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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Range-finding study
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Luperox F
- Substance type: organic peroxide
- Physical state: solid
- Analytical purity: 96.7%
- Purity test date: 22/10/2013
- Lot/batch No.: 337131015
- Expiration date of the lot/batch: End of Oct-2014
- Storage condition of test material: At room temperature (20 ± 5 °C)

Test animals

Species:
rat
Strain:
other: RccHanTM: WIST(SPF)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Kreuzelweg 535961 NM Horst / Netherlands
- Age at D0 post coitum: 11 weeks
- Weight at Day 0 Post Coitum: 169 to 203 g
- Fasting period before study: no
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2914C rodent maintenance diet
- Water (e.g. ad libitum): community tap-water
- Acclimation period: 5 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:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily. Vehicle was pre-warmed to a temperature of approximately 40 °C. Luperox F was weighed into a glass beaker on a tared precision balance and approximately 80% of the warm vehicle was added (w/v). The mixture was homogenized using an electrical homogenizer until a clear to colloid-like yellowish solution was obtained. Having obtained a homogeneous mixture, remaining vehicle was added until the required final volume. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 25, 75 and 250 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg
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. Samples of about 2 g of each concentration were taken from the middle to confirm the stability (4 hrs and 8 days at room temperature).
The samples were analyzed by HPLC coupled to an UV detector. The test item was used as the analytical standard.
The application formulations investigated during the study were found to comprise Luperox F in the range from 85.0% to 91.5% and, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of the test item in the preparations was approved because maximal coefficient of variation from the corresponding mean was 0.8% (<15%).
In addition, the test item was found to be stable in application formulations when kept for four hours at room temperature due to recoveries which met the variation limit of 10% from the time zero (homogeneity) mean.
Details on mating procedure:
After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.
The day of mating was designated day 0 post coitum.
Duration of treatment / exposure:
GD 6 to GD 20
Frequency of treatment:
daily
Duration of test:
up to GD 21
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
8
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on a previous OECD 422 study in Han Wistar rats, using dose levels of 100, 300, and 1000 mg/kg/day, resulting in a NOAEL of 100 mg/kg/day based on effects on kidney in parental generation at 300 mg/kg/day. Moreover treatment at 1000 mg/kg was associated with a smaller number of pregnant dams that were noted with less corpora lutea, less implantation sites, less live embryos at first litter check, and a higher postnatal loss. The living pups at 1000 mg/kg and at 300 mg/kg were also noted with less body weight gain until day 4 post partum.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily,during acclimatization and up to day of necropsy).

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Daily from day 0 until day 21 post coitum.

FOOD CONSUMPTION: Yes
- For the following periods: days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 21 post coitum.

WATER CONSUMPTION : No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21
- Organs examined: gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora lutea count and position of fetuses in the uterus
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Head examinations: No
- Soft tissue examinations:No
- Skeletal examinations: No
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 and included in the report.
• 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.
Indices:
Pre- and post-implantation losses, embryonic and fetal deaths, live and dead fetuses, abnormal fetuses, fetal sex ratios and fetal body weights.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Viability / Mortality
All females survived scheduled study period.

Clinical Signs
No clinical signs were noted in females at any dose level.

Food Consumption
Mean food consumption from day 6 to 21 of the gestation period was 20.1, 21.0, 19.9 and 18.4 g/animal/day at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.
Treatment with the test item at the dose level 1000 mg/kg bw/day caused a reversible reduction in food consumption. The reduction was statistically significant from day 9 to 15 post coitum. Afterwards it recovered and although remained slightly lower until the completion of the study, the differences to the control values were only minor.
No effects on food consumption were noted in remaining dose groups.

