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Key value for chemical safety assessment

Effects on fertility

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 December 06, 2016 to January 30, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were used for dose range finding study.
Sex:
male/female
Details on test animals and environmental conditions:
Species and strain: Crl:WI Wistar rats
Source: Charles River Laboratories, Research Models and Services, Germany, from SPF colony
Hygienic conditions: standard laboratory conditions
Number of animals: 48 male, 48 female rats, 12 animals/sex/group, 4 groups
Age of animals: young adult rats, approximately 10 weeks old at start and 12 weeks old at mating
Body weight range: males: 378-462 g, females: 236-297 g; did not exceed ±20 % of the mean weight for each sex at onset of treatment
Acclimation period: 5 days

Husbandry
Animal health: only healthy animals were used for the test, as certified by the clinical veterinarian. Females were nulliparous and non-pregnant.
Cage type: type II polycarbonate
Bedding: LIGNOCEL ¾ S certified wooden chips produced by J. Rettenmaier & Söhne GmbH+Co.KG, Germany
Nesting: Arbocel nest building material produced by J. Rettenmaier & Söhne GmbH+Co.KG, Germany
Light: 12 hours daily
Temperature: 21.0 – 24.9 °C (target range: 22±3°C)
Relative humidity: 30 – 60% (target range: 30-70%)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: rodents were group-housed, up to 4 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or individually housed, respectively.

Food and water supply
Animals received ssniff SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, Germany, ad libitum, and tap water from the municipal supply from a 500 mL bottle, ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Animal identification
Each parental/adult animal (P Generation) was identified by a unique number within the study, written with indelible ink on the tail.
Route of administration:
oral: gavage
Vehicle:
other: methyl cellulose
Details on exposure:
1% (w/v) aqueous methyl cellulose solution (abbreviated as 1% methyl cellulose or 1% MC in the raw data and report) was selected as vehicle of the study based on the formulation and analytical trials.
Details on mating procedure:
Mating began after the animals had attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male from the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred, for up to 5 days. A vaginal smear was prepared daily during the mating period and stained with 1 % aqueous methylene blue solution. The smears were examined with a light microscope. The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were housed individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test substance formulations for concentration and homogeneity was performed using an HPLC-UV method. Top, middle and bottom duplicate samples were taken and analysed from test substance formulations at three times during the study, one set to analyse (which were collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken on each occasion from the middle of the vehicle control formulation.
Acceptance criteria of the concentration analysis were set at 100 ± 10% of the mean nominal concentration. Acceptance criteria of the homogeneity was that the CV of replicates (top, middle and bottom of test substance formulations) must be less than 10%.
The measured concentrations of the test substance evaluated for each test substance containing formulation varied between 94% and 108% of the nominal value. No test substance was detected in the control samples at any occasion. All test substance formulation samples were found to be homogeneous. Formulations were considered to be adequately stable under the study conditions.
Duration of treatment / exposure:
Dosing of both sexes began after 5 days acclimatisation and 2 weeks before mating, during the mating, and were continued up to and including the day before the necropsy.
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating) and then euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (4-day post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum (PPD 0).
Frequency of treatment:
daily
Details on study schedule:
Dosing scheme, Male animals:
Acclimatisation: 5 days
Pre-mating period: 14 days
Mating/Post-mating period: at least 14 days (Last week of treatment: FOB, Day 24, 5 animals/group, Prior to/at necropsy examinations

Dosing scheme, Female animals:
Acclimatisation period: 5 days
Pre-mating period: 14 days
Mating: up to 5 days
Gestation: 22-24 days
Delivery
Lactation period: at least 4 days, FOB: (PND 4, 5 animals/group); Pups necropsy, (PND 4)
PND 5: Prior to/at necropsy examinations

All F1 offspring were terminated on Day 4 post-partum. In order to allow for overnight fasting of dams prior to urine collection on PPD 5, the offspring were euthanized on PPD/PND 4 and the dams on PPD/PND 5.
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
Observation of the delivery process, offspring and nursing instinct:

Females were allowed to litter and rear their offspring. The delivery process was observed as carefully as possible. Any evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy until the completion of parturition.
Dams were observed for signs of nest building with the bedding material and for covering their new-borns. Evidence of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded.
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups) and to detect the presence of gross abnormalities.
Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND 0 or 1) and on PND 4, with accuracy of 0.01 g. All litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded. All pups were culled on PND 4.







Parental animals: Observations and examinations:
The reproductive performance, pregnancy, parturition and post-partum/lactation period were monitored in the adult animals.
For the adult animals, a detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups (5 animals/group) and liver, kidney and spleen samples of all groups. Additionally, all organs showing macroscopic findings as well as retained reproductive organs of those animals in the Control group which failed to sire / deliver pups were examined microscopically.

Sperm parameters (parental animals):
Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Litter observations:
The viability, clinical signs and development were evaluated in their F1 offspring until post-natal Day (PND) 4. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals.
Postmortem examinations (parental animals):
Organ weight measurements:
At the time of termination, body weight and the weight of the following organs from all adult animals were determined:
- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids
Testes and epididymides were weighed individually.

