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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:
25 September 2012 - 22 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1995
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy
- Age/mean body weight at study initiation: on the first day of treatment, the males were approximately 10 weeks old and had a mean body weight of 367 g (range: 327 g to 410 g) and the females were approximately 9 weeks old had a mean body weight of 228 g (range: 197 g to 263 g).
- Fasting period before study: no
- Housing: the females were individually housed, except during pairing and lactation, in polycarbonate cages. The males were individually housed, except during pairing, in wire-mesh cages.
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 7 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 09 October 2012 to 23 November 2012.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. It was mixed with the required quantity of vehicle. No correction factor was applied.
The test item dose formulations were prepared on a daily basis and delivered to the study room at room temperature and protected from light.

VEHICLE
- Justification for use and choice of vehicle: test item soluble in corn oil which is commonly used for this type of study
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurred
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred to as day 0 post-coitum
- After successful mating each pregnant female was caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: GC-FID
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: homogenous
Stability: not assessed, dose formulation prepared daily
Duration of treatment / exposure:
In the males:
- 2 weeks before mating,
- during the mating period,
- until sacrifice (i.e. at least 5 weeks in total),

In the females:
- 2 weeks before mating,
- during the mating period,
- during pregnancy,
- during lactation until day 4 post-partum inclusive,
- until sacrifice for females which had not delivered.
Frequency of treatment:
Daily
Details on study schedule:
- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: 11-12 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
10 animals per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, following the results of a previous 4-week toxicity study performed in the same species and strain. In this study, three groups of five males and five females received the test item by gavage at 100, 300 or 1000 mg/kg/day as a solution in corn oil. Another group of five males and five females received the vehicle only. There were no deaths and no test item-related effects on mean body weight, mean body weight gains, food consumption and macroscopic findings. Most of the animals treated at 300 and 1000 mg/kg/day had ptyalism. The slight variations seen in some hematology and biochemistry parameters were considered to be non adverse. There were variations in some mean organ weights at necropsy particularly at 1000 mg/kg/day but generally without statistical level and not severe. Mean thymus weight were increased in females treated at 1000 mg/kg/day (up to +42%).
Therefore, the same dose-levels as in the 4-week toxicity study were used in this study.

- Rationale for animal assignment: computerized stratification procedure.
Positive control:
no (not required)

Examinations

Parental animals: Observations and examinations:
MORTALITY/MORBIDITY:
- Time schedule: at least twice a day during the treatment period.

CLINICAL OBSERVATIONS:
- Time schedule: once a day during the treatment period.

BODY WEIGHT:
- Time schedule: Males: on the first day of treatment, then once a week until sacrifice. Females: on the first day of treatment, then once a week until mating, on days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION:
- Time schedule: Males: on the first day of treatment, then once a week until the start of the pairing period. Females: on the first day of treatment, then once a week until the start of the pairing period, on days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded.
Oestrous cyclicity (parental animals):
fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until females are mated.
Sperm parameters (parental animals):
Parameters examined in males of parental generation:
- testis weight (all groups) + microscopic evaluation (control and high-dose groups)
- epididymis weight (all groups) + microscopic evaluation (control and high-dose groups)
- microscopic evaluation of stages of the spermatogenic cycle and testicular interstitial cells (control and high-dose groups).
Litter observations:
STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs

GROSS EXAMINATION OF DEAD AND SURVIVING PUPS:
- external and internal abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals after the end of the mating period and at least after 5 weeks of treatment in total.
- Female animals: all surviving animals = day 5 post-partum or, for females which had not delivered yet, day 25 post-coitum.

GROSS NECROPSY
Macroscopic post-mortem examination of the principal thoracic and abdominal organs.

HISTOPATHOLOGY
A microscopic examination was performed on:
- epididymes, testes, ovaries from all animals of the control and high-dose groups (groups 1 and 4) sacrificed at the end of the treatment period,
- all macroscopic lesions of all groups.

Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

ORGAN WEIGHTS: epididymes, testes, ovaries.
Postmortem examinations (offspring):
SACRIFICE: on day 5 post-partum

GROSS NECROPSY: on all pups (surviving and found dead)

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No
Statistics:
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions). PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01).
Reproductive indices:
Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live pups) / Number of implantation sites
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)
Offspring viability indices:
Live birth index = 100 * (Number of live born pups / Number of delivered pups)
Viability index on day 4 p.p. = 100 * (Number of surviving pups on day 4 p.p. / Number of live born pups).

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ptyalism was regularly noted in all animals at 300 and 1000 mg/kg/day and was also observed at 100 mg/kg/day mostly in males.
Half of the males treated at 1000 mg/kg/day had piloerection in week 5 of treatment for up to 3, 4 or 6 days. This was considered to be non adverse as it was transient, not associated with adverse findings in the other study parameters and not observed in the 15 other animals of the group.
Soiled urogenital area was recorded for about 5 days in week 3 for 4/10 males at 1000 mg/kg/day. A relationship with the test item treatment was considered to be unclear as this clinical sign was transient, not associated with relevant clinical signs in this week and can be commonly observed in rats.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
At 1000 mg/kg/day, there was one male found dead on day 10 without any clinical signs beforehand precluding the death. At necropsy, cryptorchidism and a translucent content in thoracic cavity were noted. In the absence histopathological changes in testes or epididymides, it was considered that the position of testes seen at necropsy was more probably due to an agonal cremaster contraction moving the testes through the open inguinal canal rather than to a real crypthorchidism.
At 100 mg/kg/day, there was one female found dead on day 23 p.c. without clinical signs prior to death. Dead and alive pups were found in the bedding. Dead fetuses, placentae, one early resorption and one scar were seen in uterus at necropsy. At the histopathological examination no specific changes suggesting a test item-related effect were observed. The death was considered to be related to dystocia.
There were no other unscheduled deaths during the study.
These deaths were considered not to be test item-related but incidental.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Overall in the pre-mating period for females or during the study for males, there was a trend towards higher mean body weight gains in test item-treated groups and in particular at 300 mg/kg/day. However, there was no dose-relationship and this had no toxicological impact on mean body weights; a relationship with the test item treatment was considered to be unlikely.
There were no effects during gestation and lactation.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no effects on mean food consumption.
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:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no changes in the mean organ weights which were indicative of a test item-related effect.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes were seen at necropsy in terminal sacrificed animals.
Reduced size of testes and epididymides was seen unilaterally in one male given 1000 mg/kg/day (associated with deformation of the testis), and bilaterally in one male given 300 mg/kg/day. These isolated observations were considered to be incidental.
Scabs seen on the neck of one male were considered without relationship with the test item and part of the normal background commonly seen in rats.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes were seen in testes or ovaries from animals given the test item at 1000 mg/kg/day.