Body Weights
Mean body weight gain from day 6 to 21 post coitum was 50%, 55%, 53% and 48% whereas mean corrected body weight gain was 14.5%, 16.6%, 12.6% and 7.1% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.
Treatment with the test item at the dose level of 1000 mg/kg bw/day caused a reduction in body weight gain and reduction in corrected body weight gain (body weight gain corrected for gravid uterus weight) in the absence of an effect on absolute body weights. Body weight gain was slightly reduced with statistical significance on days 8, 13, 14 and 15 of the gestation period. Also reduction in corrected body weight gain at this dose level was statistically significant if compared to the control value. Despite the reduction in body weight gain, mean absolute body weights at this dose level were similar to the respective control values during the most of the study period.
In one female at the high dose level (no. 28) a body weight loss by 10% was recorded after uterus weight subtraction. Because no body weight loss was recorded in any further female, observation in female no. 28 was considered probably not to be a result of the treatment with the test item but incidental.
In remaining dose groups body weights, body weight gain and corrected body weight gain were not affected by the treatment.
At the dose level of 100 mg/kg bw/day, absolute body weights were statistically significantly higher at the end of the gestation period if compared to the control value. In the absence of similar effect at the higher dose levels, this observation was considered not to be related to the treatment.

Reproduction Data
With exception for one female (no. 9) at the dose level of 100 mg/kg bw/day, all females were pregnant and had living fetuses at termination.
Mean post implantation loss was 0.5, 0.6, 1.0 and 0.3 per dam whereas mean number of living fetuses at termination was 10.4, 11.3, 12.1 and 12.3 per dam, both cited in order of ascending dose levels.
The relevant reproduction data, post-implantation loss and number of fetuses, were not affected by the treatment.

Macroscopic Findings
During macroscopical examination no findings were noted at any dose level.

Effect levels (maternal animals)

Remarks on result:
other: dose range finding study for definitive study

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
External Abnormalities and Variations
No findings were noted during external examination of fetuses.

Sex Ratios
Percentage of male fetuses was 45.8%, 48.1%, 53.6% and 60.2% at the dos levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.
In the high-dose group higher portion of male than female fetuses was found; increase in percentage of male fetuses was statistically significant. However, due to the low group size, the significance of the difference may be incidental. Furthermore, at the high dose level a single female (no. 26) had 11 male fetuses while only 2 female fetuses, which contributed considerably to the shift in the fetal sex distribution. For these reasons, increased percentage of male fetuses was considered most probably not to be related to the treatment with the test item.
No significant differences in fetal sex distribution were noted in remaining dose groups.

Body Weights
Mean body weights of fetuses were 5.0 g, 5.1 g, 4.9 g and 4.9 g at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.
Fetal body weights were not affected by the treatment with the test item at any dose level.

Effect levels (fetuses)

Remarks on result:
other: dose range finding study for definitive study

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Treatment with the test item at the dose level of 1000 mg/kg bw/day caused a reversible reduction in food consumption accompanied by slightly reduced body weight gain and a reduction in corrected body weight gain. These effects were not accompanied by any effects on absolute body weight gain and therefore considered not to be adverse. No further signs of general toxicity were observed at any dose level.
The relevant reproduction data, post-implantation loss and number of fetuses, were not affected by the treatment. No indication of an effect on fetal development was noted at any dose level.
Executive summary:

The purpose of this study was to detect effects on the pregnant rat and development of the embryo and fetus consequent to exposure of the female to the Luperox F from day 6 post coitum (implantation) to day 20 post coitum. Luperox F was administered to female rats at the following dose levels: Group 1: 0 mg/kg body weight/day (control group) Group 2: 100 mg/kg body weight/day Group 3: 300 mg/kg body weight/day Group 4: 1000 mg/kg body weight/day. Each group consisted of eight females. 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).

All females survived scheduled study period. No clinical signs were noted in females at any dose level. At the dose level of 1000 mg/kg bw/day, a reversible reduction in food consumption was observed. At the dose levels of 100 and 300 mg/kg bw/day, food consumption was not affected by the treatment with the test item. At the dose level of 1000 mg/kg bw/day, a reduction in body weight gain and in corrected body weight gain was noted in the absence of an effect on absolute body weights. At the dose levels of 100 and 300 mg/kg bw/day, body weight gain and absolute body weights were not affected by the treatment with the test item. One female at the low dose group was not pregnant. All remaining females were pregnant and had living fetuses at termination. The relevant reproduction data, post-implantation loss and number of fetuses, were not affected by the treatment. During macroscopical examination no findings were noted at any dose level. No findings were noted during external examination of fetuses. Fetal sex ratio was considered not to be affected by the treatment with the test item. No effects on fetal body weights were noted at any dose level.