Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral (gavage) administration of the substance to male and female Wistar rats at dose levels of 0 (control), 60, 250, or 500 mg/kg body weight/day for up to 8 weeks (including a two week maturation phase, pairing, gestation, and early lactation for females) did not result in any test article-related effects on fertility parameters (i.e., mean number of corpora lutea and implantation sites, mating indices, post-implantation losses, and sex ratio) at the highest dose tested.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 11 to November 25, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 421 and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
High dose level was reduced from 1000 mg/kg bw/day to 500 mg/kg bw/day on Day 3 due to toxicity
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on July 19-21, 2011/ signed on August 31, 2011)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Wister HanTM: RccHanTM: WIST strain
Details on species / strain selection:
The 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.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: Approximately 12 weeks
- Weight at study initiation: (P) Males: 326-360 g; Females: 189-221 g
- Housing: Animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a 1 male: 1 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.
- Diet (e.g. ad libitum): A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK), ad libitum
- Water (e.g. ad libitum): Mains drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 55 ± 15%
- Air changes (per hr): At least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. Formulations were prepared based on available stability data either daily, weekly or fortnightly and stored at approximately 4 ºC in the dark under nitrogen.

VEHICLE
- Concentration in vehicle: 0, 15, 62.5, 250*/125 mg/mL (* Following the early termination of three high dose level animals on Day 3 of the study the high dose level was reduced from 1000 mg/kg bw/day to 500 mg/kg bw/day. Treatment was discontinued for the high dose animals on Day 4 of the study and recommenced on Day 5 at the adjusted dose level. As for the majority of the duration of this study high dose level of 500 mg/kg bw/day was used it is reported as such.)
- Amount of vehicle (if gavage): 4 mL/kg bw/day
Details on mating procedure:
- M/F ratio per cage: 1 male: 1 female basis
- Length of cohabitation: For a maximum of 14 days
- Proof of pregnancy: Vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy.
- After successful mating each pregnant female was caged (how): Mated females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test item formulation were taken and analysed for concentration of test item. The results indicate that the prepared formulations were within ± 2% of the nominal concentration.
Duration of treatment / exposure:
Up to 8 weeks (including a two week maturation phase, pairing, gestation, and early lactation for females)
Male: Days 1-43 (mating on Days 15/16)
Female: Days 1 to post partum Day 5 (mating on Days 15/16)

After the initial decline of animal health, the high dose level was reduced from 1000 mg/kg bw/day to 500 mg/kg bw/day following agreement with the Sponsor. Animals in the high dose group were not dosed on Day 4 of the study, and re-started dosing on Day 5 at the lower dose of 500 mg/kg bw/day. Afterwards, animals at 500 mg/kg bw/day level showed recovery of the previously observed clinical signs.
Frequency of treatment:
Daily
Details on study schedule:
Chronological Sequence of Study:

i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for 1000 mg/kg bw/day animals which were not dosed on Day 4 prior to a dose reduction to 500 mg/kg bw/day and females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.

ii) On Day 15 (Treatment Groups: Control, Low and Intermediate) or Day 16 of the study (Treatment Group: High), 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. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.

v) The male dose groups were killed and examined macroscopically on Day 43 of the study.

vi) At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically.
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
initially 1000 mg/kg bw/day, reduced to 500 mg/kg bw/day on Day 5
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of previous toxicity work, i.e., the oral 28-day repeat dose toxicity study in Sprague-Dawley rats (Study Number FIR/062).
- Rationale for animal assignment (if not random): The animals were allocated to dose groups using a randomisation procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups.
Positive control:
None.
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one and five hours after dosing, during the working week (one hour after dosing at weekends (except for females during parturition where applicable).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- During the pre-mating 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-mating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

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

OTHERS:
Pregnancy and Parturition: Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Number of offspring born (both live and stillborn), Number of offspring alive recorded daily and reported on Days 1 and 4 post partum, Sex of offspring on Days 1 and 4 post partum, Clinical condition of offspring from birth to Day 5 post partum, Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)
All live offspring were assessed for surface righting reflex on Day 1 post partum.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43.
- Maternal animals: Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

GROSS NECROPSY
- Gross necropsy consisted of a full external and internal examination. For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded.
- All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ Weights: The epididymides and testes were removed from terminal kill adult males dissected free from fat and weighed before fixation. No other organs were weighed.
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated: Coagulating gland, Prostate, Epididymides**, Seminal vesicles, Ovaries, Testes**, Mammary gland (females only), Uterus/Cervix, Pituitary, Vagina.
** = preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later.