Tubular atrophy/degeneration was seen unilaterally in the right testis of one male given 1000 mg/kg/day and bilaterally in one male given 300 mg/kg/day correlating with macroscopic changes seen at necropsy. This was graded moderate at 1000 mg/kg/day and severe at 300 mg/kg/day and was associated with an apparent Leydig cell hyperplasia (minimal and unilateral or slight and bilateral respectively) that could be confused with tubular atrophy.
In both animals reduced sperm (moderate and unilateral in the male given 1000 mg/kg/day and severe and bilateral in that given 300 mg/kg/day) was observed in epididymides, correlating with macroscopic changes seen at necropsy.
In the absence of a dose-related trend and as testicular atrophy may be occasionally seen in rats, these isolated observations, which correlated with macroscopy, were considered to be incidental.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
There were no effects on pairing, mating and fertility data.
Two females treated at 300 mg/kg/day were sacrificed on day 25 p.c. because of no delivery. They were non-pregnant. This was considered to be incidental.
All delivery data from test item-treated groups were comparable to the control data and/or historical control data, including the statistically higher mean pre-implantation loss at 1000 mg/kg/day. This variation was thus considered to be of limited toxicological relevance.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
(F0)
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
Dose descriptor:
NOAEL
Remarks:
(reproductive performance)
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

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test item-related clinical signs in pups.
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no test item-related deaths in pups.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicological relevant effects on mean pup body weights and mean pup body weight gains. In the absence of a dose-level relationship and of similar effect in the opposite sex, the statistically higher mean body weight gain in males at 100 mg/kg/day was considered to be incidental.
Food consumption and compound intake (if feeding study):
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):
There were no test item-related macroscopic findings at pup necropsy.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOEL
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

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The test item was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before pairing, during pairing, gestation and until day 4 p.p., at dose-levels of 100, 300 or 1000mg/kg/day.
 
Based on the experimental conditions of this study:
.         the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day,
.         the NOAEL for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day,
.         the NOEL for toxic effects on progeny was considered to be 1000 mg/kg/day.
Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until day 4 post-partum (p.p.), based on the OECD guideline No. 421, 27 July 1995.

This study provides information on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

 

Methods

Three groups of ten male and ten female Sprague-Dawley rats received the test item daily by oral (gavage) from before pairing, through pairing and, for the females, through gestation until day 4 p.p. The test item was administered as a solution in the vehicle, corn oil, at dose-levels of 100, 300 or 1000 mg/kg/day.

Another group of ten males and ten females received the vehicle alone under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used.

The concentration of the dose formulation was checked in study weeks 1, 4 and 6.

 

The animals were checked at least once daily during the dosing period for mortality, morbidity and clinical signs. Body weight and food consumption were recorded approximately once a week. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The pups were observed daily for clinical signs, abnormal behaviour and external abnormalities and weighed on days 1 and 5 p.p.

The males were sacrificed after 5 weeks of treatment and the dams on day 5 p.p. Final body weights and selected organs weights (epididymides, testis, ovaries) were recorded and a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on selected organs (epididymides, testis, ovaries) from the control and high-dose groups and on all macroscopic lesions.

The pups were sacrificed on day 5 p.p. and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs.

Results 

The test item concentrations in the administered dose formulations analyzed in weeks 1, 4 and 6 were within the acceptance criteria (± 10% of the nominal concentrations).

 

There were no test item-related deaths. Ptyalism was regularly noted in all animals at 300 and 1000 mg/kg/day and was also observed at 100 mg/kg/day mostly in males; half of the males treated at 1000 mg/kg/day had also piloerection in week 5 of treatment. These clinical signs were considered to be non-adverse. Soiled urogenital area was recorded in week 3 for 4/10 males at 1000 mg/kg/day. A relationship with the test item treatment was unclear.

 

There were no toxicologically relevant effects on mean body weights, mean food consumption or on mean reproductive data.The higher mean pre-implantation loss at 1000 mg/kg/day (7.0% vs. 1.0, p<0.05) was considered to be of limited toxicological relevance.


At pathology, there were no changes in testes and epididymides of males and in ovaries of females ascribed to test item treatment.

 

There were no test item-related deaths or clinical signs in pups andno toxicological relevant effects on mean pup body weights and mean pup body weight gains or on the percentage of male pups at birth. There were no test item-related macroscopic findings at pup necropsy.

 

Conclusion

The test item was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before pairing, during pairing, gestation and until day 4 p.p., at dose-levels of 100, 300 or 1000 mg/kg/day.

 

Based on the experimental conditions of this study:

.         the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day,

.         the NOAEL for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day,

.         the NOEL for toxic effects on progeny was considered to be 1000 mg/kg/day.