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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-06-28 to 2013-03-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted under GLP according to OECD TG 422.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories BV, Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: 290 to 326 g (males), 162 to 222 g (females)
- Fasting period before study: no
- Housing: in groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: at room temperature (20 ± 5 °C) in glass beakers

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 0, 12.5, 75, 250 mg/mL
- Amount of vehicle: 4 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days maximum
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (8 days) samples of about 2 g of each concentration were taken from the middle of each aliquot used on day 7 of the treatment. During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to analytic laboratory and stored there at -20 ± 5 °C until analysis. The samples received were dissolved in TBME by sonication for at least 5 minutes and then diluted to volume with TBME. Sample solutions were further diluted with TBME into the calibration range. The samples were analysed with gas chromatography coupled to an FID.
Duration of treatment / exposure:
The test substance was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 3 post-partum, one day before necropsy.
Frequency of treatment:
daily
Details on study schedule:
not relevant
Doses / concentrationsopen allclose all
Dose / conc.:
50 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:
11
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous non-GLP dose range-finding toxicity study in Han Wistar rats, using dose levels of 0, 50, 200 and 1000 mg/kg/day.
Positive control:
no data

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioural abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: In males, it was performed once prior to the first administration of the test item and weekly thereafter. In females, it was prepared once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.

BODY WEIGHT: Yes
- Time schedule for examinations: daily from treatment start to day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Males: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 13; after pairing period weekly.
- Females: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 13; gestation days 0 - 7, 7 - 14 and 14 - 21 and days 1 - 4 of the lactation.

OTHER: HEMATOLOGY, CLINICAL CHEMISTRY, NEUROBEHAVIOURAL EXAMINATION
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 and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies

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 sacrificed after treatment for 28 days
- Maternal animals: All surviving animals were sacrificed on day 4 post-partum

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in attached document [Organ Weights and Preservation] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to post-mortem examinations (macroscopic and/or microscopic examination) as follows: no data

GROSS NECROPSY
no data

HISTOPATHOLOGY / ORGAN WEIGTHS
no data
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
• Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
From the on-line recorded reproduction data, the following parameters were calculated: mean precoital time, percentage mating, fertility index, conception rate, post-implantation loss, gestation index, birth index and viability index.
Offspring viability indices:
From the on-line reproduction data, the following parameters were calculated: dead/live pups at first litter check, pup sex ratio and viability index.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
please see Details on results (parentals animals)
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects on absolute body weight or body weight gain were noted in males and females at any dose level.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effects on absolute body weight or body weight gain were noted in males and females at any dose level.
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, 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
Description (incidence and severity):
please see Details on results (parent animals)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
During macroscopical examination enlarged liver was found in one female at the dose level of 1000 mg/kg bw/day. This finding was considered to be related to the treatment with the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
please see Details on results (parent animals)
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: gavage

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
At the dose level of 1000 mg/kg bw/day, bedding in mouth and salivation in males starting from day 6 of the pairing period and in females starting from day 1 of the pairing period. At the dose level of 300 mg/kg bw/day, bedding in mouth in females starting from day 1 of the gestation period was observed. These observations were considered to be sign of discomfort caused by the treatment with the test item.

BODY WEIGHT AND FOOD CONSUMPTION
No effects on absolute body weight or body weight gain were noted in males and females at any dose level.

TEST SUBSTANCE INTAKE
No effects (gavage) observed.

REPRODUCTIVE PERFORMANCE
Mating performance, fertility, corpora lutea count, duration of gestation, implantation rate and post-implantation loss, litter size or postnatal loss were not affected by the treatment with the test item at any dose level.

ORGAN WEIGHTS
At the dose level of 1000 mg/kg bw/day, statistically significantly increased liver weights were noted in males. This finding was considered to be due to adaptive changes in the liver metabolism caused by the treatment with the test item and not adverse.

GROSS PATHOLOGY
During macroscopically examination enlarged liver was found in one female at the dose level of 1000 mg/kg bw/day. This finding was considered to be related to the treatment with the test item.

HISTOPATHOLOGY
Microscopic changes related to the treatment with the test item were observed in the liver of males and females at the dose level of 1000 mg/kg bw/day and in the kidneys of males at the dose levels of 1000 and 300 mg/kg bw/day.

Liver and thyroid gland
Centrilobular hypertrophy of the hepatocytes was seen in males and females at the dose level of 1000 mg/kg bw/day with a higher incidence and severity present in males. In males at the dose level of 1000 mg/kg bw/day, hypertrophy of the follicular epithelium of the thyroid gland was also noted. This finding was considered to be very likely secondary to the increased metabolism of T4 and T3 in the liver that in turn stimulated the secretion of TSH from the pituitary and subsequent follicular epithelial hypertrophy of the thyroid.

