

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, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

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.

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.

Postmortem examinations (offspring):

- All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

See below

Reproductive indices:

PRE-COITAL INTERVAL
- Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

FERTILITY INDICES
- Calculated for each group.
- Mating Index (%) = Number of animals mated / number of animals paired * 100
- Pregnancy Index (%) = Number of pregnant females / number of animals mated * 100

GESTATION AND PARTURITION DATA
- The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
(i) Gestation length = number of days of gestation including the day for observation of mating and the start of parturition.
(ii) Parturition Index (%) calculated for each group = Number of females delivering live offspring / number of pregnant females * 100

Offspring viability indices:

LITTER RESPONSES
- The standard unit of assessment was considered to be the litter; therefore values were first calculated for each litter and the group mean was calculated using their individual litter values.
- Group mean values included all litters reared to termination (Day 13 of age).
- Group mean percentile post-implantation loss was calculated for each female/litter using the equation Post-implantation loss (%) = [(Number of implantation sites – Total number of offspring born) / Number of implantation sites] * 100

LIVE BIRTH AND VIABILITY INDICES
- Live Birth Index (%) = Number of offspring alive on Day 1 / Number of offspring born * 100
- Viability Index 1 (%) = Number of offspring alive on Day 4 / Number of offspring alive on Day 1 * 100
- Viability Index 2 (%) = Number of offspring alive on Day 13 / Number of offspring alive on Day 4 * 100
- Viability index 2 takes into consideration the offspring used for blood sampling on Day 4 post-partum

SEX RATIO (% MALES)
- Sex ratio was calculated for each litter value on Days 1, 4, 7 and 13 post-partum, using the equation Sex Ratio (% males) = Number of male offspring born / Total number of offspring * 100

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):

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:

no effects observed

Description (incidence and severity):

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.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

- 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.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

MATING
- Mating performance as assessed by the number of paired animals that mated and pre-coital interval was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.
- At 1000 mg/kg bw/day, one pair of animals did not show any evidence of mating. Macroscopic necropsy did not show any obvious underlying reason for this lack of mating and this isolated occurrence was considered to be incidental and unrelated to treatment.

FERTILITY
- There was no obvious effect on fertility, as assessed by the number of females that achieved pregnancy, at 100, 300 or 1000 mg/kg bw/day.

GESTATION LENGTH
- The intergroup distribution of gestation lengths observed during the study did not indicate any obvious effect of treatment.

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

Mortality / viability:

no mortality observed

Description (incidence and severity):

LITTER RESPONSES
- One female at each dosage including control was non-pregnant. Additionally, one female at 300 mg/kg bw/day showed evidence of pregnancy (two implantations) but no litter was observed to be born.
- The following assessment is generally based on the 11, 11, 10 and 10 females successfully rearing young to days 13 of age at 0 (Control), 100, 300 or 1000 mg/kg bw/day respectively.

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 at birth/Day 1 and subsequent offspring survival to Day 13 of age at 100, 300 or 1000 mg/kg bw/day.
- Sex ratio for the offspring was similar in all groups and did not indicate any selective effect of maternal treatment on survival for either sex at any of the dosages investigated.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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 of age 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.
- Clinical signs apparent for the offspring during the study were typical of the age observed and the distribution and low incidence did not indicate any obvious effect of maternal treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

NECROPSY
- 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.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

other: absence of test item-related effects at any maternal dose level

Critical effects observed:

no

Key result

Reproductive 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 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Additional information

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 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.

 

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.  

Effects on developmental toxicity

Description of key information

Screening: Treatment at dosages of up to 1000 mg/kg bw/day was well tolerated and the No Observed Effect Level (NOEL) for reproduction, including the survival, growth and development of the offspring was considered to be 1000 mg/kg bw/day (OECD 422).

