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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:
2015-04-03 to 2015-05-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: young adult females, males: 20-21 weeks
- Housing: Before mating: 1-3 females per cage 1-2 males per cage
Mating: 1 male and 1-3 females / cage
During gestation: 2 sperm positive females per cage, if not possible 1 sperm positive female per cage
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany, ad libitum
- Water. ad libitum
- Acclimation period: 22 days for females, 85 days for males

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:
other: sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 10 mg/mL, 32.5 mg/mL and 100 mg/mL. Formulations were prepared in the laboratory of Toxi-Coop Zrt. daily or according to the stability data of the formulations.

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Amount of vehicle: A constant treatment volume of 2 mL dose preparation/kg body weight was administered.
- Lot/batch no.: 1410-5159
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
MEKP content was determined in sunflower oil formulations using the previously validated reverse phase HPLC method with UV detection on a Kinetex 2.6u C18 100A column (100x4.6 mm). Detector: 205 nm.
MEKP concentrations in the samples varied in the range from 95% to 100 % in comparison to the nominal values.
Recovery of MEKP from sunflower oil formulations at two concentration levels (~10 and ~200 mg/mL) was 96 and 102 %.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: one male: one to three females
- Length of cohabitation: in the mornings for two to four hours
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
The sperm positive females were treated from gestational day 5 to 19. The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis every day at similar time.
Frequency of treatment:
daily
Duration of test:
gestational day 5 to 19, gross pathology afterwards
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
65 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
only females were treated, at least 24 per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting is based on findings obtained in a 28-Day Repeated Dose Oral Toxicity Study (OECD 407) in the Rat.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g).

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
Food consumption was measured between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet (accuracy: 1 g).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: viscera, uterus with cervix and the ovaries

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]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [ half per litter]
- Head examinations: Yes: [half per litter]
Statistics:
The statistical evaluation of data was performed with the program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant Duncan’s Multiple Range test was used to access the significance of inter-group differences. If significance is the result of the Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Indices:
Pre-implanation loss:
((number of corpora lutea)-(number of implantations))/(number of corpora lutea)*100

Post-implanation loss:
((number of implantations)-(number of live fetuses))/(number of implantations)*100

Sex distribution:
(number of male (female)fetuses)/(number of fetuses)*100

External abnormalities/ litter:
(number of fetuses with abnormality/(number of fetuses)*100

Visceral abnormalities/litter:
(number of fetuses with abnormality/(number of fetuses examined)*100

Skeletal abnormalities/litter
(number of fetuses with abnormality/(number of fetuses examined)*100
Historical control data:
Findings were compared to historical control data.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
The death of one as well as clinical signs of three further pregnant females (squish breath, dyspnoea and breath with open mouth started in all cases immediately after drawing the gavage out of the esophagus) in the 200 mg/kg bw/day group were suspected to be due to an aspiration or inhalation of the test item hence as a consequence of a technical reason probably due to the properties of the test item at this concentration.
The stomach and intestines of the dam which died before scheduled necropsy were empty and filled with gas. The necropsy findings (reddish mottled lungs, dark red liver) of the non-pregnant female in the 200 mg/kg bw/day group that died before scheduled termination might confirm the suspect of aspiration/asphyxia.
There was no mortality and treatment related clinical signs and necropsy findings in the 65 and 20 mg/kg bw/day dose groups.
A slight, but statistically significant reduction in the body weight gain was observed in the food consumption and body weight gain of the dams in the 200 mg/kg bw/day group between gestation days 11 and 17 which was attributed to an effect of the test item. There were no treatment related differences in the food consumption and body weight of the animals in the 65 and 20 mg/kg bw/day groups

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
65 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
The mean number of implantations, intrauterine mortality and sex distribution of the fetuses was not influenced by the treatment. There were no test item related differences in the fetal- and placental weight, body weight retardation and other external, visceral and skeletal variations. There were no fetal malformations found at external and skeletal examination. External hydrocephaly was found in one fetus as a malformation at visceral examination in the 200 mg/kg bw/day dose group which was judged to be unrelated from the treatment based on the historical control data

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment related adverse effects observed up to the highest dose tested.

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAELs) in this read across study were determined as follows:
NOAEL (maternal toxicity): 65 mg/kg bw/day
NOAEL (developmental toxicity): 200 mg/kg bw/day
Executive summary:

The read across substance Reaction mass of butane-2,2-diyl dihydroperoxide and dioxydibutane-2,2-diyl dihydroperoxide (CAS 1338-23-4) was examined for its possible prenatal developmental toxicity in this read across study. Groups of 24 sperm-positive female Hsd. Brl. Han: Wistar rats were treated with the test substance by oral administration daily at three dose levels of 20, 65 and 200 mg/kg bw/day from day 5 up to and including day 19 post coitum. A control group of 24 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test substance in sunflower oil was stable at room temperature for 4 hours and in a refrigerator (5 ± 3 °C) for 3 days at the concentrations of 10 and 200 mg/mL. Analytical control of dosing solutions was performed on the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 95 and 100 % of nominal concentrations at both analytical occasions confirming proper dosing. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day when sperm was detected in the vaginal smear was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

In total, there were 23 evaluated litters each in the control and 20 mg/kg bw/day group, 24 and 21 in the 65 and 200 mg/kg bw/day groups respectively.

The death of one as well as clinical signs of three further pregnant females (squish breath, dyspnoea and breath with open mouth started in all cases immediately after drawing the gavage out of the esophagus) in the 200 mg/kg bw/day group were suspected to be due to an aspiration or inhalation of the test item hence as a consequence of a technical reason probably due to the properties of the test item at this concentration.

The stomach and intestines of the dam which died before scheduled necropsy were empty and filled with gas. The necropsy findings (reddish mottled lungs, dark red liver) of the non-pregnant female in the 200 mg/kg bw/day group that died before scheduled termination might confirm the suspect of aspiration/asphyxia.

There was no mortality and treatment related clinical signs and necropsy findings in the 65 and 20 mg/kg bw/day dose groups.

A slight, but statistically significant reduction in the body weight gain was observed in the food consumption and body weight gain of the dams in the 200 mg/kg bw/day group between gestation days 11 and 17 which was attributed to an effect of the test item. There were no treatment related differences in the food consumption and body weight of the animals in the 65 and 20 mg/kg bw/day groups.

The mean number of implantations, intrauterine mortality and sex distribution of the fetuses was not influenced by the treatment.

There were no test item related differences in the fetal- and placental weight, body weight retardation and other external, visceral and skeletal variations. There were no fetal malformations found at external and skeletal examination. External hydrocephaly was found in one fetus as a malformation at visceral examination in the 200 mg/kg bw/day dose group which was judged to be unrelated from the treatment based on the historical control data.

As a conclusion,based on these observations the No Observed Adverse Effect Level (NOAELs) weres determined as follows:

NOAEL (maternal toxicity): 65 mg/kg bw/day

NOAEL (developmental toxicity): 200 mg/kg bw/day