Kidneys
At the dose level of 1000 and 300 mg/kg bw/day, hyaline droplets accumulation was present in the proximal convoluted tubules in the kidneys of males, which was associated to an increased incidence of tubular basophilia and to the presence of granular casts in one male at the high-dose level. In one female at the dose level of 1000 mg/kg bw/day (no. 88), an unilateral nephroblastoma was observed in the kidney and considered to be of spontaneous occurrence and therefore unrelated to the treatment with the test item. The remainder of findings noted was within the range of normal background lesions which may be recorded in rats of this strain and

OTHER FINDINGS
The examination of the ovaries and the feminine genital tract did not reveal any difference in estrus cycling between control and treated animals. The qualitative assessment of the male genital organs did not reveal any treatment-related effect.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: The highest applied dose
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The highest applied dose

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
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:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not specified
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

VIABILITY
No effects observed.

CLINICAL SIGNS
No effects observed.

BODY WEIGHT
No effects observed.

SEXUAL MATURATION
No effects observed.

ORGAN WEIGHTS
No effects observed.

GROSS PATHOLOGY
No effects observed.

Effect levels (F1)

Dose descriptor:
NOEL
Remarks:
reproductive/developmental
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The highest applied dose

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the study findings in the rats, the relevant NOAEL for human toxicity is 1000 mg/kg bw/day. The NOEL for reproduction/developmental toxicity is considered to be 1000 mg/kg bw/day.
Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the substance on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition. The substance was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 3 post-partum, one day before necropsy.

The following dose levels were applied:

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 50 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).

Parent Animals

Mortality and General Tolerability

All animals survived scheduled study period. Bedding in mouth and salivation were noted in males and females at the dose level of 1000 mg/kg bw/day. Bedding in mouth alone was noted in females at the dose level of 300 mg/kg bw/day. These observations were considered to be sign of discomfort caused by the treatment with the test item. No further clinical signs or observations were noted in males or females at any dose level.

Functional Observational Battery

With exception for salivation noted in one male and one female at the dose level of 1000 mg/kg bw/day, no further test item-related findings were noted during the tests of functional observational battery or measurement of locomotor activity.

Food Consumption

At the dose level of 1000 mg/kg bw/day in females, transient reduction in food consumption was noted at the beginning of the treatment. No test item-related effects on food consumption were noted in males at any dose level or females at dose levels of 50 and 300 mg/kg bw/day.

Body Weights

No effects on absolute body weights or body weight gain were noted in males or females at any dose level.

Clinical Laboratory Investigations

In males statistically significantly higher creatinine concentration was noted in all dose groups. This finding was accompanied by statistically significantly reduced concentration of phosphorus in high- and mid-dose groups. These findings were considered to most probably be secondary to lesions in the kidneys which were considered sex- and species-specific for male rats and therefore not relevant for humans. Further findings were statistically significantly lower concentrations of total bilirubin and statistically significantly lower concentration of bile acids in males at the dose level of 1000 mg/kg bw/day. These findings were considered to possibly be secondary to the adaptive changes in the liver metabolism caused by the treatment with the test item and not to be adverse. No further test item-related changes in hematology or biochemistry parameters were noted in males or females at any dose level.

Reproduction and Breeding Data

Mating performance, fertility, corpora lutea count, duration of gestation, implantation rate and post-implantation loss, litter size or postnatal loss were not affected by the treatment with the test item at any dose level.

Organ Weights

At the dose level of 1000 mg/kg bw/day, statistically significantly increased liver weights were noted in males. This finding was considered to be due to adaptive changes in the liver metabolism caused by the treatment with the test item and not adverse.

Macroscopical Findings and Histopathological Examinations

During macroscopical examination enlarged liver was found in one female at the dose level of 1000 mg/kg bw/day. This finding was considered to be related to the treatment with the test item.

During microscopic examinations, test item-related findings were noted in the liver of males and females at the dose level of 1000 mg/kg bw/day. These changes consisted of centrilobular hypertrophy of the hepatocytes and were considered to be an adaptive change due to the increased metabolism of the liver. In addition, hypertrophy of the follicular epithelium of the thyroid gland was noted in males at the dose level of 1000 mg/kg bw/day. This finding was considered to be very likely secondary to the increased metabolism in the liver. Further findings related to the treatment were noted in the kidneys of males at the dose levels of 1000 and 300 mg/kg bw/day; hyaline droplets associated with tubular basophilia and presence of granular casts. These findings were considered to be specific for male rats and adverse in this species but not toxicologically relevant for humans.

Litter Data - F1 Pups

Findings at First Litter Check and during Lactation

No test item-related findings were noted in pups during first litter check and during lactation at any dose level. Pups sex ratio was not affected by the exposure to the test item at any dose level.

Pup Weights to Day 4 Post-Partum

Body Weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.

Macroscopical Findings

No macroscopical findings were noted in pups at any dose level.

Conclusion

Based on the lesions in the kidneys found in males at the dose levels of 1000 and 300 mg/kg bw/day which were considered to be adverse, general NOAEL (No Observed Adverse Effect Level) for males was established at 50 mg/kg bw/day. However, lesions in the kidneys were considered to be specific for male rats and therefore not toxicologically relevant for humans. NOAEL for females was considered to be 1000 mg/kg bw/day, the highest dose level used in the study. The NOEL for reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level used in the study.