 

Development toxicity: oral administration of test item to pregnant rats by oral gavage from gestation Day 3 to 19 at dose levels of 100, 300 and 1000 mg/kg bw/day, resulted in no treatment related effects. Therefore, the No Observed Effect Level (NOEL) for the pregnant female was considered to be 1000 mg/kg bw/day. No treatment-related changes were detected in the uterine parameters measured or on embryofetal development. Therefore the No Observed Effect Level (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg bw/day (OECD 414).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

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:

other: 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

Details on test animals or test system and environmental conditions:

ANIMAL WELFARE
- The study was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Envigo Research Limited, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom’s Animals (Scientific Procedure) Act 1986 Amendment Regulations 2012. The conduct of the study may be reviewed, as part of the Envigo Research Limited, Shardlow, UK Ethical Review Process.
- The study was conducted in accordance with the UK Home Office Guidance document on Regulatory Toxicology and Safety Evaluation Studies and the OECD guidance document on recognition, assessment and use of clinical signs as humane endpoints for experimental animals used in safety evaluation.

ANIMAL INFORMATION
- A sufficient number of male and female Wistar Han:RccHan:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury.
- 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
- Initially, all animals were housed in groups of three (Non-recovery) or five (Recovery) in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, non-recovery animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- 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. Certificates of analysis of the batches of diet used are given in Annex 7. 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) except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. 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. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Deviations from the target ranges did occur on the study but were considered not to have affected the purpose or scientific integrity of the study (see Deviations from Study Plan).
- 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 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.

Details on mating procedure:

MATING
- Non-recovery animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days.
- Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded.
- The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages.
- Mated females were housed individually during the period of gestation and lactation.

PREGNANCY AND PARTURITION
- Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition.
- Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays.
- The following was recorded for each female:
(I) Date of pairing
(ii) Date of mating
(iii) Date and time of observed start of parturition
(iv) Date and time of observed completion of parturition

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.

Maternal examinations:

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.

ESTROUS CYCLE ASSESSMENT
- Vaginal smears were taken daily throughout the two-week pre-pairing treatment period and the stage of the oestrous cycle was recorded for each day.

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.

THYROID HORMONE ANALYSIS
- Where possible, blood samples were taken into EDTA, centrifuged, and the plasma from each blood sample stored frozen at -70 ºC ±10 °C for possible evaluation of thyroid hormones from the following:
(a) Two offspring from each litter at Day 4 post-partum (offspring samples from the same litter were pooled when animals were of the same sex).
(b) One male and one female offspring from each litter at Day 13 post-partum.
(c) All adult males at termination.
(d) All adult females at termination.
- Where possible, additional blood samples were taken, allowed to clot, centrifuged, and the serum from each blood sample stored frozen at lower than -60 °C for possible evaluation of thyroid hormones from the following:
(a) Two offspring (one male and one female where possible) at Day 13 post-partum.
(b) All adult males at termination.
(c) All adult females at termination.
- Analysis of samples was conducted at the Test Sites under the supervision of relevant Principal Investigators for the study phase. Initially only the plasma from adult non-recovery males and serum from Day 13 offspring were analysed for Thyroxine (T4).
- Analysis of plasma samples was conducted at the Test Site (Envigo CRS Limited, Huntingdon).
- Analysis of serum samples was conducted at the Test Site (Envigo CRS Limited, Huntingdon) for this study phase.

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.

SPERM
- Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
- Since there were no indications of treatment-related changes, examinations were not extended to include animals from the low, intermediate or recovery groups.

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.

Ovaries and uterine content:

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.

Fetal examinations:

LITTER DATA
- On completion of parturition (Day 0 post-partum), the number of live and dead offspring was recorded.
- Offspring were individually identified within each litter by tattoo on Day 1 post-partum.
- The following was recorded for each litter:
(i) Number of offspring born
(ii) Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post-partum
(iii) Sex of offspring on Days 1, 4, 7 and 13 post-partum
(iv) Clinical condition of offspring from birth to Day 13 post-partum
(v) Individual offspring weights on Days 1, 4, 7 and 13 post-partum (litter weights were calculated retrospectively from this data).

PHYSICAL DEVELOPMENT
- All live offspring were assessed for ano-genital distance on Day 1 post-partum.
- Additionally, visible nipple counts were performed for all male offspring on Day 13 post-partum.