The tissues from control and high dose animals, any animals dying during the study, 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 Haematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes and epididymides from all control and 500 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Postmortem examinations (offspring):
SACRIFICE
- Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. These animals were subjected to postmortem examinations (macroscopic examination) on Day 5 post partum.

GROSS NECROPSY
- Gross necropsy consisted of a full external and internal examination.
Statistics:
The following parameters were subjected to statistical analysis:
Body weight and body weight change
Food consumption for females during gestation and lactation
Pre-coital interval and gestation length
Litter size and litter weights
Sex ratio
Corpora lutea and implantation sites
Implantation losses and viability indices
Offspring body weight and body weight change
Offspring surface righting
Adult absolute and body weight-relative organ weights (Males)
See Section "Any other information on materials and methods incl. tables" for more information on statistical procedures.
Reproductive indices:
Mating Performance and Fertility:
The following parameters were calculated from the individual data during the mating period of the parental generation:
i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii) Fertility Indices
For each group the following were calculated:
Mating Index (%) = (number of animals mated/number of animals paired) x 100
Pregnancy Index (%) = (number of pregnant females/number of animals mated) x 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
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii) Parturition Index
The following was calculated for each group:
Parturition Index (%) = (number of females delivering live offspring/number of pregnant females) x 100
Offspring viability indices:
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 5 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)/number of corpora lutea] x100
% post–implantation loss = [(number of implantation sites - total number of offspring born)/number of implantation sites] x 100
ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (number of offspring alive on Day 1/number of offspring born) x 100
Viability Index (%) = (number of offspring alive on Day 4/ number of offspring alive on Day 1) 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/total number of offspring) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Two females and one male treated with 1000 mg/kg bw/day were killed in extremis on Day 3 of treatment due to clinical signs that exceeded the severity limits for this type of study. The remaining animals of either sex treated with 1000 mg/kg bw/day showed either ataxia, lethargy, noisy respiration, pilo-erection or prostration during the first three days of treatment only and subsequently the dose level was reduced. Following the reduction of the high dose level (decreased to 500 mg/kg bw/day starting on Day 5 of the study after one day non-dosed), animals of either sex did not show any toxicologically significant clinical observations. The only clinical sign at 250 mg/kg bw/day was salivation in males, which was considered to be of no toxicological importance given that this is often observed following administration of unpalatable formulations. There were no clinical observations in animals at 60 mg/kg bw/day.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two females and one male when treated at 1000 mg/kg bw/day were killed in extremis on Day 3 prior to the reduction of this dose level to 500 mg/kg bw/day. There were no further unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight reductions were evident for the 1000 mg/kg bw/day animals of either sex during the first week of the study. Following the reduction of the high dose level to 500 mg/kg bw/day body weight there was recovery and body weight development was comparable to controls. Overall bodyweight gain was reduced in the high dose group compared to control in both sexes.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption and food efficiency were reduced for the 1000 mg/kg bw/day animals when compared to the controls. Recovery was evident thereafter following the reduction of the high dose level to 500 mg/kg bw/day. There was no body weight, food consumption, or food efficiency changes at 250 or 60 mg/kg bw/day.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Food consumption and food efficiency were reduced for the 1000 mg/kg bw/day animals when compared to the controls. Recovery was evident thereafter following the reduction of the high dose level to 500 mg/kg bw/day. There was no body weight, food consumption, or food efficiency changes at 250 or 60 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspections of water bottles did not reveal any overt changes.
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
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No toxicologically significant microscopic abnormalities were detected for terminal kill adults.
There were adverse abnormalities detected during microscopic examinations in the three high dose animals killed in extremis at the beginning of the study. Ulcerations and inflammation with submucosal edema or focal hyperkeratosis were recorded in the forestomach of those animals.
There were no treatment related abnormalities recorded in reproductive organs during microscopic examinations.
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis. All findings recorded were within the range of normal background alteration.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating: There were no treatment related effects detected on mating performance in treated animals when compared to controls. All animals mated within the first five days of pairing (i.e. at the first oestrus opportunity). One control female did not mate due to no apparent vaginal opening. In isolation and as this finding occurred in a control female only, it is considered fortuitous. There were no treatment related effects detected in corpora lutea and implantation counts or sex ratio in any treated groups when compared to control females/litters.

