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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
07/09/2007-13/11/2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented report of a guideline study conducted to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): BCQ (60% aqueous solution of APTAC)
- Substance type: organic
- Physical state: liquid
- Analytical purity: 59.4%
- Impurities (identity and concentrations): water (40.4%)
- Composition of test material, percentage of components: 100%
- Purity test date: 04.04.2008
- Lot/batch No.: 0000260407 (part of the main batch 0050012509)
- Expiration date of the lot/batch: April 2008
- Stability under test conditions: stable
- Storage condition of test material: +4°C protected from light
- Other:

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l. San Pietro al Natisone (UD), Italy
- Age at study initiation: 6 to 7 weeks old (P)
- Weight at study initiation: (P) Males: 191.4 - 207.8 g; Females: 155.1 - 166.3 g
- Fasting period before study: no
- Housing: From arrival to pairing, animals were housed up to 5 of one sex to a cage, in clear
polycarbonate cages with a stainless steel mesh lid and floor. The males, after mating, were re-caged as they were before mating. After mating, the females were transferred to individual clear polycarbonate solid bottomed cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 10 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 07/09/2007 To: 13/11/2007

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of BCQ (60% aqueous solution of APTAC) was dissolved in the vehicle (distilled water). The formulations were prepared daily (concentrations of 25, 50 and 100 mg/ml). Concentrations were calculated and expressed in terms of test item as supplied.
VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 0, 25, 50 and 100 mg test substance/ml
- Amount of vehicle (if gavage): The test item was administered orally by gavage at a dose volume of 10 ml/kg body weight.
- Purity: distilled
Details on mating procedure:
- M/F ratio per cage: Mating was monogamous (one male to one female).
- Length of cohabitation: until sperm identification and/or copulation plugs were found on the cage tray or in situ
- Proof of pregnancy: A vaginal smear was taken from the day after the start of pairing until sperm identification and/or copulation plugs were found
on the cage tray or in situ
- Unsuccessful pairing replacement of first male by another male with proven fertility: after 14 days (for 3 females only)
- Further matings after two unsuccessful attempts: no (not required)
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment the proposed formulation procedure was checked by chemical analysis to confirm that the method was acceptable. Stability over a 24-hour period at room temperature was previously assessed for content check (performed in RTC study no. 68770 for concentration of 30 mg/ml). Samples of the formulations prepared during week 1 and the last week of treatment were analysed to check the concentration. Results of all the analyses were within the limits of acceptance (95-105%). Results of these analyses, carried out by the Analytical Chemistry Department at RTC, are presented in Addendum II of the report.
Duration of treatment / exposure:
Males: 7 days a week for 2 consecutive weeks prior to pairing and thereafter through the day before necropsy.
Females: 7 days a week for 2 consecutive weeks prior to pairing and thereafter during gestation and post partum periods, until the day before necropsy (Day 3
post partum).
Frequency of treatment:
Animals were dosed once a day.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 250, 500, 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: The oral route was selected, as it is a possible route of exposure of the test item in man. The dose levels of 250, 500 and 1000 mg/kg/day were chosen in consultation with the Sponsor based on information from the RTC study number 68770.
- Rationale for animal assignment: The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Animals with the pre-test numbers 86 and 92 showed slight hairloss at allocation and were considered to be healthy
for the study.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Clinical signs and cage tray observations All clinical signs were recorded for individual animals. Once before commencement of treatment and at least once daily during treatment, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day; the interval was selected taking into consideration the presence of post-dose reactions. No signs were seen during the pairing period, therfore no data were presented. Each cage tray held absorbent paper which was inspected at least 3 times a week, for presence of soft faeces. The observations of the cage tray were performed starting from day 1 of dosing up to the day of sacrifice. Data recorded were not tabulated.
BODY WEIGHT:
- Males
- Animals were weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.
