Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 09 July 2015 First day of treatment:27 August 2015 Final day of necropsy: 10 October 2015 Experimental Completion Date: 17 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han™:RccHan™:WIST strain rats
Details on species / strain selection:
male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for seven days during which time their health status was assessed. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 313 to 358g, the females weighed 175 to 225g, and were approximately twelve weeks old.
Sex:
male/female
Details on test animals and environmental conditions:
Animal Care and HusbandryInitially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). DuringThe pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. 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 5. 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 the final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment were 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 computerized 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; there were no deviations from the range for temperature and deviations from the range for relative humidity were considered not to have affected the purpose or integrity of the study (see deviations from Study Plan).The animals were randomly allocated to treatment groups using a stratified body weight randomization 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 randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in dried Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least eighteen days. Formulations were therefore prepared in batches of up to 14 days daily doses and stored at approximately 4 °C in the dark.
Details on mating procedure:
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 estrus 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 ParturitionEach 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 pairingii.Date of matingiii.Date and time of observed start of parturitioniv.Date and time of observed completion of parturition
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test item formulation were taken and analyzed for concentration of test substance at Envigo Research Limited, Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in the attached Annex 2. The results indicate that the prepared formulations were within 98% to 102% of the nominal concentration and were considered to acceptable for the purpose of this study.
Duration of treatment / exposure:
The study was performed according to the study plan and was designed to screen for potential adverse effects on reproduction, including offspring development, by repeated oral administration to the Wistar Han™:RccHan™:WIST strain rat for approximately six weeks
Frequency of treatment:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe.
Details on study schedule:
Dose AdministrationAnimals were allocated to treatment groups as followsTreatment GroupDose Level (mg/kg bw/day)Treatment Volume (mL/kg)Concentration (mg/mL)Animal NumbersMaleFemaleControl04012 (1-12)12 (13-24)Low20045012 (25-36)12 (37-48)Intermediate400410012 (49-60)12 (61-72)High800420012 (73-84)12 (85-96)The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group.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 4 mL/kg of dried 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.Chronological Sequence of Studyi.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). The first day of dosing was designated as Day 1 of the study.ii.On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.iii.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.iv.Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.v.The male dose groups were killed and examined macroscopically on Day 43.vi.At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically.
Doses / concentrationsopen allclose all
Dose / conc.:
800 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control animals, they were treated in an identical manner with 4 mL/kg of dried Arachis oil BP
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
The study was performed according to the study plan and was designed to screen for potential adverse effects on reproduction, including offspring development, by repeated oral administration to the Wistar Han™:RccHan™:WIST strain rat for approximately six weeks at dose levels of 200, 400 and 800 mg/kg bw/day. A control group was dosed with vehicle alone (dried Arachis Oil BP) over the same treatment period.The dose levels were chosen based on the results of previous toxicity work including a Twenty-Eight Day Rat Toxicity Study (Harlan Laboratories Report No.: 41200575). 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.The study was performed between 09 July 2015 and 17 May 2016. The in-life phase of the study was conducted between 27 August 2015 (first day of treatment) and 10 October 2015 (final day of necropsy).JustificationThe rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
General Observations/MeasurementsClinical ObservationsAll animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (see Deviations from Study Plan). All observations were recorded.Body WeightIndividual 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 and 4 post partum.Normal range data for body weight changes in pregnant and lactating females are shown in the attached Annex 6.Food ConsumptionDuring the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. 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 during the lactation period (Days 1-4).Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated during gestation and lactation.Normal range data for pregnant and lactating females are presented in the attached Annex 6.Water ConsumptionWater intake was observed daily by visual inspection of water bottles for any overt changes.Reproductive PerformanceNormal range data for reproductive parameters and offspring are presented in the attached Annex 6 and Annex 7.Organ WeightsThe epididymides and testes were removed from terminal kill adult males dissected free from fat and weighed before fixation.Normal ranges for organ weights are given in the attached Annex 8.HistopathologySamples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:Coagulating glandProstateEpididymides *Seminal vesiclesOvaries Testes *Mammary gland (females only)Uterus/CervixPituitary VaginaAll tissues were dispatched to the histology processing Test Site (Envigo CRS Limited, Eye, Suffolk) for processing (Principal Investigator: J Schofield). The tissues from control and 800 mg/kg bw/day dose group animals, the single adult animal that died during the study, and any animals which 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. In addition, sections of testes from all control and 800 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.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* preserved in Modified Davidsons fluid 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.PathologyMicroscopic examination was conducted by the Study Pathologist (Wendy Henderson). A peer review of the findings observed was conducted by Peter Millar at the histopathology peer review test site (Peter Millar Associates Ltd., Edinburgh). A complete histopathology phase report is presented in Annex 1 and represents the consensus view of both pathologists.
