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Administrative data

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 October 03, 2016 to May, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation:
Males: approximately10 weeks.
Females: approximately 11 weeks.
- Housing: Animals were housed in groups of 5 animals of the same sex in Macrolon plastic cages
(MIV type, height 18 cm).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten
GmbH, Soest, Germany).
- Water (e.g. ad libitum): tap water
- Acclimation period: At least 5 days prior to start treatment.
ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of
40 to 70%, at least 10 room air changes/hour, and a 12-hour light/12-hour dark cycle. The light/dark
cycle was interrupted for study related activities.
Route of administration:
oral: gavage
Details on exposure:
Dose volume: 5 mL/kg body weight.
Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.

Detection of mating was not confirmed for animal no. 72 which delivered live offspring. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and
accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature
protected from light was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were
90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation
was ≤ 10%. Formulations were considered stable if the relative difference before and after storage
was maximally 10%.
Duration of treatment / exposure:
- Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to and including
the day prior to scheduled necropsy.
- Females that delivered were exposed for 42-47 days (most females) or 54 days (one female), i.e.
two weeks prior to mating (with the objective of covering at least two complete estrous cycles), the
variable time to conception, the duration of pregnancy and at least four days after delivery, up to and
including the day before scheduled necropsy. Females which failed to deliver healthy offspring were
treated for 42 days.
Routinely, females that are littering are left undisturbed.The omission of one day of dosing over a period of several weeks is considered not to affect the toxicological evaluation
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes
Details on study design:
- Dose selection rationale: Based on a preliminary dose range-finder
- Rationale for animal assignment (if not random): random
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortalitty/viability checked at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals immediately after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1 and 4.

FOOD CONSUMPTION :
- Time schedule: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1 and 4.

WATER CONSUMPTION : Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes (F0 generation only)
- Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m.
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION (FUNCTIONAL OBSERVATIONS): Yes
The following functional observations tests were performed on each individual animal of the selected 5 animals/sex/group:
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), and static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
- fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
- locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
The selected males were tested during Week 4 of treatment and the selected females were tested during the lactation period from lactation Day 4 onwards (all before blood sampling). These tests were be performed after observation for clinical signs (incl. arena observation, if applicable).

IMMUNOLOGY: No


Oestrous cyclicity (parental animals):
No
Sperm parameters (parental animals):
Parameters examined in male parental generations:
- testis weight, epididymis weight, prostate weight, seminal vesicle weight
- testes slides stained with PAS/haematoxylin to examine staging of spermatogenesis
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:

Mortality / Viability: The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated.

Clinical signs At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective tables.

- Body weights: Live pups were weighed on PND 1 and 4.

- Sex: Sex was determined for all pups on PND 1 and 4.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY / ORGAN WEIGHTS: Yes
- Including number of corpora lutea and former implantation sites for all paired females.
Postmortem examinations (offspring):
Pups surviving to planned termination were killed by decapitation on Days 4-7 of lactation.
All pups were sexed and descriptions of all external abnormalities were recorded.
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated. Pups found dead during the weekend were fixed in an identified container containing 70% ethanol (Klinipath, Duiven, The Netherlands) as these pups were not necropsied on the same day.
Statistics:
The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-toone
t-test) based on a pooled variance estimate was applied for the comparison of the treated groups
and the control groups for each sex.
• The Steel-test (Ref. 3; many-to-one rank test) was applied if the data could not be assumed to follow
a normal distribution.
• The Fisher Exact-test (Ref. 4) was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test (Ref. 5) was applied to motor activity data to de
termine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. G
roup means were calculated for continuous data and medians were calculated for discrete data (score
s) in the summary tables. Test statistics were calculated on the basis of exact values for means an
d pooled variances. Individual values, means and standard deviations may have been rounded off
before printing. Therefore, two groups may display the same printed means for a given parameter,
yet display different test statistics values.
Reproductive indices:
- Mating index (%)
- Fertility index (%)
- Conception index (%)
- Gestation index (%)
- Duration of gestation (d)
Offspring viability indices:
- % live males at first litter check
- % live females at first litter check
- % postnatal loss
- Viability index (%)
Clinical signs:
no effects observed
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):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and spermatogenic staging profiles were normal for all males examined.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index was not affected by treatment. All females showed evidence of mating.
Fertility and conception index were not affected by treatment.
Two females treated at 100 mg/kg (no. 51 and 53) were not pregnant. These cases of non-pregnancy, which occurred in absence of related histopathology changes in reproductive organs, were considered to be unrelated to treatment as the incidence of non-pregnancy showed no dose-related trend.
Precoital time was not affected by treatment.
Numbers of corpora lutea and implantation sites were not affected by treatment.
For female no. 67 (300 mg/kg) the number of pups was slightly higher than the numbers of corpora lutea and implantation sites. This was considered to be due to normal resorption of these areas as these enumerations were performed on Day 6 of lactation.
Gestation index and duration of gestation were not affected by treatment.
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.


Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (sperm measures)
reproductive performance
Remarks on result:
other: no adverse effects observed
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment.
The incidental clinical signs observed remained within the range considered normal for pups of this age and showed no dose-related trend. They were therefore considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Viability index (number of offspring surviving until planned necropsy as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 97% (control group) or 100% (treatment groups).
Three of the remaining four pups of litter no. 47 were missing on PND 2 and were likely cannibalized. The remaining (female) pup was small, which was corroborated by a low pup weight on PND 4. As this was control group litter, this was not related to treatment with the test item.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted in body weights of pups.
At PND 4, mean pup weights in the test groups were higher than those in the control group (statistically significant at 100 and 300 mg/kg). This finding was considered to be unrelated to treatment because the differences showed no dose-related response and could be explained by relatively low pup weights in two litters of the control group.
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 macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the few findings noted remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Sex ratio was not affected by treatment.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Reproductive effects observed:
no
Conclusions:
Based on these results, the parentalNOAEL was determined to be 300 mg/kg bw/day (considering histopathological renal changes at 1000 mg/kg bw/day in males), the reproduction NOAEL was ≥1000 mg/kg bw/day and the developmental NOAEL was also ≥1000 mg/kg bw/day.
Executive summary:

A combined 28 d repeated dose toxicity study with a reproduction/developmental toxicity screening was conducted with the test substance in rat according to OECD Guideline 422, in compliance with GLP. The substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. Males were treated for 2 weeks prior to mating, during mating, and up to and including the day before scheduled necropsy (for 29 d). The females that delivered were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 d of lactation (mostly for 42-47 d). Females that failed to deliver offspring were treated for 42 d. Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 5 h at room temperature protected from light.

In the parental generation, no toxicologically relevant changes were noted in the in-life parameters examined in this study up to 1000 mg/kg bw/day (i.e. mortality, clinical appearance, functional observations, body weight and body weight gain, food consumption). Also, no treatment-related or toxicologically relevant changes were noted in haematology parameters or findings at macroscopic examination. However, effects were noted on organ weights and histopathology.

No reproduction or developmental toxicity was observed up to the highest dose level tested.

Based on these results, the parental NOAEL was determined to be 300 mg/kg bw/day (considering histopathological renal changes at 1000 mg/kg bw/day in males), the reproduction NOAEL was ≥1000 mg/kg bw/day and the developmental NOAEL was also ≥1000 mg/kg bw/day (Hartman-Van Dycke, 2017).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Good quality database
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 combined 28 d repeated dose toxicity study with a reproduction/developmental toxicity screening was conducted with the test substance in rat according to OECD Guideline 422, in compliance with GLP. The substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. Males were treated for 2 weeks prior to mating, during mating, and up to and including the day before scheduled necropsy (for 29 d). The females that delivered were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 d of lactation (mostly for 42-47 d). Females that failed to deliver offspring were treated for 42 d. Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 5 h at room temperature protected from light.

In the parental generation, no toxicologically relevant changes were noted in the in-life parameters examined in this study up to 1000 mg/kg bw/day (i.e. mortality, clinical appearance, functional observations, body weight and body weight gain, food consumption). Also, no treatment-related or toxicologically relevant changes were noted in haematology parameters or findings at macroscopic examination. However, effects were noted on organ weights and histopathology.

No reproduction or developmental toxicity was observed up to the highest dose level tested.

Based on these results, the parental NOAEL was determined to be 300 mg/kg bw/day (considering histopathological renal changes at 1000 mg/kg bw/day in males), the reproduction NOAEL was ≥1000 mg/kg bw/day and the developmental NOAEL was also ≥1000 mg/kg bw/day (Hartman-Van Dycke, 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

Based on the results of a combined 28 d repeated dose toxicity study with a reproduction/developmental toxicity screening conducted in rats, the test substance does not warrant classification for reproductive or developmental toxicity according to the EU CLP criteria (EC 1272/2008).

Additional information