Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Justification for study design:
- Premating exposure duration for parental (P0) animals: 14 days prior to mating
- Basis for dose level selection:
- Route of administration: oral

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
No information on test material available. Endpoint study information provided by ECHA from migrated NONS data and specific information not included.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on species / strain selection:
Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: At least 7 weeks
- Weight at study initiation: Males 372 to 438g and females 217 to 261g.
- Fasting period before study: None
- Housing: Stainless hanging type bracket cages. For females during gestation and lactation periods polymethlypentene cages were used.
- Diet (e.g. ad libitum): Pelleted rodent diet fed adlibitum.
- Water (e.g. ad libitum): Well water provided adlibitum.
- Acclimation period: At least 20 days (quarantine and acclimation period)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 to 24.4°C
- Humidity (%): 44.3 to 58.5%
- Air changes (per hr): 10 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The dose formulations were prepared once every 4 days (based on obtained stability data). The appropriate amount of the substance was weighed and added to corn oil whilst mixing with a magnetic stirrer. This was then added to a cylinder and made up to volume to prepare the dosing formulations. These were added to brown glass vials ready for use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil is a standard vehicle for tests of these types.
- Concentration in vehicle: 3.75 mL/kg (dosing volume). Individual volume was calculated on the basis of the most recently measured bodyweights.



Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: Maximum of two weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as GD day 0 of pregnancy
- After successful mating each pregnant female was caged: Individually
- Any other deviations from standard protocol: None
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test substance concentration in dosing formulations:

- At the initial dosing preparation, 20 mL of each sample was taken from the whole dosing formulation at each concentration (except controls). The analytical determination was based on a previously developed HPLC method with UV Detection.
- The test material itself was analysed by FTIR prior to use and at the end of the dosing period which confirmed stability.
- Stability of the test material in the vehicle was confirmed for up to 4 days (20 to 200 mg/mL formulations) at room temperature.
Duration of treatment / exposure:
Males: from 14 days prior to mating until the day before necropsy though the mating period (35 days in total).
Females: From 14 days before mating until day 12 of lactation through the mating, gestation and partuition periods. Non-mating females, non-copulation females and females that failed to product litters were maintained until the day before scheduled necropsy.
Frequency of treatment:
Once daily via oral gavage
Details on study schedule:
- As a screening study only parental animals were mated. The necropsy of the F1 pups was performed on Day 13 of the lactation period.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
120 mg/kg bw/day
Dose / conc.:
240 mg/kg bw/day
Dose / conc.:
480 mg/kg bw/day
No. of animals per sex per dose:
12 males and 12 females per sex per dose. This is a total of 96 animals.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a preliminary study. This dosed rats at 0, 225, 450 or 750 mgkg/day. In this study, 4/5 females that received 750 mg/kg died. In animals that received 450 mg/kg there was a decreasing trend of bodyweight gain in males indicating slight toxicity. Accordingly the upper dose in the main study was set as 480 mg/kg/day in which signs of toxicity were expected to occur in dams. A geometric ratio of two was used to set the remaining dose levels.

- Rationale for animal assignment (if not random): On the day prior to dosing 10 females showing 5-day or more estrous cycles were excluded from the group assignment based on the results of estrous cycle determination. The other animals were assigned to each group by stratified randomisation on the basis of bodyweights.
Positive control:
None included as is not standard of studies of this type

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed twice a day (before and after dosing) during the dosing period, and once a day in other periods.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed on Days 1, 8, 15, 22, 29 and 36. Females were weighed on Days 1, 8 and 15 before mating. Days 0, 7, 14 and 20 during gestation and on lactation days 0, 4, 7 and 13. Non-mating females (including one non-cohabiting to to the death of its chosen partner) and non-copulating females were also weiged on Days 22, 29 and 36.

FOOD CONSUMPTION: Yes
- Time schedule: For males the food was weighed on Days 2, 8, 15 and 36. For females this was Days 2, 8 and 15 prior to mating. During lacation this was weighed on Days 1, 7, 14 and 20. During lacation this was weighed on Days 1, 4, 7 and 13. No measurements were carried out during the mating period or for non-mating or non-copulation females. A feeder containg the diet was weighed and added to the animal cage in the morning . On the following morning the remaining diet was weighed to calculate food consumption on the day. The day of measurement of the remaining diet was designated as the day of measurement of food consumption.