Litter observations:
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.For each litter the following was recorded:i.Number of offspring bornii.Number of offspring alive recorded daily and reported on Days 1 and 4 post partumiii.Sex of offspring on Days 1 and 4 post partumiv.Clinical condition of offspring from birth to Day 5 post partumv.Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)Physical DevelopmentAll live offspring were assessed for surface righting reflex on Day 1 post partum.
Postmortem examinations (parental animals):
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 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 on or after Day 25 post coitum.For all 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). The corpora lutea were also counted.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.
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:
Data EvaluationData were processed to give summary incidence or group mean values and standard deviations where appropriate. All data were summarized in tabular form.Data shown in the appendices are frequently rounded values for presentation purposes. Group mean values are generally calculated using non-rounded values therefore is it not always possible to calculate the exact group values from the individual values presented in the appendices.For body weights and food consumptions during gestation, group mean values were calculated using data from females which were observed to give birth to offspring.For body weights and food consumptions during lactation, group mean values were calculated using data from females with live young at Day 4 of lactation.
Reproductive indices:
Reproductive IndicesMating Performance and FertilityThe following parameters were calculated from the individual data during the mating period of the parental generation:i.Pre-coital IntervalCalculated as the time elapsing between initial pairing and the observation of positive evidence of mating.ii.Fertility IndicesFor each group the following were calculated:Mating Index (%) = Number of animals mated divided by Number of animals paired x 100 Pregnancy Index (%) = Number of pregnant females divided by Number of animals mated x 100 Gestation and Parturition DataThe following parameters were calculated from individual data during the gestation and parturition period of the parental generation:i.Gestation LengthCalculated as the number of days of gestation including the day for observation of mating and the start of parturition.ii.Parturition IndexThe following was calculated for each group:Parturition Index (%) = Number of females delivering live offspring divided by Number of pregnant females x 100
Offspring viability indices:
Litter ResponsesThe 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 Day 4 of age.i.Implantation Losses (%)Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows: Pre–implantation loss (%) = Number of corpora lutea - Number of implantation sites divided by Number of corpora lutea x100Post–implantation loss (%)= Number of implantation sites - Total number of offspring born divided by Number of implantation sites x100 ii.Live Birth and Viability IndicesThe following indices were calculated for each litter as follows:Live Birth Index (%) = Number of offspring alive on Day 1 divided by Number of offspring born x 100 Viability Index (%) = Number of offspring alive on Day 4 divided by Number of offspring alive on Day1 x 100 iii.Sex Ratio (% males)Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:Number of male offspring divided by Total number of offspring x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
A summary incidence of daily clinical observations is given in the attached Table 2. Individual data is presented in the attached Appendix 1.Clinical signs for surviving animals were generally restricted to increased post-dosing salivation in all animals (both sexes) at 200, 400 and 800 mg/kg bw/day. At 800 and 400 mg/kg bw/day, this clinical sign was first observed during the firstweek of treatment with the incidence tending to increase as the study progressed. At 200 mg/kg bw/day, the clinical sign was not apparent until Day 20 (males) and Day 22 (females) and the incidence of this sign tended to be sporadic and less consistent than observed at the higher dosages. Increased post-dosing salivation is frequently observed when unpalatable/slightly irritant test items are dosed via the oral gavage route and is generally considered to be of little toxicologicalsignificance and not to indicate any systemic effect of treatment.The incidence and distribution of the other clinical signs observed throughout the study did not indicate any obvious effect of treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no mortalities on the study that were considered to be related to treatment.At 800 mg/kg bw/day, one female was killed in extremis after showing loss of righting reflex, piloerection, lethargy, apparent hypothermia (cold to touch) and pallor of the extremities on Day 40 (around the time of expected parturition). Necropsy revealed a “twisted” left uterine horn that appeared to have prevented the animal successfully giving birth; this horn contained one dead fetus and was filled with red fluid. This was considered to be the underlying reason for the decline in the condition of the animal and, as such, this death was considered to be incidental and unrelated to treatment with the Test Item. Other necropsy findings revealed pale brain, enlarge adrenals, pallor of the liver and red discoloration of the lungs; some of these findings may have been a result of suspected blood loss in the left uterine horn.