Fertility: In total 8, 9, 10 and 6 females from the control, 60, 250, and 500 mg/kg bw/day dose groups respectively, gave birth to a live litter and successfully reared young to Day 5 of age. There were no treatment related effects detected on fertility in treated animals when compared to controls. Two females treated at the high dose level and one control female did not achieve pregnancy following evidence of mating. No correlating histopathology was evident in the female reproductive organs, however, the male partners for these females showed testicular tubular atrophy and/or aspermia or oligospermia in the epididymides. Both microscopic findings could be correlated macroscopically to small and flaccid testes and small epididymides. In addition, in one high dose male multinucleated spermatid giant cells were recorded. All these findings are naturally occurring background changes in rats and therefore, are considered not to be related to treatment.

Gestation Lengths: There were no treatment related effects detected on gestation length. There was a slightly longer gestation length evident at 500 mg/kg bw/day in comparison to control females. In the control group the majority of females showed gestation lengths of 22 ½ days whilst at 500 mg/kg bw/day the majority of females showed gestation lengths of 23 ½ days, however, all the individual values were within the normal ranges for rats of the strain and age used in this study (21.8 to 23.5). In the view of this fact the effect is considered not related to treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no offspring clinical observations that were attributable to the test item.
Description (incidence and severity):
On Days 1 and 4 post partum, the number of live offspring per litter was slightly lower at the 500 mg/kg bw/day dose level compared to controls; however, this finding was not statistically significant. This was partially reflected by a statistically significant lower live birth index at the 500 mg/kg bw/day dose level compared to controls (P<0.05). By Day 4 post partum, 3 of the 6 litters had lost more than 3 offspring after birth at the 500 mg/kg bw/day dose level. In contrast, there were no litters in the controls or other treatment groups that lost more than 1 offspring in the same time period.
Consistent with the observed slight decrease in litter size, the number of offspring at the 500 mg/kg bw/day dose level that were noted to be found dead or missing was higher than those reported at other dose levels. Incidence of clinical signs was slightly higher in litters from females treated with 500 mg/kg bw/day when compared to control litters. The number of pups that were reported to be “found dead” or “missing” was slightly higher at the 500 mg/kg bw/day dose level compared to the others, with a reported incidence of 2, 5, 2 and 12 for the control, 60, 250 and 500 mg/kg bw/day dose groups, respectively. These findings are consistent with the slightly smaller post partum litter sizes reported at the 500 mg/kg bw/day dose level. There were no other clinical signs that were indicative of a treatment related effect.
No treatment related effects were evident in sex ratio.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects in total litter weights, offspring body weight and body weight changes.
Mean litter weight values were slightly reduced in 500 mg/kg bw/day litters due to smaller litter size values recorded in this treatment group. In view of the fact that mean offspring weight values at Day 1 and 4 assessments were comparable to controls this finding is considered not related to treatment.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No obvious adverse macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.
Histopathological findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Surface righting mean values appeared slightly reduced in 500 mg/kg bw/day litters when compared to control values. This effect did not reach statistical significance and a true dose related response was not evident. All values were also within the normal ranges for the strain used on this study, therefore, the intergroup difference was considered not to be treatment related.
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect observed
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