- Females
- Animals were weighed on the day of allocation to treatment groups, weekly from the first day of treatment to mating, on Days 0, 7, 14 and 20 post coitum and on Days 1 and 4 post partum. Body weight of females performed at weekly intervals during the pairing period was not reported.
FOOD CONSUMPTION:
Food consumption was recorded at weekly intervals from allocation to pairing. Food consumption of females was also recorded on Days 7, 14 and 20 post coitum, starting from Day 0 post coitum and on Day 4 post partum (starting from Day 0 post partum). For female no. 45 food consumption during post partum period was recorded from day 1 post partum (protocol deviation) and was excluded from group mean calculation.
MORTALITY:
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning from the first day of treatment until a positive identification of mating was made or a maximum period of pairing allowed. The vaginal smear data were examined to detect: a) anomalies of the oestrous cycle; b) the pre coital interval (i.e., the number of paired nights prior to the detection of mating).
Litter observations:
General litter observations
As soon as possible after parturition (Day 0 or 1 post partum), the total litter size (live and dead) was counted, live pups were individually identified within the litter, sexed and examined for external abnormalities. All litters were weighed on Day 1 post partum and examined daily for dead and abnormal pups. The pups were also weighed on Day 4 post partum. All pups found dead were necropsied.
Postmortem examinations (parental animals):
SACRIFICE
- Euthanasia: All animals were killed with carbon dioxide. All viable pups were euthanised by intrascapular injection of Tanax on Day 4 post partum.
- Parental males: The males were killed after the mating of females. Sacrifice procedure started on study day 43 and ended on study day 44 (after 42 and 43 days of dosing respectively).
- Parental females: The females with live pups were killed on Day 4 post partum. Females, which had not given birth after mating, were killed after 25 days of the post coitum period. Female 57, which did not mate was sacrificed on day 36 of the pairing period.
GROSS NECROPSY
The clinical history of the animal was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination. All females killed at term (including the non pregnant and the female which did not mate) were examined for:
- a) external and internal abnormalities;
- b) number of visible implantation sites;
- c) number of corpora lutea (if detectable).
Uteri of females with no visible implantations and of the female which did not mate were immersed in a 20 % solution of ammonium sulphide to reveal evidence of implantation. All pups found dead in the cage were necropsied. All live pups were killed on Day 4 post partum and examined for :
- a) external and internal abnormalities;
- b) sex confirmation by gonadal inspection.
ORGAN WEIGHTS
From all animals completing the scheduled test period the following organs were dissected free of fat and weighed:
- Ovaries
- Testes
- Epididymides
The ratios of organ weight to body weight were calculated for each animal.
HISTOPATHOLOGICAL EXAMINATIONS
The tissues required for histopathological examination are listed in section 4.4.6 of the report. A detailed histopathological examination was performed on testes and epididymides of the control and high dose group male animals. After dehydration and embedding in paraffin wax, sections of
the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin. In addition, sections of the tissues from testes and epididymides of control and high dose males were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS) and the morphological evaluation of the seminiferous epithelium (staging of the spermatogenetic cycle) was performed. The examination was restricted as detailed below:
- a) Tissues specified in section 4.4.6 from all animals in the control and high dose group killed after 4 weeks of treatment.
- b) All abnormalities in all groups.
Postmortem examinations (offspring):
Necropsy findings in decedent pups, or in pups sacrificed on Day 4 post partum were considered as incidental with no indication of a possible F0 treatment effect.
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett's test or a modified t test, depending on the homogeneity of data. The homogeneity of the data was assessed by Bartlett’s test. Statistical analysis of non-continuous variables was carried out by means of the
Kruskal-Wallis test and intergroup differences between the control and treated groups
assessed by a non-parametric version of the Williams test.
The mean values, standard deviations were calculated from actual values in the computer
without rounding off.
Statistical analysis of histopathological findings was carried out by means of the nonparametric
Kolmogorov-Smirnov test.
