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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 06 December 2011 to 02 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline 422. GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (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
Details on test material:
- Physical state: Clear colourless to slightly yellowish viscous liquid
- Purity: UVCB
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Age at start F0-treatment: Approximately 10 weeks.
- Weight at study initiation: Males: Mean: 284-286 g. Females: Mean: 196-197 g
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females
were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left
without their dam or a bottle filled with warm water for longer than 30-40 minutes.
General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri,
Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: Acclimatization F0: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 ºC
- Humidity (%): 40 to 70%
- Air changes (per hr): approximately 15 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle.

IN-LIFE DATES: From: 06 December 2011 To: 02 April 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for
density (1.084 g/cm3) of the test substance.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations analysed in the formulations at dose concentrations of 10, 30, 100 and 200 mg/ml were in agreement with target concentrations
(i.e. mean accuracies between 85% and 115%).

The formulations at the entire range were homogeneous (i.e. coefficient of variation ≤ 10%).

Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.
Details on mating procedure:
- M/F ratio per cage: 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.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.

Females were exposed for 41-54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
Frequency of treatment:
Once daily for 7 days per week.
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes
Details on study design:
- Dose selection rationale: Based on the results of a 10-day dose range finding study, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg bw/day.

Examinations

Maternal examinations:
MORTALITY / VIABILITY: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals. Once prior to start of
treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed
on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Weekly, except for males and females which were housed together for mating and for females without evidence of
mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

OTHER:
General reproduction data:
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Fetal examinations:
PARAMETERS EXAMINED
Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined.
Clinical signs: At least once daily, detailed clinical observations were made for all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-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 was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

The following additional methods of statistical analysis were used:
Locomotor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.

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.
Indices:
Mating index (%), Fertility index (%), Conception index (%), Gestation index (%), Duration of gestation.
Percentage live males at First Litter Check, Percentage live females at First Litter Check, Percentage of postnatal loss Days 0-4 of lactation, Viability index.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period.

No clinical signs of toxicity were noted during the observation period. Incidental findings that were noted for control and/or treated animals included
alopecia and scabbing on different parts of the body. These findings occurred within the range of background findings to be expected for rats of this
age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

BODY WEIGHT AND WEIGHT GAIN
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
Food consumption before or after allowance for body weight was similar between treated and control animals.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Staging of spermatogenesis did not provide any evidence of test article related impairment to the spermatogenetic cycle.

REPRODUCTIVE DATA
The mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites were considered to be unaffected by treatment up to 1000 mg/kg bw/day.

There were 7, 8, 10 and 10 litters available for evaluation in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. In the control group, one female was not mated and two other females were not pregnant. At 100 mg/kg, one female was not mated and another female was not pregnant. In the absence of a treatment-related distribution, these findings were not considered toxicologically relevant.

ORGAN WEIGHTS
There were no toxicologically relevant changes in organ weights or organ to body weight ratios seen with treatment up to 1000 mg/kg.

Statistically significant changes included lower seminal vesicle organ weights (absolute and relative to body weight) for males at 100, 300 and 1000
mg/kg bw/day, higher relative brain weights for males at 300 mg/kg bw/day and higher relative adrenal weights for males at 1000 mg/kg bw/day. These findings were considered not to be toxicologically relevant as changes were very slight, a treatment-related trend was absent, no corroborative
histopathological changes were evident and/or the reproductive ability of the males was not affected.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

Incidental findings seen for control and/or treated animals included pelvic dilation of the kidneys (unilateral), greenish, soft nodule on the tail of the
epididymides (both unilateral and bilateral), redbrown discoloration of the thymus, enlarged mandibular lymph nodes (unilateral), tan discoloration of
the clitoral glands, and alopecia on different sides of the body. The incidence of these macroscopic findings was within the background range of
findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. They were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic observations attributed to the test article.
No significant abnormalities were seen in the reproductive organs of the paired animals that failed to conceive.

GESTATION
Gestation index and duration of gestation were in the same range for treated groups and controls.

PARTURITION / MATERNAL CARE
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
No deficiencies in maternal care were observed.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING)
The number of pups found dead or went missing during the first days of lactation were 1, 1, 3 and 1 in the control, 100, 300 and 1000 mg/kg bw/day groups. Pups that went missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS (OFFSPRING)
There were no clinical signs noted.

BODY WEIGHT (OFFSPRING)
Body weights of pups were considered to have been unaffected by treatment.

SEXUAL MATURATION (OFFSPRING)
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

GROSS PATHOLOGY (OFFSPRING)
Incidental macroscopic findings of pups that were found dead included no milk in the stomach and partial cannibalism. There were no macroscopic findings noted among surviving pups. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Effect levels (fetuses)

Remarks on result:
not measured/tested

Fetal abnormalities

Abnormalities:
not examined

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day was derived.
Executive summary:

The test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300, or 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41- 54 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Parental results: No parental toxicity was evident up to the highest dose level tested (1000 mg/kg bw/day). The statistically significantly higher locomotor activity (total movements and ambulations) noted for females at 1000 mg/kg bw/day as compared to controls was not considered adverse, since changes were relatively slight, and occurred in the absence of concurrent clinical signs or changes in other functional observation tests. No treatment-related effects were noted on any of the remaining parental parameters investigated in this study (i.e. clinical appearance, hearing ability, pupillary reflex, static righting reflex and grip strength, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

Reproductive results: No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites).

Developmental results: No developmental toxicity was observed up to the highest dose level tested 1000 mg/kg bw/day). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day was derived.