Statistics:

See below

Indices:

LITTER RESPONSES
- The standard unit of assessment was considered to be the litter; therefore values were first calculated for each litter and the group mean was calculated using their individual litter values.
- Group mean values included all litters reared to termination (Day 13 of age).
- Group mean percentile post-implantation loss was calculated for each female/litter using the equation Post-implantation loss (%) = [(Number of implantation sites – Total number of offspring born) / Number of implantation sites] * 100

LIVE BIRTH AND VIABILITY INDICES
- Live Birth Index (%) = Number of offspring alive on Day 1 / Number of offspring born * 100
- Viability Index 1 (%) = Number of offspring alive on Day 4 / Number of offspring alive on Day 1 * 100
- Viability Index 2 (%) = Number of offspring alive on Day 13 / Number of offspring alive on Day 4 * 100
- Viability index 2 takes into consideration the offspring used for blood sampling on Day 4 post-partum

SEX RATIO (% MALES)
- Sex ratio was calculated for each litter value on Days 1, 4, 7 and 13 post-partum, using the equation Sex Ratio (% males) = Number of male offspring born / Total number of offspring * 100

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):

- 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.

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:

no effects observed

Description (incidence and severity):

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.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

- One female at each dosage including control was non-pregnant. Additionally, one female at 300 mg/kg bw/day showed evidence of pregnancy (two implantations) but no litter was observed to be born.
- The assessment is generally based on the 11, 11, 10 and 10 females successfully rearing young to days 13 of age at 0 (Control), 100, 300 or 1000 mg/kg bw/day respectively.

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): GESTATION LENGTH
- The intergroup distribution of gestation lengths observed during the study did not indicate any obvious effect of treatment.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

FERTILITY
- There was no obvious effect on fertility, as assessed by the number of females that achieved pregnancy, at 100, 300 or 1000 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: absence of test item-related effects at any dose level

Abnormalities:

no effects observed

Fetal body weight changes:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): - There was no obvious effect of maternal treatment on offspring body weight on Day 1 and subsequent body weight gain to Day 13 of age at 100, 300 or 1000 mg/kg bw/day.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

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

Changes in sex ratio:

no effects observed

Description (incidence and severity):

- Sex ratio for the offspring was similar in all groups and did not indicate any selective effect of maternal treatment on survival for either sex at any of the dosages investigated.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

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

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

- Clinical signs apparent for the offspring during the study were typical of the age observed and the distribution and low incidence did not indicate any obvious effect of maternal treatment.

External malformations:

no effects observed

Description (incidence and severity):

- 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.

Skeletal malformations:

no effects observed

Description (incidence and severity):

- 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.

Visceral malformations:

no effects observed

Description (incidence and severity):

- 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.

Other effects:

no effects observed

Description (incidence and severity):

- 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.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

other: absence of test item-related effects at any maternal dose level

Abnormalities:

no effects observed

Developmental 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 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 August 2018 to 07 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22 January 2001

Deviations:

yes

Remarks:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

yes

Remarks:

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

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 Nousan No 8147

Version / remarks:

24 November 2000

Deviations:

yes

Remarks:

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

Qualifier:

according to guideline

Guideline:

other: Test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008

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:

Sprague-Dawley

Remarks:

Crl:CD (SD) IGS BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Day 0 or Day 1 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation.
- Weight at study initiation: 190 g to 299 g
- Fasting period before study:
- Housing: Individual housing in solid-floor polypropylene cages with stainless steel
mesh lids furnished with softwood flakes. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels.
- Diet: Free access to pelleted diet.
- Water: Free access to mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: Day 1 and/or Day 2 of gestation depending on the delivery in which time-mated females were received.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12 (low intensity fluorescent lighting)