None

Conclusions:
Under the test conditions, the NOAEL for adult toxicity is 250 mg/kg bw/day and NOAEL for reproductive/developmental toxicity is 250 mg/kg bw/day in rats.
Executive summary:

In a Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 421 and in compliance with GLP, test item was administered to groups of Wister HanTM: RccHanTM: WIST strain rats (10/sex/dose) at 0, 60, 250, 1000/500 mg/kg bw/day by oral (gavage) for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 60, 250 and 500 mg/kg bw/day (Male: Days 1-43 (mating on Days 15/16); Female: Days 1 to post partum Day 5 (mating on Days 15/16)). A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Pairing of animals within each dose group was undertaken on a 1 male: 1 female basis within each treatment group following at least fourteen days of treatment, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. All animals were subjected to a gross necropsy examination, selected organs were weighed and histopathological evaluation of selected tissues was performed. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken.

 

Two females and one male when treated at 1000 mg/kg bw/day were killed in extremis on Day 3 prior to the reduction of this dose level to 500 mg/kg bw/day. The remaining animals of either sex treated with 1000 mg/kg bw/day showed either ataxia, lethargy, noisy respiration, pilo-erection or prostration during the first three days of treatment only. There were no further unscheduled deaths. Following the reduction of the high dose level (decreased to 500 mg/kg bw/day starting on Day 5 of the study after one day non-dosed), animals of either sex did not show any toxicologically significant clinical observations. Body weight reductions were evident for the 1000 mg/kg bw/day animals of either sex during the first week of the study. Following the reduction of the high dose level to 500 mg/kg bw/day body weight development was comparable to controls. Overall bodyweight gain was reduced in both males and females in the high dose group when compared to the control group. Food consumption and food efficiency were reduced for the 1000 mg/kg bw/day animals when compared to the controls. Recovery was evident thereafter following the reduction of the high dose level to 500 mg/kg bw/day. There were no treatment related effects detected on mating performance, fertility and gestation length in treated animals when compared to controls.

 

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability: No treatment related effects were evident in sex ratio. On Days 1 and 4 post partum, the number of live offspring per litter was slightly lower at the 500 mg/kg bw/day dose level compared to controls; however, this finding was not statistically significant. This was partially reflected by a statistically significant lower live birth index at the 500 mg/kg bw/day dose level compared to controls (P<0.05). By Day 4 post partum, 3 of the 6 litters had lost more than 3 offspring after birth at the 500 mg/kg bw/day dose level. In contrast, there were no litters in the controls or other treatment groups that lost more than 1 offspring in the same time period.

Offspring Growth and Development: No treatment related effects were detected in offspring growth and development.

Offspring Observations: Consistent with the observed slight decrease in litter size, the number of offspring at the 500 mg/kg bw/day dose level that were noted to be found dead or missing was higer than those reported at other dose levels. There were no other offspring clinical observations that were attributable to the test item.

 

Pathology: The 1000 mg/kg bw/day male and two 1000 mg/kg bw/day females that were killed in extremis had a raised limiting ridge in the stomach at necropsy, considered as evidence of local irritation. No toxicologically significant macroscopic, microscopic abnormalities and changes in organ weights were detected for terminal kill adults and no obvious adverse macroscopic findings were detected for offspring.

Under the test conditions, the NOAEL for adult toxicity is 250 mg/kg bw/day and NOAEL for reproductive/developmental toxicity is 250 mg/kg bw/day in rats.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is GLP-compliant and of high quality (Klimisch score = 1)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A Good Laboratory Practices (GLP)-compliant reproductive/developmental toxicity screening assay was conducted according to OECD Test Guideline No. 421 (Harlan Laboratories, 2012). The substance was administered orally (by gavage) to male and female Wistar rats at dose levels of 0 (arachis oil control), 60, 250, or 500 (1000 for first 3 days) mg/kg body weight/day for up to 8 weeks (including a two week maturation phase, pairing, gestation, and early lactation for females).