Oestrous cyclicity (parental animals):
Vaginal smears were collected from females in the mornings from the start of dosing until the day of confirmed copulation or the end of the mating period. These smears were plated and smeared. The estrus cycles were classified as either DPEM. The mean estrous cycle and the length of the estrous period were calculated.
Sperm parameters (parental animals):
Parameters examined in male parental generation:

Testis weight and epididymis weight were measured in parental males. No further assessment of sperm parameters were taken.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: On Day 4 the litter size was standardised to 8 (4/sex/litter) by random removal of F1 offspring. Litters with less than 8 offspring were maintained and those with sexes less than 4 were also maintained.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities, anogenital distance (AGD) and presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS:
Offspring found dead were fixed and still borns were discarded. They were all assessed for external abnormalities initially.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not conducted.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Hormone concentration (total T4) was assessed on postnatal Day 13. Blood samples were taken from discarded pups on postnatal day 4 and kept in reserve.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed on Day 36.
- Maternal animals: All surviving animals were sacrificed on lactation Day 13.
- The animals were scarificed by exsanguination from the abdominal aorta after blood sampling.

GROSS NECROPSY
- Gross necropsy consisted of examination of the thoracoabdominal organs/tissues. Non-mating females, non-copulation females and females that failed to deliver were treated in the same way.

HISTOPATHOLOGY / ORGAN WEIGHTS

Organs sampled: Thyroid (including parathyroid), Testis, Epididymides, Seminal vesicle (including dorsolateral and coagulation gland), prostate, levator ani/bulbocavernous muscle, cowper's gland, glans penis, ovary, uterus and other gross lesions.

Organs weighed: Thyroid (including parathyroid), Testis, Epididymides, levator ani/bulbocavernous muscle, cowper's gland and glans penis. Where relevant both organs were weighed together (e.g. testis). Absolute organ weights were measured and the ratio of organ weight to bodyweight was calculated.

Organs examined histopathologically: Testis, Epididymides ovary and other gross lesions. Examinations were conducted for the control and high dose groups and the ovaries of non-pregnant females.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrified on post natal Day 13. The litters were culled on post natal day 4. The animals subject to blood sampling were sacrified after blood sampling decapitation after anaesthetic was provided. Other animals were sacrified by exsanguination from the lateral iliac artery under anaesthesia.

GROSS NECROPSY
- Gross necropsy consisted of external examination of the thoracoabdominal organs/tissues. The tyroid glands of 1 male/female per litter were collected, fixed and preserved.

HISTOPATHOLOGY / ORGAN WEIGTHS
None determined.
Statistics:
Multiple comparison test: Body weights, food consumption, absolute and relative organ weights, estrous cycle, number of estrus, days intil copulation, gestation length, number of implantations, number of delivered offspring, number of live offspring, bodyweight of offspring and AGD.

Chi-squared test: Copulation index, fertility index, gestation index, delivery index and sex ratio.

Wilcoxon's rank sum test: Stillborn index, external anomaly index, external anomaly typing index, live birth index, viability index on Day 4, viability index on Day 13, and nipple development anomaly index.

The data of offspring were analyzed on the basis of litter mean values. Moreover, the body weight and food consumption of non-pregnant females as well as the body weight of non-mating or non-copulation females on and after the start of mating were excluded from the evaluation.
Reproductive indices:
Duration of mating, copulation index, fertility index, mean estrous cycle, number of estrus periods, gestation length, gestation index and delivery index.
Offspring viability indices:
Birth index, stillborn index, viability index (Day 4 and 13) sex ratio, external anomaly index and external anomaly typing index.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In parental males, salivation was observed in a number of animals across all dosing groups. This was observed sporadically from Day 8 in treated animals. The same effect was observed in a number of treated females from Day 9.

These findings were not considered to be related to treatment but rather the palability of the test substance itself.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No treatment-related deaths were observed on study. A single animal in the high dose group showed decrease in locomotor activity, prone position, bradypnea, and hypothermia and was found dead on Day 8. This death was determined to be as a result of a miss-dose as edema of the lungs was observed at necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males bodyweights on Day 36 in animals receiving 240 mg/kg and on Days 8, 22, 29 and 36 in animals receiving 480 mg/kg were significantly lower than controls suggesting a treatment related effect on bodyweight gain. In females bodyweight was significantly lower in animals receiving 480 mg/kg compared with controls on day 4 of lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There were no significant effects on food consumption in males. In females food consumption was significantly lower in animals receiving 480 mg/kg compared with controls on day 13 of lactation. There was also a decrease in food consumption on Day 8 in females receiving 120 mg/kg, although this was not considered treatment related as no dose-dependancy was observed.
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
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Description (incidence and severity):
The plasma total T4 concentration of test substance treated groups was equivalent to that of the control group in parental animals.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes in organ weights were observed in any dose group.

In males, absolute and relative testes weight was significantly higher in the 120 mg/kg group than that in the control group. However, it was not considered to be treatment-related because there was no dose-dependency.
Relative testes weight was significantly higher in the 480 mg/kg group than that in the control group. However, this change was judged to be toxicologically insignificant because no changes were observed histopathologically.
Absolute epididymides weight was significantly lower in the 480 mg/kg group than that in the control group. However, this change was judged to be toxicologically insignificant because no changes were observed in relative epididymides weight.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related changes were observed in any animal.