IN-LIFE DATES: From: 11 August 2018 To: 30 August 2018

Route of administration:

oral: gavage

Vehicle:

arachis oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

TEST ITEM PREPARATION AND ANALYSIS
- For the purpose of the study the test item was prepared at the appropriate concentrations as a straw coloured solution in arachis oil. Due to the nature of the test item, it was necessary to warm the test item (maximum temperature approximately 70 °C).
- In this study, stability at 12.5 and 125 mg/mL was confirmed at ambient temperature for up to 4 hours. Formulations were therefore prepared daily and dosed within this known stability period.
- Analytical results indicated that the prepared formulations were within 89 % to 103 % of the nominal concentration.DEVIATIONS FROM STUDY PLAN
- Animal Husbandry - Environment
- The Study Plan target value for relative humidity (RH) was 50 + 20%.
- A review of the data outputs showed that the target humidity values were exceeded on 21 occasions. A number of these incidents occurred on the same day but at different time points over five separate days.
- The extent of the deviations was minor and no abnormal health issues were reported for any animal as a consequence of the changes. It was therefore concluded that there was no impact upon the integrity or validity of the study.
- Terminal Investigations - Necropsy
- For the female number 36, an additional fetus was found at post life examination. This fetus was not weighed/sexed and no placental weight was recorded. Individual litter data values have been excluded from group means where appropriate to reflect this error. The loss of data, whilst regrettable has been concluded to have not affected the overall assessment of the study.
- The gravid uterus of female number 63 was not weighed in error. Group mean values have been adjusted to account for this.
- The loss of this data is regrettable but it is considered that sufficient data is available for this dose group to allow for assessment and therefore the validity and integrity of the study is not affected.

TEST ITEM PREPARATON AND ANALYSIS
- The stability and homogeneity of the test item formulations were determined by Covance CRS Research Limited, Shardlow, UK Analytical Services in previous toxicity work.
- Samples were taken of each test item formulation on two occasions and were analyzed for
achieved concentration at 100, 300 and 1000 mg/kg bw/day at Covance Analytical Laboratory, Shardlow. The method used for analysis of formulations and the results obtained were provided in Annex 1 of the full study report.

TEST ANIMALS
- Source: Ninety-six time-mated females were obtained from Charles River (UK) Limited, Margate, Kent.
- Housing: Single air-conditioned room within the Covance CRS Research Limited, Shardlow, UK Barrier Maintained Rodent Facility.
- Diet: Pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK).
- Environmental enrichment: Softwood flakes, wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).

MAJOR COMPUTERIZED SYSTEMS
- Delta BMS
- R Environment for Statistical Computing
- Chemstation

Details on mating procedure:

Time-mated female animals were received and used in the study.

Duration of treatment / exposure:

Day 3 to Day 19 of gestation

Frequency of treatment:

Daily

Duration of test:

Day 0 or Day 1 of gestation (animal receipt) to Day 20 of gestation (sacrifice).

Dose / conc.:

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

Remarks:

Control group: animals 1 to 24 (treatment volume 8 mL/kg; concentration 0 mg/mL)

Dose / conc.:

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

Remarks:

Low dose treatment group: animals 25 to 48 (treatment volume 8 mL/kg; concentration 12.5 mg/mL)

Dose / conc.:

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

Remarks:

Intermediate dose treatment group: animals 49 to 72 (treatment volume 8 mL/kg; concentration 37.5 mg/mL)

Dose / conc.:

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

Remarks:

High dose treatment group: animals 73 to 96 (treatment volume 8 mL/kg; concentration 125 mg/mL)

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on available toxicity data including Oral (Gavage) Combined Repeat Dose Toxicity Study (with Recovery Groups) with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422) and a preliminary pre-natal developmental toxicity study. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
- Rationale for animal assignment: The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in the laboratories.
- Other: The dose levels were chosen based on available toxicity data including Oral (Gavage) Combined Repeat Dose Toxicity Study (with Recovery Groups) with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422) and a preliminary pre-natal developmental toxicity study. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the dosing period, observations were recorded immediately before and soon after dosing and one-hour post dosing. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or
behavioral changes once daily during the gestation period. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of the water bottles for any overt changes.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20 by carbon dioxide asphyxiation followed by cervical dislocation.
- Organs examined: Ovaries and uteri of pregnant females were removed and examined. The following data were recorded: Number of corpora lutea, number, position and type of intrauterine implantation, fetal sex, external fetal appearance, fetal weight, placental weight, gravid uterus weight. The uteri of any apparently non-pregnant females were immersed in 0.5 % ammonium polysulphide solution to reveal evidence of implantation.
- Implantation types were divided into early death (no visible distinction between placental/decidual tissue and embryonic tissue), late death (separate embryonic/fetal and placental tissue visible) and dead fetus (a fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes). All implantations and viable fetuses were numbered according to their intrauterine position.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes (number, position and type of intrauterine implantation)
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Placental weight