Adverse effects on systemic toxicity in adult animals were evident during the first three days of treatment. Predominantly 1000 mg/kg bw/day animals were affected, showing reductions in body weights and food consumption values as well as excessive clinical observations. Three high dose animals (two females and one male) were killed  in extremis  on Day 3 of the study; subsequently, administration of the test item was withdrawn on Day 4 for 1000 mg/kg bw/day animals and re-commenced on Day 5 at the reduced dose level of 500 mg/kg bw/day. Following the reduction in high dose level, the initial decline in good health of the animals showed some recovery as all the affected parameters were comparable to controls thereafter exept for overall bodyweight gain. Clinical signs at the 250 mg/kg bw/day dose level included salivation in males (also observed at 500 mg/kg bw/day), which was consistent with the administration of an unpalatable substance and considered to be of no toxicological importance. There were no clinical signs of note at 60 mg/kg bw/day and no bodyweight findings at 250 or 60 mg/kg bw/day. Macroscopic and microscopic findings for adult animals were generally unremarkable and did not indicate any adverse effect of treatment. There were statistically significant differences in male reproductive organ weights, however, in the absence of any evidence of histopathological change, these changes were considered to be of no toxicological significance.

The substance did not result in any test article-related effects on the following fertility parameters: mating, fertility, mean number of corpora lutea, mean number of implantation sites, pre-implantation losses, and sex ratio. While the percentage of post-implantation losses was slightly increased (10.3%) at the high-dose level compared to controls (6.6%), the result was not statistically significant and did not appear to be biologically relevant (as the total number of high-dose offspring born was similar to that of controls).  In total, 8, 9, 10, and 6 females gave birth to a live litter in the control, 60, 250, and 500 mg/kg bw/day groups (It should be noted that in the high dose group 2 females and 1 male died prematurely and 1 male was non-fertile for non-treatment related reasons, so the 6 females that were pregnant was equivalent to a preganacy rate of 100%) . Slight reductions were noted in litter size and live birth index values (p<0.05) on Days 1 and 4 post partum at the high-dose level; a loss of 3 or more offspring between birth and Day 4 post partum was noted in 3 of 6 high-dose litters. In contrast, no litter had lost more than 1 pup during the same time period in the remaining groups, including controls.  While statistical significance achieved was only of low probability (P) value and the viability index was not significantly different compared to control values, a potential adverse treatment related effect cannot be entirely excluded. However, the small group size in the high dose group (only six females) and the early toxic effects of the treatment on the females means that maternal toxicity cannot be excluded for the small effects on pup survival, or that the minor effects were spurious and not related to treatment.

In summary, oral administration of the substance to rats by gavage at dose levels of 60, 250 and 500 mg/kg bw/day (highest dose reduced from 1000 mg/kg bw/day on Day 5 of treatment), resulted in treatment-related effects at the 1000 mg/kg bw/day dose level such as ataxia, lethargy, pilo-erection and body weight loss. The overall bodyweight gain was reduced in both sexes at the reduced high dose 500 mg/kg bw/day, therefore the level of 250 mg/kg bw/day is classified as a NOAEL for adult toxicity.

Oral administration of the substance was associated with a slight reduction in postnatal survival as evidenced by slightly smaller litter sizes on Days 1 and 4 post partum, a reduced live birth index and increased incidence of offspring clinical observations (“found dead” and “missing”) at the 500 mg/kg bw/day dose level. However, it should be noted that the number of litters observed at the 500 mg/kg bw/day dose level (6) was quite small and thus, a conclusive judgement regarding the effects of the substance on reproductive and developmental toxicity cannot be made on this study alone. In addition, there was no difference in pup weights on Day 4 between control and the high dose group, which indicates no clear evidence for developmental toxicity. Nevertheless, for conservative reasons, the NOAEL for reproductive/developmental toxicity was considered to be 250 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