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean weekly body weights and standard deviations are given in the attached Table 3 and are presented graphically in the attached Figure 1 and Figure 2. Group mean weekly body weight gains and standard deviations are given in the attached Table 4 (statistically significant differences are indicated). Individual data are given in the attached Appendix 2 and Appendix 3.At 800 mg/kg bw/day, body weight gain of males was lower than control for the first four weeks of the study, with differences attaining statistical significance, and overall body weight gain to Day 43 was also lower than control.There was no obvious effect of treatment on body weight gain for males at 200 or 400 mg/kg bw/day.There was no obvious adverse effect of treatment on body weight gain for females during the pre-pairing, gestation and lactation phases of the study at 200, 400 or 800 mg/kg bw/day.For females at 400 and 800 mg/kg bw/day, body weight gain was statistically significantly higher than control during the first week of the study which resulted in a slightly higher overall body weight gain compared to control for the two week pre-pairing period. Females in this study design are not in a steep growth curve and the differences observed were considered most likely to reflect normal biological variation.At 800 mg/kg bw/day, body weight gain was slightly lower than control during the final week of gestation although differences did not attain statistical significance. The differences in weight gain were considered to reflect a lower contribution to maternal weight by the litter rather than any effect on the parental female, particularly as maternal body weight at Day 1 of lactation was similar to control.At 400 and 800 mg/kg bw/day, maternal body weight gain during Days 1 to 4 of lactation was lower than control although differences failed to attain statistical significance. Body weight gain of the females at this particular phase of the study is very variable and the differences observed were considered most likely to reflect normal biological variation.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean food consumptions are given in the attached Table 5 and are presented graphically in Figure 3 and Figure 4. Individual values for females during gestation and lactation are presented in Appendix 4. Food efficiency for males and for females during the pre-mating phase is given in Table 6.At 800 mg/kg bw/day, food consumption and food conversion efficiency for males was lower than control during the two week pre-pairing period. There was no obvious effect of treatment on food consumption during the subsequent post-pairing phase of the study.At 200 and 400 mg/kg bw/day, food consumption and food conversion efficiency for males was considered to be similar to control throughout the study.There was no obvious effect of treatment on food consumption and food conversion efficiency for females during the pre-pairing phase of the study or on food consumption during the gestation and lactation phases of the study at 200, 400 or 800 mg/kg bw/day.For females at 400 and 800 mg/kg bw/day, food conversion efficiency was slightly superior to control during the first week of the study but these differences showed no dosage relationship and were considered to reflect normal biological variation.Water ConsumptionDaily visual inspection of water bottles did not reveal any overt intergroup differences.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Group mean absolute and body weight-relative male reproductive organ weights and standard deviations for test and control group animals are presented in the attached Table 15 (statistically significant differences are indicated). Individual data are given in Appendix 13 and Appendix 14.No statistically significant differences were apparent for male reproductive organ weights (testes and epididymides) compared to control at 200, 400 and 800 mg/kg bw/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
A summary incidence of necropsy findings for offspring and adults is given in the attached Tables 13 and 14. Individual data are given in Appendices 11 and 12. OffspringThe necropsy findings observed for both decedent offspring and those terminated on Day 5 of age were typical for the age observed and did not indicate any adverse effect of maternal treatment on offspring development at 200, 400 and 800 mg/kg bw/day.AdultsNeither the type, incidence nor distribution of necropsy findings for adult animals surviving to scheduled termination indicated any adverse effects of treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A complete histopathology phase report is presented in the attached Annex 1.At 800 mg/kg bw/day, there was an increase in the appearance of vacuolated/pale cells in the pituitary compared to control. These vacuolated cells probably reflect hypertrophy of thyroid-stimulating hormone-producing cells (thyrotrophs), a commonfinding following administration of liver enzyme inducers where the underlying mechanism is considered to be increased hepatic clearance of thyroid hormones followed by a compensatory increase in the pituitary secretion of TSH (Capen et al., 2002, Zabka et al., 2011). As this correlates with changes in the liver and thyroid gland noted in the previous 28 day rat toxicity study (Harlan Laboratories Report No.: 41200575) carried out at this laboratory it is considered that this is consistent and the most likely explanation for the change.
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive performance:
no effects observed
Description (incidence and severity):
MatingA summary of adult performance is presented in the attached Table 1. A summary incidence for mating performance is presented in Table 7. Individual data are given in Appendix 5.There was no effect of treatment on mating performance with all animals mating within the first four days of pairing.FertilityA summary of adult performance is presented in the attached Table 1. Group values for fertility, litter data and implantation losses are given in Tables 7, 8 and 9. Individual data are given in Appendices 5 to 7.Although all animals showed evidence of mating, a total of 0, 3, 1 and 2 females at 0 (Control), 200, 400 and 800 mg/kg bw/day failed to attain pregnancy; the intergroup distribution of non-pregnancy did not indicate any association with treatment.Gestation LengthA summary of gestation lengths is presented in the attached Table 7. Individual lengths are given in Appendix 5.Gestation length at 200, 400 and 800 mg/kg bw/day were within the normally expected range and appeared unaffected by treatment.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks:
The No Observed Adverse Effect Level (NOAEL) for adult toxicity for this OECD 421 study is considered to be 800 mg/kg bw/day
Effect level:
800 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
neuropathology
reproductive function (oestrous cycle)
reproductive performance
Remarks on result:
other: The No Observed Adverse Effect Level (NOAEL) for adult toxicity for this OECD 421 study is considered to be 800 mg/kg bw/day