Fetal examinations:

- External examinations: Yes:
- Soft tissue examinations: Yes: [half per litter]. Alternate fetuses were identified using an indelible marker and placed in Bouin's fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin.
- Skeletal examinations: Yes: [half per litter]. Remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed into 70 % IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50 % glycerol for examination of skeletal development and anomalies and storage.
- Head examinations: No

Statistics:

- The following parameters were analyzed statistically, where appropriate, using the test methods outlined.
- Female body weight change, food consumption and gravid uterus weight, fetal, litter and placental weight: Shapiro Wilk normality test and Bartlett's test for homogeneity of variance and one way analysis of variance, followed by Dunnett's multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
- All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney 'U' test, where significance was seen.
- Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney 'U' test.
- Probability values (p) were presented as ** (p < 0.01); * (p < 0.05); not significant (p ≥ 0.05).

Indices:

- All data was summarized in tabular form, including reproductive indices. Group mean values were calculated to include data from all females with live fetuses on Day 20 of gestation. Values given in appendices of the full study report may represent rounded values for presentation purposes. Group mean values were generally calculated using unrounded values therefore it is not always possible to calculate the exact group mean values from values presented in the appendices of the full study report.
- As the litter was standard unit of assessment, values were first calculated within the litter and group mean values represent the mean of these individual litter values.
- Percentage pre-implantation loss was calculated as [(number of corpora lutea – number of implantations) / number of corpora lutea] * 100
- Percentage post-implantation loss was calculated as [(number of implantations – number of live fetuses) / number of implantations] * 100
- Sex ratio was calculated as % male fetuses = [number of male fetuses / total number of fetuses] * 100

Historical control data:

Normal range data for pre-natal studies were provided in annexes to the full study report.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- There were no clinical signs of toxicity detected at any dose level.
- In isolation one 300 mg/kg bw/day female exhibited noisy respiration and one 1000 mg/kg bw/day female had staining around the mouth on Day 5 of gestation.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths on the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- There were no adverse effects on body weight development at any dose level.
- Females treated with 300 or 1000 mg/kg bw/day had slightly lower overall body weight gains compared to controls. This resulted in overall body weight gains 7% and 2 % lower compared to controls respectively and this was considered to be due to biological variation rather than an effect of treatment. Therefore, these reductions would be associated with the slightly lower adjusted body weights for the gravid uterus weight for Day 20.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- There was no effect on food consumption during gestation at 100, 300 or 1000 mg/kg bw/day.

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

no effects observed

Description (incidence and severity):

- Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Gross pathological findings:

no effects observed

Description (incidence and severity):

- No macroscopic abnormalities were detected in treated females.
- In total there were three females found to be non-pregnant; one female each from the control, 300 and 1000 mg/kg bw/day dose groups.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

- There was no obvious effect of maternal treatment on litter data as assessed by numbers of implantations or pre and post-implantation losses at 100, 300 or 1000 mg/kg bw/day.

Early or late resorptions:

no effects observed

Description (incidence and severity):

- There was no obvious effect of maternal treatment on litter data as assessed by in utero offspring survival (as assessed by the mean numbers of early or late resorptions).

Dead fetuses:

no effects observed

Description (incidence and severity):

- There was no obvious effect of maternal treatment on litter data as assessed by live litter size.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

- With the exception of one female each from the control, 300 and 1000 mg/kg bw/day dose group, all females were found to be pregnant at necropsy.
- The assessments were therefore based on 23, 24, 23 and 23 females with live young at Day 20 of gestation at 0 (control), 100, 300 or 1000 mg/kg bw/day, respectively.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

- At 1000 mg/kg bw/day the mean placental weights were statistically higher (p < 0.05) in relation to controls. All individual placental weights were within the background control ranges; albeit at the higher end of this range.
- This increase in weights did not effect the total placenta weights for this dose level. As there were observations of large placentas within this dose group, it is considered to be the un-related to treatment and a result of biological variation.