Oral administration of the substance was associated with a slight reduction in postnatal survival as evidenced by slightly smaller litter sizes on Days 1 and 4 post partum, a reduced live birth index and increased incidence of offspring clinical observations (“found dead” and “missing”) at the 500 mg/kg bw/day dose level. However, it should be noted that the number of litters observed at the 500 mg/kg bw/day dose level (6) was quite small and thus, a conclusive judgement regarding the effects of the substance on reproductive and developmental toxicity cannot be made on this study alone. In addition, there was no difference in pup weights on Day 4 between control and the high dose group, which indicates no clear evidence for developmental toxicity. Nevertheless, for conservative reasons, the NOAEL for reproductive/developmental toxicity was considered to be 250 mg/kg bw/day.

 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is GLP-compliant and of high quality (Klimisch score = 1)
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A Good Laboratory Practice (GLP)-compliant reproductive/developmental toxicity screening assay was conducted according to OECD Test Guideline No. 421 (Harlan Laboratories, 2012). The substance was administered orally (by gavage) to male and female Wistar rats at dose levels of 0 (arachis oil control), 60, 250, or 500 (100 for first 3 days) mg/kg body weight/day for up to 8 weeks (including a two week maturation phase, pairing, gestation, and early lactation for females). Information on the effects of the substance on the male and female parental animals and on the absence of effects on fertility parameters is found in the Discussion of Effects on Fertility (above).

In total, 8, 9, 10, and 6 females gave birth to a live litter in the control, 60, 250, and 500 mg/kg bw/day groups, the low number in the hish dose was caused by the premature death of two females and one male on Day 3 of dosing and the sterility of one male caused by a non-treatment related cause, so the pregnancy rate was 100% in the high dose group. The total number of offspring born in all treatment groups was similar to that of control litters, however, high dose litter sizes at Days 1 and 4 were slightly reduced, but not related to effects on fertility. There were 3 out of the 6 litters in the high dose group that had lost 3 or more offspring after birth; however, in the remaining groups, there was not more than 1 offspring lost per litter during the same time period. This small reduction in litter size resulted in a statistically significant reduction in live birth index values (P<0.05) and a reduction in litter weight values at the high dose (500 mg/kg bw/day). 

In offspring, there were no treatment-related effects noted in growth and development and no macroscopic observations were reported. Consistent with the decrease in litter size observed, a higher number of offspring were reported to have been found dead or missing in the high dose group (three of six litters had lost three or more offspring after birth). Specifically, 1 male and 1 female pup reported to be missing/found dead in the control group compared to 3 males and 5 females and 3 males and 1 female reported to have been found dead or missing, respectively, in the high dose group.  This is consistent with the smaller postnatal litter sizes reported at the high-dose level.

In summary, oral administration of the substance to rats by gavage at dose levels of 60, 250 and 500 mg/kg bw/day (highest dose reduced from 1000 mg/kg bw/day on Day 5 of treatment), was associated with a slight reduction in postnatal survival as evidenced by slightly smaller litter sizes on Days 1 and 4 post partum, a slightly reduced live birth index and increased incidence of offspring clinical observations (“found dead” and “missing”) at the 500 mg/kg bw/day dose level. However, it should be noted that the number of litters observed at the 500 mg/kg bw/day dose level (6) was quite small and thus, a conclusive judgement regarding the effects of the substance on reproductive and developmental toxicity cannot be made on this study alone. Nevertheless, for conservative reasons, the NOAEL for reproductive/developmental toxicity was considered to be 250 mg/kg bw/day.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008.

Self-classification:

While there was a slight reduction in postnatal survival (as evidenced by slightly smaller litter sizes on Days 1 and 4 post partum), a reduced live birth index, and increased incidence of offspring clinical observations (“found dead” and “missing”) at the 500 mg/kg bw/day dose level, the number of litters observed at the 500 mg/kg bw/day dose level (6) was quite small and thus, a conclusive judgement regarding the effects cannot be made on this study alone. As a result, the substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008 or the GHS.