Reproductive indices:
Group mean values were calculated for all parameters. Data from non-pregnant females were excluded as appropriate. All derived values (e.g., means, percentages, ratios) were calculated within the litter and the group values expressed as a mean of individual litter values.
- Copulatory Index (%) (males and females) = No. of animals mated x 100/No. of animals paired
- Fertility Index (%) (males and females) = No. of animals fertile x 100/No. of animals paired
- Copulatory Interval = Mean number of days between pairing and mating.
Offspring viability indices:
- Pre-birth loss was calculated as a percentage from the formula:
- (No. of visible implantations-total litter size) x 100/No. of visible implantations
- Pup loss at birth was calculated as a percentage from the formula:
- (Total litter size-live litter size) x 100/Total litter size

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- Clinical signs were unaffected by treatment. Soft faeces were noted on the cage tray of the high dose animals during the whole dosing period. Soft faeces were also noted in low dose group animals on day 2 of treatment.
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Body weight and body weight gain were similar between groups A statistically significant body weight increase was noted in low dose females on post partum day 4. This increase was considered to be incidental.
FOOD CONSUMPTION (PARENTAL ANIMALS)
- Food consumption was unaffected by treatment.
REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- No treatment-related anomalies were noted in the oestrus cycle of treated females when compared to controls. Irregular cycle (oestrus never observed) was noted in one control female, no.17.
- Male nos. 12 (control group), 28 (low dose group) and 74 (high dose group) did not induce
pregnancy
- Eight out of 10 paired males of the mid-dose group had positive identification of mating; each one inducing pregnancy. Male no. 58 (paired with female no.57) did not mate after the maximum period allowed (14 days).
- The mean number of copulation plugs and mean pre-coital day’s interval was similar between groups. The number of females, which littered, was nine in each group.
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The number of implantation, pre-birth loss data and gestation length was similar between control and the treated groups.
ORGAN WEIGHTS (PARENTAL ANIMALS)
- Terminal body weight and absolute and relative testes, epididymides and ovaries weights were similar between groups.
GROSS PATHOLOGY (PARENTAL ANIMALS)
- No macroscopic observations were reported in the treated animals that could be considered as treatment-related.
HISTOPATHOLOGY (PARENTAL ANIMALS)
- The histopathological examination of the testes and epididymides did not reveal any evident difference between the findings observed in the treated and control animals that could be considered as treatment-related. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. The abnormalities noted were considered to be incidental and not clearly treatment-related.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Litter size and percentage pup loss at birth and on Day 4 post partum were similar between groups. Sex ratios, calculated as the percentage of males, at birth and on Day 4 post partum did not show differences between groups.
CLINICAL SIGNS (OFFSPRING)
Clinical signs observed in pups during the lactation were considered to be incidental with no relation to parental F0 treatment
BODY WEIGHT (OFFSPRING)
Litter weight and mean pup weight at birth and throughout the lactation period were similar between groups.
GROSS PATHOLOGY (OFFSPRING)
Necropsy findings in decedent pups, or in pups sacrificed on Day 4 post partum were considered as incidental with no indication of a possible F0 treatment effect.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Effects on reproduction and offspring development were evaluated after oral administration of BCQ (60% aqueous solution of APTAC) in male and female rats and their offspring during 4 days of lactation period. The test item was given before and during mating and throughout the gestation period until Day 3 post partum at dosages of 250, 500 and 1000 mg/kg/day.
Clinical signs, body weight and food consumption were not affected by treatment in male or female animals. Presence of soft faeces was noted in all high dose animals during the entire treatment period.
Reproductive parameters such as fertility index, pre-coital interval and copulatory index did not show significant differences in treated groups compared to controls.
Gestation length litter data, sex ratios and implantation losses (pre and post) were unaffected by treatment.
At macroscopic and microscopic examination no treatment related lesions were seen.
On the basis of the results obtained, the dosage of 1000 mg/kg/day could be considered as the NOAEL.