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: no treatment-related effects at highest dose level tested.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

- Intergroup differences for mean fetal weights did not indicate any adverse effects of maternal treatment at 100, 300 or 1000 mg/kg bw/day.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

- There was no obvious effect of maternal treatment on litter data as assessed by live litter size.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

- There was no obvious effect of maternal treatment on litter data as assessed by sex ratio.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

- There was no obvious effect of maternal treatment on litter data as assessed by live litter size.
- Intergroup differences for mean litter weights did not indicate any adverse effects of maternal treatment at 100, 300 or 1000 mg/kg bw/day.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

- Neither the type, incidence nor distribution of external findings apparent for fetuses at Day 20 of gestation indicated an adverse effect of maternal treatment on fetal development at 100, 300 or 1000 mg/kg bw/day.
- External findings were confined to isolated offspring across all dose groups which were considered to be small or large in fetal or placenta size or both. Statistical analysis showed there were no inter-group differences for these findings and therefore they were considered to be of no toxicological significance.

Skeletal malformations:

no effects observed

Description (incidence and severity):

- Neither the type, incidence nor distribution of skeletal findings apparent for fetuses at Day 20 of gestation indicated an adverse effect of maternal treatment on fetal development at 100,
300 or 1000 mg/kg bw/day.
- Statistical analysis showed no significant intergroup differences at any dose level.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

- At 100, 300 and 1000 mg/kg bw/day statistically significant reduction in the incidence of increased renal pelvic cavitation (p < 0.05 or p < 0.01) was observed in relation to the controls.
- These statistical findings were predominantly due to the number of control litters showing a higher percentage of litters showing increased renal pelvic cavitation. The percentage incidence found in the treated groups were within the background control ranges and in isolation is considered to be due to normal biological variation and therefore, unrelated to treatment with the test item.
- There was a statistically significant reduction of the total number of fetuses affected from litters at 300 and 1000 mg/kg bw/day (p < 0.05). The statistical significance was influenced by the increased number of visceral findings from the control litters and therefore of no toxicological significance.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

other: no treatment-related changes detected in uterine parameters measured or embryofetal development at highest maternal dose tested.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

DISCUSSION

- The administration of test item was well tolerated. There were no effects of treatment with the test item at any dose level on maternal body weight development, clinical

observations or associated food consumption.

- Additionally, there were no macroscopic findings detected for any of the animals on the study. The uterine and fetal examination also did not reveal any effect of the treatment on uterine parameters examined or fetal development.

Conclusions:

The oral administration of test item to pregnant rats by oral gavage from gestation Day 3 to 19 at dose levels of 100, 300 and 1000 mg/kg bw/day, resulted in no treatment related effects. Therefore, the No Observed Effect Level (NOEL) for the pregnant female was considered to be 1000 mg/kg bw/day. No treatment-related changes were detected in the uterine parameters measured or on embryofetal development. Therefore the 'No Observed Effect Level' (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg bw/day.

Executive summary:

GUIDELINE

The study was designed to comply with US EPA Health Effects Test Guideline OPPTS 870.3700,‘Prenatal Developmental Toxicity Study’(August 1998), Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 Nousan No 8147, (24 November 2000), OECD Guidelines for Testing of Chemicals, No 414,‘Prenatal Developmental Toxicity Study’(adopted 22 January 2001) and Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

METHODS

The study was performed to investigate the effects of the test item on embryonic and fetal development, following repeated administration by gavage at dose levels 100, 300 and 1000 mg/kg bw/day to the Sprague-Dawley Crl:CD (SD) IGS BR strain rat during gestation, including the period of organogenesis. The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD (SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels 100, 300, and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (arachis oil) over the same treatment period to serve as a control. Clinical signs, body weight change, food and water consumptions were monitored during the study. All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

 

RESULTS

Mortality: There were no unscheduled deaths during the study.

