Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 August 2016 - 22 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
yes
Justification for study design:
The doses were based on a 10-day dose range finding study in which no dose limiting effects were seen up to 1000 mg/kg.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Appearance: Colourless to light yellow viscous liquid
Batch: 16020201
Purity/Composition: 99.56%
Test item storage: At room temperature
Stable under storage conditions until: 21 March 2021 (expiry date)

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han) (outbred, SPF-Quality)
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 11 wks

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (10 October 2016) according to a validated method.
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). 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%.
Duration of treatment / exposure:
Males were dosed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females that delivered were dosed for 50-55 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for 38 or 42 days. Pups were not dosed directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Positive control:
No.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes (subject examination)

OTHER: Mortality, clinical signs, body weight, food consumption, haematology, clinical chemistry, organ weights, or gross pathology and histopathology.
Estrous cyclicity (parental animals):
Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.
During pretest, this was done for 48 females. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight and gross and histopathology, epididymis weight and gross and histopathology. Seminal vesicles including coagulating glands weights.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups (PND 1), stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain (on PND 1, 4, 7 and 13), physical abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups (PND 13).

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.]

Postmortem examinations (parental animals):
Mortality, clinical signs, body weight, food consumption, haematology, clinical chemistry, organ weights, or gross pathology and histopathology.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
All pups were examined for external abnormalities.
Blood samples were drawn from 2 pups/litter for thyroid hormone analysis.
The thyroid from 2 pups/litter were fixed for macroscopic examination.
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk.
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 (many-to-one 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 (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and 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, Post-implantation survival index, Live birth index, Percentage live males at First Litter Check, Percentage live females at First Litter Check.
Offspring viability indices:
Viability index, Live birth index, Percentage live males at First Litter Check, Percentage live females at First Litter Check, Lactation index.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were noted during daily clinical observations or during weekly arena observations. Clinical findings noted incidentally occurred within the range of background findings to be expected for rats of this strain and age which are housed and treated under the conditions in this study. At the incidence observed, these were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
One female (no. 64) at 300 mg/kg was sacrificed on PND 1 because of total litter loss.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control animals.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological parameters were not affected by treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical biochemistry parameters were not affected by treatment.
The statistically significant variations noted in potassium (females) were unrelated to treatment because they occurred in the absence of a dose-related response. A few outlying values were noted in treated females (high urea and creatinine at 1000 mg/kg; high bile acids at 300 and 1000 mg/kg). As these outlying values occurred in only a single animal of a group and in the absence of corroborative changes indicative of renal or hepatic toxicity, they were considered not to reflect adverse effects of the test item.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment. The statistically significant differences noted in hind limb grip strength (higher in males at 100 mg/kg and in females at 300 mg/kg) occurred
in the absence of a dose-related response and were therefore considered to be unrelated totreatment. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period. Mean values for total movements and/or ambulations were lower in males at 300 mg/kg and higher in females at 300 and 1000 mg/kg. The differences from controls were not statistically significant and showed no (clear) dose-related response. Moreover, values in treated animals were within normal limits. Therefore, these findings were considered to be unrelated to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related alterations in organ weights. The statistically significant change noted in relative spleen weights for females at 300 mg/kg was unrelated to treatment because this occurred in the absence of a dose-related response and no corroborative findings were observed.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross
observations encountered in rats of this strain and age, and did not show a dose-related incidence trend. These findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic change was noted in the heart of 3/5 males at 1000 mg/kg, consisting of myofiber necrosis (in the apex and/or left papillary muscle) up to slight degree.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment. All females had regular cycles of 4 days.
Reproductive function: sperm measures:
not 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. All females were pregnant.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
cardiovascular
Organ:
heart
myofibres
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The incidence of litters including one or two pups with a lean appearance was higher in the 1000 mg/kg group than in the control group (6 lean pups, all females, from 5 litters at 1000 mg/kg versus none in controls). The lean pups had lower body weights than those with a normal appearance. Mean pup weights at 1000 mg/kg were similar to the control weights. At the low incidence observed (within normal limits) and in the absence of treatment-related changes in pup body weights, the occurrence of lean pups at 1000 mg/kg was considered not to be toxicologically relevant. Other clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices across the groups were 98-100%. Post-natal loss up to PND 4 was limited to two pups of the control group (litter nos. 44 and 46) which went missing on PND 2, and one pup at 1000 mg/kg (litter no. 79) which went missing on PND 3. The missing pups were most likely cannibalized. This incidental postnatal loss was unrelated to treatment with the test item.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights of pups were not affected by treatment.
A statistically significantly higher body weight of female pups was noted at 300 mg/kg on PND 13. In the absence of a dose-related response, this finding was considered to be unrelated to treatment.
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:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 13-15 pups were not affected by treatment.
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 toxicologically relevant.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment.
The slight, statistically significant differences in anogenital distance (absolute and/or normalized) noted in male pups at 100 and 1000 mg/kg occurred in the absence of a doserelated trend, and as a slight increase in male anogenital distance is not considered toxicologically relevant and all values were within normal limits, the differences were therefore not considered to be attributed to treatment.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
clinical biochemistry
gross pathology
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
No reproduction or developmental toxicity was observed up to the highest dose level tested (1000 mg/kg). Thus, a NOAEL of >1000 mg/kg bw/day could be identified.
Executive summary:

In a reproductive and developmental screening study the substance was administered to 10 rats/sex/dose by oral gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day for a duration of 28 days. Dosing of the females was continued during lactation. There were no substance-related effects on parental mortality, clinical signs, body weight, food consumption, haematology, clinical chemistry, organ weights, or gross pathology. Histopathological examination revealed a degenerative change in the heart (myofiber necrosis in the apex and/or left papillary muscle) of males treated at 1000 mg/kg. The parental NOAEL is 300 mg/kg bw/day based on the degenerative change in the heart at the top dose in males. The reproduction and developmental NOAEL is >1000 mg/kg bw/day based on no treatment-related effects being observed in the reproductive and developmental parameters.This study in the rat is acceptable and satisfiesthe guideline requirement for a subacute oral study OECD 422 in rats with screening for reproductive and developmental effects.