In males, defect of cowper’s gland (unilateral) was observed in 1 male in the 120 mg/kg group and small of testis (unilateral) and epididymis (unilateral) was observed in 1 male in the 240 mg/kg group. However, these changes were not considered to be treatment-related because there was no dose-dependency. In females, enlargement of thyroid (bilateral) was observed in 1 female in the 480 mg/kg group. However, this finding was not considered to be treatment-related because same changes were not observed in the other females.

In the animal receiving 480 mg/kg that was found dead on day 8 of study, edema of the lung, a dark reddish patch in the mucosa of the glandular stomach and a small thymus were observed. The edema was considered to be a finding indicative of aspiration of the test material into the lungs by a misdose which caused the animals death. The other findings are considered to be stress related as a result of the lung edema.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related changes were observed in any animal.

Degeneration of seminiferous tubular epithelium of testis and cell debris in the lumen of the epididymal duct were observed in 1 male in the 480 mg/kg group. However, these changes were not judged to be related to the test substance treatment, as such changes were also observed in 1 male in the control group.

In the animal receiving 480 mg/kg that was found dead on day 8 of study, minimal alveolar edema, moderate erosion in the glandular stomach mucosa, and moderate atrophy of the cortex of the thymus were observed. The suspected causes of these changes are noted as for the gross pathological findings and are not considered treatment related.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes in estrous cycle were observed in any female.

The count of estrus was significantly lower in the 120 mg/kg group than that in the control group. However, it was not considered to be treatment-related because there was no dose-dependency.

Individually, irregular estrous cycle (3 or 6-day cycles) were observed in females in the 240 and 480 mg/kg groups. However, it was not considered to be treatment-related because no statistically significant change was observed in the mean estrous cycle between the control group and any test substance treated group.
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
Not directly examined, although there were no treatment related effects on organ weights, gross pathology or histopathology of sex organs in treated males.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Fertility index (45.5%) in the 480 mg/kg group was significantly lower than that (100%) in the control group. Although there was no statistical significance, fertility index (75%) in the 240 mg/kg group was also lower than that in the control group.

No statistically significant difference was observed in the copulation index between the control group and any test substance treated group. Non-copulation was found in 1 pair of the control group and 1 pair of the 120 mg/kg/day group. Almost all mating pairs of each group copulated during the first estrus stage of females after the start of mating.

Three females in the 240 mg/kg group and 6 females in the 480 mg/kg group did not become pregnant. Accordingly, the copulation indices were 91.7%, 91.7%, 100%, and 100% for the control, 120, 240, and 480 mg/kg groups, respectively, and the fertility indices were 100%, 100%, 75.0%, and 45.5% for the control, 120, 240, and 480 mg/kg groups, respectively.

Although there was no statistical significance, number of implantation was lower in the 480 mg/kg group (12.60) than that in the control group (14.73). No statistically significant difference was observed in the gestation length, delivery index, or gestation index between the control group and any test substance treated groups. No abnormalities were observed in the delivery or nursing conditions of the dams.

No statistically significant difference was observed in birth index, sex ratio, number of stillborn, stillborn index, or viability index on Day 4 or 13 between the control group and any test substance treated groups. No external anomaly was observed in any offspring. Although there was no statistical significance, number per litter (11.4) and number of live newborns (11.4) were lower in the 480 mg/kg group than that in the control group.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No effects on offspring were observed (as assessed by viability index on post natal Days 4 and 13).
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No mortality in pups observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In male offspring, body weight on post natal day 13 was significantly lower in the 240 and 480 mg/kg groups than that in the control group. In female offspring, body weight on post natal day 13 was significantly lower in the 480 mg/kg group than that in the control group. As the only effect on offspring observed was on bodyweight gain, this is likely to be as a result of maternal toxicity rather than the potential for inherant toxicity to developing offspring.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
No statistically significant differences were observed in the AGD or nipple development anomaly index between the control and test substance treated groups. No external abnormalities of the offspring were observed on postnatal day 13.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No abnormalities at necropsy were observed in any offspring.
Histopathological findings:
not examined
Other effects:
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
The plasma total T4 concentration of test substance treated groups was equivalent to that of controls in offspring.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
120 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
240 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
A reproductive screening study on rats conducted in accordance with the OECD 421 guideline and GLP provided a NOAEL of 120 mg/kg for effects on parental animals (including reproductive indices) and offspring. This was predominantly based on a decreased fertility index and number of implantations observed at higher doses, but also bodyweight and food consumption effects at the higher doses in parental animals. These effects although adverse are considered to be of low toxicological significance.

As the only effect on offspring observed was on bodyweight gain, this is likely to be as a result of maternal toxicity rather than the potential for inherant toxicity to developing offspring.