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 800 mg/kg bw/day, there was a higher overall incidence of clinical signs observed for the offspring to termination on Day 5 at this dosage compared to control.At 200 and 400 mg/kg bw/day there was no obvious effect on the incidence of clinical signs
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 800 mg/kg bw/day, initial body weight of offspring on Day 1 of age and subsequent body weight gain to Day 4 was slightly lower than control; however although both sexes were affected only values for females attained statistical significance. The lower body weight, in combination with the smaller litter size, resulted in lower litter weights at Day 1 and, to a greater extent, Day 4 of ageAt 200 and 400 mg/kg bw/day there was no obvious effect of maternal treatment on mean body weight on Day 1 of age or on the incidence of clinical signs and subsequent body weight gain to Day 4 of age.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
The necropsy findings observed for both decedent offspring and those terminated on Day 5 of age were typical for the age observed and did not indicate any adverse effect of maternal treatment on offspring development at 200, 400 and 800 mg/kg bw/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The necropsy findings observed for both decedent offspring and those terminated on Day 5 of age were typical for the age observed and did not indicate any adverse effect of maternal treatment on offspring development at 200, 400 and 800 mg/kg bw/day.

Details on results (F1)

Litter ResponsesLitter responses are generally assessed using the 12, 9, 11 and 8 litters reared to Day 4 of age at 0 (control), 200, 400 and 800 mg/kg bw/day respectively. At 800 mg/kg bw/day, one pregnant female was killed in extremis around the time of parturition (this death was not related to treatment) and one female showed total litter loss post partum.Offspring Litter Size, Sex Ratio and ViabilityGroup mean corpora lutea and implantation counts, litter size, implantation losses, survival indices and sex ratio are given in Tables 8 to 10. Individual data are given in Appendices 6 to 8.At 800 mg/kg bw/day, there was no obvious effect of maternal treatment on corpora lutea count, pre-implantation loss or the number of implantations. Subsequent mean post- implantation loss was slightly higher than control but this did not attain statistical significance or result in any clear difference in the number of offspring born or alive on Day 1 of age. However, although mean live birth index was similar to control, subsequentviability of the offspring was inferior to control resulting in lower litter size at Day 4. While these differences from control did not attain statistical significance, they have to be seen in the context of a further female at this dosage showing total litter loss post partum. There was considered to be no effect of treatment on the corpora lutea count, pre- implantation loss, numbers of implantations, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to Day 4 of age at 200 or 400 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 200, 400 or 800 mg/kg bw/day.Offspring Growth and DevelopmentGroup mean values for total litter weights, offspring body weights and body weight changes, surface righting reflex and a summary incidence of clinical signs are given in Table 8, Table 11 and Table 12. Individual values and observations are given in Appendices 6, 9 and 10.At 800 mg/kg bw/day, initial body weight of offspring on Day 1 of age and subsequent body weight gain to Day 4 was slightly lower than control; however although both sexes were affected only values for females attained statistical significance. The lower body weight, in combination with the smaller litter size, resulted in lower litter weights at Day 1 and, to a greater extent, Day 4 of age. Additionally, the success rate at assessment of surface right for the offspring at Day 1 of age was lower than control and there was a higher overall incidence of clinical signs observed for the offspring to termination on Day 5 at this dosage compared to control.At 200 and 400 mg/kg bw/day there was no obvious effect of maternal treatment on mean body weight on Day 1 of age or on the incidence of clinical signs and subsequent body weight gain to Day 4 of age. The success rate at assessment of surface righting for the offspring at Day 1 of age was lower than control but these differences were considered most likely to represent normal biological variation rather than any underlying effect of treatment.

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
gross pathology
Remarks on result:
other: No Observed Effect Level (NOEL) for reproduction, including the growth, development and survival of the offspring, was considered to 400 mg/kg bw/day

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
400 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

The No Observed Adverse Effect Level (NOAEL) for adult toxicity for this OECD 421 study is considered to be 800 mg/kg bw/day and the No Observed Effect Level (NOEL) for reproduction, including the growth, development and survival of the offspring, was considered to 400 mg/kg bw/day.

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for adult toxicity for this OECD 421 study is considered to be 800 mg/kg bw/day and the No Observed Effect Level (NOEL) for reproduction, including the growth, development and survival of the offspring, was considered to 400 mg/kg bw/day.
Executive summary:

Introduction

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).

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

Methods

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 (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 200, 400 and 800 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (dried Arachis oil BP) over the same treatment period.

Clinical signs, body weight change, dietary intake 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 5 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 surface righting reflex.

Adult males were terminated on Day 43, and all surviving littering females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

 

Results

Adult Responses Mortality

At 800 mg/kg bw/day, one female was killed in extremis after showing adverse clinical signs around the time of expected parturition. Necropsy revealed a “twisted” left uterine horn that appeared to have prevented the animal successfully giving birth; this horn contained one dead fetus and was filled with red fluid. This was considered to be the underlying reason for the

decline in the condition of the animal and, as such, this death was considered to be incidental and unrelated to treatment with the Test Item.

 

Clinical Observations

Clinical signs for surviving animals were generally restricted to increased post-dosing salivation in all animals (both sexes at 200, 400 and 800 mg/kg bw/day). At 800 and 400 mg/kg bw/day, this clinical sign was first observed during the first week of treatment

with the incidence tending to increase as the study progressed. At 200 mg/kg bw/day, the clinical sign was not apparent until Day 20 (males) and Day 22 (females) and the incidence of this sign tended to be sporadic and less consistent than observed at the higher dosages.

 

Body Weight

At 800 mg/kg bw/day, body weight gain of males was lower than control for the first four weeks of the study, with differences attaining statistical significance, and overall body weight gain to Day 43 was also lower than control.

There was no obvious effect of treatment on body weight gain for males at 200 or 400 mg/kg bw/day.

There was no obvious adverse effect of treatment on body weight gain for females during the pre-pairing, gestation and lactation phases of the study at 200, 400 or 800 mg/kg bw/day.

Food Consumption and Food Conversion Efficiency

At 800 mg/kg bw/day, food consumption and food conversion efficiency for males was lower than control during the two week pre-pairing period. At 200 and 400 mg/kg bw/day, food consumption and food conversion efficiency for males was considered to be similar to control throughout the study.

There was no obvious effect of treatment on food consumption and food conversion efficiency for females during the pre-pairing phase of the study or on food consumption during the gestation and lactation phases of the study at 200, 400 or 800 mg/kg bw/day.

 

Water Consumption

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

Reproductive Performance Mating

There was no effect of treatment on mating performance with all animals mating within the first four days of pairing.

Fertility

Intergroup distribution of pregnancy rate did not indicate any association with treatment at 200, 400 and 800 mg/kg bw/day.

Gestation Length

Gestation length at 200, 400 and 800 mg/kg bw/day appeared unaffected by treatment.

 

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

At 800 mg/kg bw/day, mean post-implantation loss was slightly higher than control but this did not attain statistical significance or result in any clear difference in the number of offspring born or alive on Day 1 of age. Subsequent viability of the offspring was inferior to control resulting in lower litter size at Day 4 and one female showed total litter loss post partum.

There was considered to be no effect of treatment on the corpora lutea count, pre- implantation loss, numbers of implantations, post-implantation loss, litter size and sex ratio at birth/Day 1 and Day 4 of age at 200 or 400 mg/kg bw/day.

 

Offspring Growth and Development

At 800 mg/kg bw/day, initial body weight of offspring on Day 1 of age and subsequent body weight gain to Day 4 was slightly lower than control; the lower body weight, in combination with the smaller litter size, resulted in lower litter weights at Day 1 and Day 4 of age. The success rate at assessment of surface right for the offspring at Day 1 of age was lower than control and there was a higher overall incidence of clinical signs observed for the offspring at this dosage compared to control.

At 200 and 400 mg/kg bw/day there was no obvious effect of maternal treatment on mean body weight on Day 1 of age or on the incidence of clinical signs and subsequent body weight gain to Day 4 of age. The success rate at assessment of surface righting for the offspring at Day 1 of age was lower than control but these differences were considered most likely to represent normal biological variation rather than any underlying effect of treatment.

 

Offspring Observations Pathology

Necropsy

Offspring

The necropsy findings observed for both decedent offspring and those terminated on Day 5 of age were typical for the age observed and did not indicate any adverse effect of maternal treatment on offspring development at 200, 400 and 800 mg/kg bw/day.

 Adults

Neither the type, incidence nor distribution of necropsy findings for adult animals surviving to scheduled termination indicated any adverse effects of treatment.

 

Organ Weights

No statistically significant differences were apparent for male reproductive organ weights (testes and epididymides) compared to control at 200, 400 and 800 mg/kg bw/day.

Histopathology

At 800 mg/kg bw/day, there was an increase in the appearance of vacuolated/pale cells in the pituitary compared to control. These vacuolated cells were consistent with findings observed in a previous Twenty-Eight Day Rat Toxicity Study (Harlan Laboratories Report No.: 41200575) and were considered to reflect increased hepatic clearance of thyroid hormones followed by a compensatory increase in the pituitary secretion of TSH. As the underlying thyroid changes are considered to be species specific with no safety relevance for humans and adaptive in nature, these pituitary changes observed in this OECD 421 study were considered to be of no toxicological relevance to man.

Conclusion

The No Observed Adverse Effect Level (NOAEL) for adult toxicity for this OECD 421 study is considered to be 800 mg/kg bw/day and the No Observed Effect Level (NOEL) for reproduction, including the growth, development and survival of the offspring, was considered to be 400 mg/kg bw/day.