Clinical observations: Clinical signs did not indicate any systemic effect of treatment at 100, 300 or 1000 mg/kg bw/day.

Body weight: There were no adverse effects on body weight development at any dose level.

Food consumption: There was no effect on food consumption during gestation at 100, 300 or 1000 mg/kg bw/day.

Water consumption: There was no visual indication of an effect on water consumption at any dose level.

Post-mortem studies: No macroscopic abnormalities were detected in treated females at 100, 300 or 1000 mg/kg bw/day.

Litter data and litter placental and fetal weights: The number of implantations, pre and post implantation loss, live litter size and sex ratio on Day 20 of gestation were considered to be unaffected by maternal treatment at 100, 300 or 1000 mg/kg bw/day. Mean fetal, placental and litter weights were also considered to have been unaffected by maternal treatment at 100, 300 or 1000 mg/kg bw/day.

Fetal examination: External examination of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 100, 300 or 1000 mg/kg bw/day. Findings at detailed skeletal and visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 100, 300 or 1000 mg/kg bw/day.

 

CONCLUSION

The oral administration of test item to pregnant rats by oral gavage from gestation Day 3 to 19 at dose levels of 100, 300 and 1000 mg/kg bw/day, resulted in no treatment related effects. Therefore, the No Observed Effect Level (NOEL) for the pregnant female was considered to be 1000 mg/kg bw/day. No treatment-related changes were detected in the uterine parameters measured or on embryofetal development. Therefore the 'No Observed Effect Level' (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Additional information

Screening

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 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.

 

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. 

Development toxicity

The key study was designed to comply with US EPA Health Effects Test Guideline OPPTS 870.3700,‘Prenatal Developmental Toxicity Study’(August 1998), Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 Nousan No 8147, (24 November 2000), OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001) and Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

The study was performed to investigate the effects of the test item on embryonic and fetal development, following repeated administration by gavage at dose levels 100, 300 and 1000 mg/kg bw/day to the Sprague-Dawley Crl:CD (SD) IGS BR strain rat during gestation, including the period of organogenesis. The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD (SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels 100, 300, and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (arachis oil) over the same treatment period to serve as a control. Clinical signs, body weight change, food and water consumptions were monitored during the study. All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

 

Mortality: There were no unscheduled deaths during the study.

Clinical observations: Clinical signs did not indicate any systemic effect of treatment at 100, 300 or 1000 mg/kg bw/day.

Body weight: There were no adverse effects on body weight development at any dose level.

Food consumption: There was no effect on food consumption during gestation at 100, 300 or 1000 mg/kg bw/day.

Water consumption: There was no visual indication of an effect on water consumption at any dose level.

Post-mortem studies: No macroscopic abnormalities were detected in treated females at 100, 300 or 1000 mg/kg bw/day.

Litter data and litter placental and fetal weights: The number of implantations, pre and post implantation loss, live litter size and sex ratio on Day 20 of gestation were considered to be unaffected by maternal treatment at 100, 300 or 1000 mg/kg bw/day. Mean fetal, placental and litter weights were also considered to have been unaffected by maternal treatment at 100, 300 or 1000 mg/kg bw/day.

Fetal examination: External examination of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 100, 300 or 1000 mg/kg bw/day. Findings at detailed skeletal and visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 100, 300 or 1000 mg/kg bw/day.

 

The oral administration of test item to pregnant rats by oral gavage from gestation Day 3 to 19 at dose levels of 100, 300 and 1000 mg/kg bw/day, resulted in no treatment related effects. Therefore, the No Observed Effect Level (NOEL) for the pregnant female was considered to be 1000 mg/kg bw/day. No treatment-related changes were detected in the uterine parameters measured or on embryofetal development. Therefore the 'No Observed Effect Level' (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg bw/day.

Justification for classification or non-classification

No treatment-related effects on P0 or F1 generations were reported at dose levels of 100, 300 and 1000 mg/kg bw (OECD 422 plus OECD 414) and classification for reproductive or developmental toxicity is therefore unnecessary under the criteria of Regulation (EC) No 1272/2008 and subsequent amendments.