For the adult animals, a detailed histological examination was performed as follows:
- on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group),
- all macroscopic findings (abnormalities), except of minor order from all animals,
- retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups and additionally of the one male that failed to sire and the one female that failed to deliver healthy pups.
Postmortem examinations (offspring):
Macroscopic Findings
On PND 0, there were 3 Low Dose and 9 Mid Dose pups found dead that remained intact (not cannibalized or autolysed). Those were subjected to necropsy with macroscopic examination. No test substance-related macroscopic findings were seen. No other pups that died during the lactation period could be examined for test substance-related macroscopic findings.

Organ weights
Compared to the control, there were no statistically significant differences in mean values recorded in any of the dose groups.
Statistics:
Data was recorded on the appropriate forms from the relevant SOPs of the labolatory and then tabulated using the Microsoft Office Word and/or Excel, or using the software PROVANTIS v.9, as appropriate. Numerical data obtained during the conduct of the study was subjected as appropriate to calculation of group means
and standard deviations.
Clinical signs:
effects observed, non-treatment-related
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Reproductive organs of the Control and Low dose males (testes, epididymides, prostate, seminal vesicles with coagulation gland) that failed to mate or sire, and reproductive organs of females (uterus, cervix, ovary, oviduct and vagina) having mating problem or being non-pregnant were also examined. No test substance-related microscopic changes were observed in those cases.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
The fertility index was 83-100% in all male groups including control. The fertility index values for females was 100% except of the Low dose group where 83% was detected (there were two non-pregnant females in that group).
The gestation index for females was 100% except of the Mid dose group where 92% was detected (one pregnant females in that group did not deliver living pups). However, based on the facts that in the High dose group the fertility and gestation indices were 100%, these values were considered as animal variability, and being without toxicological relevance.
Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within up to 5 days of pairing (cohabitation) in each case. Extreme value was only detected in the Control group in two cases.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproduction
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Based on the external evaluation, most of the pups were normal. One pup in the Control group was cold to touch on PND0, it was found cannibalized one day later.
Cyanosis was noted for one Low dose pup and two High dose pup on PND0, they were cannibalized by the next day. Cyanosis was also recorded for one Mid dose pup on PND 0 and for two Mid dose pups on PNDs 0-1, but they later became symptom-free.
Paleness was observed for two Mid dose pups on PND 0 and/or PND1, but they were symptom-free after that. Paleness on the whole body was observed for two High dose pups, they were cannibalized by the next day.
Haemorrhage was recorded for one Low dose pup on the thorax dorsal area, this pups was cannibalized by the next day. Haemorrhage was also detected on PND0 on the nose of a High dose pup, on the thorax dorsal area of another High dose pup and left hind paws of a third High dose pup, but all those animals became symptom-free by the next day.
None of these events were considered as being related to the test substance treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of treatment on the offspring body weight on PND0 or PND4 following administration of test substance at 30, 100 or 300 mg/kg bw/day (Low, Mid and High dose groups).
A statistically significant difference (p<0.05) was observed in the body weight gain of Low dose pups, but due to lack of dose response and based on the absolute value (increase), this fact was considered as having no biological relevance. No significant differences in body weights (PND 0 or PND 4) or body weight gain values were noted in other dose groups when compared to the control (litter mean values).
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:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test substance-related macroscopic findings were seen in the unscheduled death (found dead or cannibalized pups).
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Under the study conditions, the NOAEL for the test substance was considered to be 100 mg/kg bw/day for parental/adult generation for systemic effects, and 300 mg/kg bw/day for reproduction effects and for the F1/pups generation.
Executive summary:

A study was conducted to determine repeated dose toxicity of the test substance when administered to Crl:WI Wistar rats according to OECD Guideline 422, in compliance with GLP. The reproductive/developmental toxicity-screening test intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to post-partum Day (PPD) 4. The details on systemic effects are summarised in section RDT: oral. Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including post-partum/lactation Day (PPD) 4. The test substance, formulated in methyl cellulose, was administered by oral gavage to test animals. One control group and three treated groups were tested (30 mg/kg bw, 100 mg/kg bw and 300 mg/kg bw), each consisting of 12 males and 12 females. The reproductive performance, pregnancy, parturition and post-partum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until post-natal Day (PND) 4. No test substance related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period in any dose groups when compared to control. There were no effects on the F1 offspring viability, clinical signs, development or at macroscopic observations in any dose groups. Under the study conditions, the NOAEL for the test substance was considered to be 100 mg/kg bw/day for parental/adult generation for systemic effects, and 300 mg/kg bw/day for reproduction effects and for the F1/pups generation (Hargitai, 2017).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
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 study was conducted to determine repeated dose toxicity of the test substance when administered to Crl:WI Wistar rats according to OECD Guideline 422, in compliance with GLP. The reproductive/developmental toxicity-screening test intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to post-partum Day (PPD) 4. The details on systemic effects are summarised in section RDT: oral. Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including post-partum/lactation Day (PPD) 4. The test substance, formulated in methyl cellulose, was administered by oral gavage to test animals. One control group and three treated groups were tested (30 mg/kg bw, 100 mg/kg bw and 300 mg/kg bw), each consisting of 12 males and 12 females. The reproductive performance, pregnancy, parturition and post-partum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until post-natal Day (PND) 4. No test substance related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period in any dose groups when compared to control. There were no effects on the F1 offspring viability, clinical signs, development or at macroscopic observations in any dose groups. Under the study conditions, the NOAEL for the test substance was considered to be 100 mg/kg bw/day for parental/adult generation for systemic effects, and 300 mg/kg bw/day for reproduction effects and for the F1/pups generation (Hargitai, 2017).

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification