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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:
2012-10-17 to 2013-02-19
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Purity batch
> 99.0 % 7.32% active oxygenBatch 247408973
99.27 % 9407226339; 247408999
99.6 % 0410105225413
> 97 % 4289070300; 0419101226056
99.1 % 11600 0010-108
99.3 % 247413988
99.4 % 7.35 % active oxygen Batch 247409800
98.9 % 0707518256
> 99.1 % 247412384
98.5 % 0707603153
99.0 % 1703465433
99.5 % 150726; 247413309
99.3 % 000055055

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: Females: Young adult and nulliparous females, 10-11 weeks of age at start of the mating period. Males: experienced males 35-37 weeks of age at start of the mating period.
- Fasting period before study: no
- Housing: Before mating: 1-3 females per cage, 1-2 males per cage. Mating: 1 male and 1-3 females / cage. During gestation: 2-3 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" ad libitum
- Water: tap water ad libitum
- Acclimation period: 20 days for females and 181 days for males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 36-46
- Air changes (per hr): 8-12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2012-10-17 To: 2012-11-13

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Helianthy Annui Oleum Raffinatum / Sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations were prepared in the laboratory of Toxi-Coop Zrt. daily or according to the stability data of the formulations (based on previous analytical measurements performed in the Laboratory of Toxi-Coop Zrt).

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble and not stable in water therefore oleum helianthy was used for preparing formulations appropriate for oral administration. Oleum helianthy /sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 100, 200, 500 mg/mL
- Amount of vehicle: 2 mL/kg bw
- Lot/batch no.: 19T3
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control (concentration, homogeneity) of dosing solutions was performed in the Laboratory of Toxi-Coop Zrt. on the first and last treatment weeks.
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation. The test item concentrations in the samples were found to be 94 – 106 % in comparison to the nominal values.

Analytical method:
- HPLC-UV
- Detector: 210 nm
- Column: HyperPrep HS C18, 250 x 4.6 mm, 8 μm,
- Mobil Phase: Acetonitrile: Water = 9 : 1 (v/v)
- Flow: 1.2 mL/min
- Injection volume: 50 μL
- Temperature: 25 °C
- Retention time: 5.3 min ± 10 %
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/3
- Length of cohabitation: in the mornings for two to four hours
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
GD 5 to GD 19
Frequency of treatment:
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.
Duration of test:
GD 5 to GD 19
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 Females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses have been chosen by the Sponsor on the basis of a previous study (GLP OECD 421 Reproduction/Developmental Toxicity Screening Study of Tert.-Butylperoxy- 2-ethylhexanoat in the Wistar Rat).
- Rationale for animal assignment: The sperm positive females were allocated to each experimental group on each mating day in such a way that the group averages of the body weight were as similar as possible on the first day of gestation. If possible, females paired with the same male were allocated to different groups on the same mating day.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for signs of morbidity and mortality were made twice daily, at the beginning of the working period and in the afternoon.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a day, after treatment at approximately the same time. Individual observation included the check of behavior and general condition.
Duration and severity of the clinical signs were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days 0, 3, 5, 8, 11, 14, 17 and 20

FOOD CONSUMPTION: Yes
- Time schedule for examinations: 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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined:
the uterus with cervix and the left ovary were removed and weighed. The right ovary was placed into a Petri dish after removal. After removing the uterus gross pathology of dams' viscera was performed. The number of corpora lutea in each ovary and implantation sites in each uterine horn, live fetuses, early and late embryonic death and fetal death were counted. Animals, in which unambiguous implantation sites, but not fetuses have been found, were considered as pregnant.

EXAMINATION OF PLACENTAL SIGNS:
All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the 13th gestational day. If negative on day 13, the examination was repeated on day 14 of gestation.
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 was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan’s Multiple Range test was used to access the significance of inter-group differences. If significance was 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.
Dams or litters were excluded from the data evaluation in cases of:
- Any disease or death of the dam unrelated to the treatment (total exclusion)
- Non pregnant females or dams with 3 or less implantations independent of their viability (total exclusion)
Although these animals were excluded from the data evaluation the final report contains all data of these animals, too.
A male/female fetus was considered as retarded in body weight, when its weight is below the average minus twofold standard deviation of the control male/female fetuses.
Indices:
- Number of corpora lutea
- Number of implantations
- Number and percent of live fetuses
- Pre-implantation loss (%):
(Number of corpora lutea - Number of implantation) / Number of corpora lutea x 100
- Post-implantation loss (%):
(Number of implantations - Number of live foetuses) / Number of implantations x 100
- Sex distribution (%):
Number of Male (Female) foetuses / Number of foetuses x 100
- External abnormalities per litter (%)
Number of fetuses with abnormality / Number of fetuses x 100
- Visceral abnormalities per litter (%)
Number of fetuses with abnormality / Number of foetuses examined x 100
- Skeletal abnormalities per litter (%)
Number of fetuses with abnormality / Number of foetuses examined x 100
Historical control data:
Historical contral data are available and were used for evaluation of study results.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Alopecia was found sporadically without a dose response in the females. Salivation was recorded in association the treatment in nine of 23 females in the 400 mg/kg bw/day group and in all of the dams of the 1000 mg/kg bw/day dose group, directly after treatment. This was judged to be in relationship with the taste of the test item.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One pregnant female in the control group died in the course of the study on gestational day 20. The death was considered to be due to the intrauterine autolyzing of dead embryos and fetuses. This dam had no clinical signs before death but lost weight.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no indication of an effect of the test item on body weight development of the dams in the 200 and 400 mg/kg bw/day dose groups.
The body weight gain was statistically significant (p<0.01) reduced on the first three days of treatment in the 1000 mg/kg bw/day group. This finding was considered as test item related but not adverse. Between gestational days 8 and 11 it turned to an increased body weight gain with a statistical significance (p<0.05).
There were no dose related differences in the corrected body weight and corrected body weight gain of the dams in the experimental groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was no indication of an effect of the test item on the food consumption of the dams in the 200 and 400 mg/kg bw/day dose groups.
There was a statistically significantly (p<0.01) reduced food consumption on the first six days of treatment in the 1000 mg/kg bw/day dose group related to the treatment with the test item. Statistical significant increases (p<0.05) were indicated in two occasions (once before the treatment period and once at the beginning of it) in the food consumption of the animals in the 200 mg/kg bw/day dose group which are not associated with the test item.
Food efficiency:
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
Behaviour (functional findings):
not examined
Immunological findings:
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 macroscopic alterations recorded for the dams during necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
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

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weight was similar in the control, 200 and 400 mg/kg bw/day groups. The slight but statistically significant (p<0.01) reduction in the mean body weight of the male and female fetuses in the 1000 mg/kg bw/day group might be attributed to the statistically significant reduced body weight gain and statistically significant lower food consumption of the dams between gestational day 5 and 8 and 5 and 11 respectively in this dose group. Although a statistical significance in the fetal weight in the 1000 mg/kg bw/day group was noted, the value was in the range of the historical control data and therefore considered to be non-adverse. Placental weight was similar in all experimental groups. There was a statistically significant increase indicated in the relative placental weight in the 1000 mg/kg bw/day dose group (p<0.05), however it was below the historical control level.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no dose related significant difference in the intrauterine mortality of the conceptuses, the number of implantations, viable fetuses and their sex distribution. The number of late embryonic death increased slightly but statistically significantly (p<0.05) in the 400 mg/kg bw/day dose group and without a statistical significance in the 1000 mg/kg bw/day group. No dose response was indicated and the values are in the range of the historical control data. There was no statistical significance indicated in the mean percentage value of the late embryonic death in the experimental groups.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
The number of evaluated fetuses was 228, 190, 239 and 192 at external and 114, 96, 120 and 97 at visceral examination in the control, 200, 400 and 1000 mg/kg bw/day groups, respectively.
The incidence of visceral abnormalities (malformations and variations) was statistically significant (p<0.05) higher in the 1000 mg/kg bw/day dose group. However, the number of affected fetuses is well within the historical control range and therefore considered to be of no biological relevance.

- Malformations
Umbilical hernia was found in one fetus as a malformation at external and visceral examination in the 1000 mg/kg bw/day dose group which was neither proven nor closed out to be in relationship with an effect of the test item. According to the experience of this laboratory and the scientific literature umbilical hernia is a rather common finding in the tester strain used. Therefore, this single event is not considered to be an indication for a test substance related effect.

- Variation
There was no increased incidence of external and visceral variations in the test item treated groups. Visceral variations such as bilateral hydroureter or hydroureter with dilated renal pelvis occurred with a very low incidence without significant difference among the experimental groups, including control.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The number of examined fetuses was 114, 94, 119 and 95 in the control, 200, 400 and 1000 mg/kg bw/day respectively.
The incidence of skeletal abnormalities (malformations and variations) increased with a statistical significance (p<0.01) due to the increase in the variations (p<0.01) in the 1000 mg/kg bw/day dose group.

-Malformation
Malformations were recorded such as a bipartite thoracic centrum and dumb-bell shaped cartilage of thoracic centrum in two fetuses in the control and in one in the 1000 mg/kg bw/day dose group without a relationship with the test item.

- Variation
Incomplete ossification of the skull, bipartite supraoccipital, incompletely ossified or misaligned sternebrae, wavy ribs, dumb-bell shaped or bipartite vertebral centra, incomplete or asymmetric ossification of sacral arches and asymmetric or incomplete ossification of metacarpal or metatarsal, were evaluated as variations during the skeletal examination. There was a slightly but statistically significant (p<0.01) increase in the incidence of fetuses with incomplete ossification of the skull-bones and metacarpal/metatarsal in the 1000 mg/kg bw/day dose group. At this dose level, reduced body weight and food consumption of the dams might explain this slight delay in ossification. Therefore, this variation is not considered to be an indication for developmental toxicity. This assumption is supported by Mylchreest et al. (2005), who stated that retarded skull bone ossification is a relatively common observation and may not be a reliable indicator for developmental toxicity.
Visceral malformations:
no effects observed
Description (incidence and severity):
The number of evaluated fetuses was 228, 190, 239 and 192 at external and 114, 96, 120 and 97 at visceral examination in the control, 200, 400 and 1000 mg/kg bw/day groups, respectively.
The incidence of visceral abnormalities (malformations and variations) was statistically significant (p<0.05) higher in the 1000 mg/kg bw/day dose group. However, the number of affected fetuses is well within the historical control range and therefore considered to be of no biological relevance.

- Malformations
Umbilical hernia was found in one fetus as a malformation at external and visceral examination in the 1000 mg/kg bw/day dose group which was neither proven nor closed out to be in relationship with an effect of the test item. According to the experience of this laboratory and the scientific literature umbilical hernia is a rather common finding in the tester strain used. Therefore, this single event is not considered to be an indication for a test substance related effect.

- Variation
There was no increased incidence of external and visceral variations in the test item treated groups. Visceral variations such as bilateral hydroureter or hydroureter with dilated renal pelvis occurred with a very low incidence without significant difference among the experimental groups, including control.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
References
- Mylchreest, E., Munley, S.M., and Kennedy Jr., G.L. (2005) Evaluation of the Developmental Toxicity of 8-2 Telomer B Alcohol. Drug and Chemical Toxicology, 28:315-328.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Oral treatment of pregnant Hsd. Brl. Han: WISTAR rats from gestation day 5 up to day 19 with Tert.-Butylperoxy- 2-ethylhexanoat at the dose levels of 200, 400 and 1000 mg/kg bw/day did not cause death and necropsy findings. The test item did not reveal any adverse effect on the pregnancy and the intrauterine mortality of the conceptuses, the number of viable fetuses and their sex distribution. Further the test substance did not increase significantly the incidence of external and visceral variations, and caused no skeletal malformations. The slight delay in ossification in fetuses of the 1000 mg/kg bw/day dose group is considered to be non-adverse.
Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL maternal toxicity: 1000 mg/kg bw/day
NOAEL developmental toxicity: 1000 mg/kg bw/day
Executive summary:

Groups of 24 sperm-positive female Hsd. Brl. Han: WISTAR rats were treated with Tert.-Butylperoxy- 2-ethylhexanoat by oral administration daily at three dose levels of 200, 400 and 1000 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.

During the study, mortality was checked for 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. A 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 21, 19, 23 and 19 evaluated litters in the control, 200, 400 and 1000 mg/kg groups, respectively. One pregnant female died in the course of the study in the control group which was found dead on gestational day 20 due to total intrauterine death.

No other clinical signs than alopecia in a few females unrelated to the treatment and salivation in the 400 and 1000 mg/kg bw/day dams immediately after treatment were observed. This was attributed to be an effect of the treatment, however as non-adverse.

There were no findings observed at necropsy.

There was no indication of an effect of the test item on body weight development and food consumption of the dams in the 200 and 400 mg/kg bw/day dose groups. The statistically significantly (p<0.01) reduced body weight gain on the first three days of treatment and the statistically significantly (p<0.01) reduced food consumption in the first week of treatment in the 1000 mg/kg bw/day dose were considered as test item related but not adverse.

There was no dose related significant difference in the intrauterine mortality of the conceptuses, the number of implantations, viable fetuses and their sex distribution. The number of late embryonic death increased slightly but statistically significant (p<0.05) in the 400 mg/kg bw/day dose group (and without a statistical significance in the 1000 mg/kg bw/day group) and was around the historical control level. There was no statistical significance indicated in the mean percentage value of the late embryonic death in the experimental groups. The statistically significant (p<0.01) reduction in the body weight of the male and female fetuses in the 1000 mg/kg bw/day group, which was in the range of historical control data, might be a consequence of the statistically significant reduction of the food consumption between gestation day 5 and 11 and lower mean body weight gain of the dams between gestation day 5 and 8. Placental weight was similar in all experimental groups. There was a statistically significant increase indicated in the relative placental weight in the 1000 mg/kg bw/day dose group, however it was below the historical control level. The distribution of external and visceral variations was homogenous in the test item treated groups, however the incidence of visceral abnormalities (variations and malformation) increased statistically significant. The occurrence of single type of variations was in the historical control range. Umbilical hernia as a malformation occurred in one fetus in the 1000 mg/kg bw/day dose group which was neither proven nor closed out to be in relationship with an effect of the test item. According to the experience of the formar laboratory of this facility and the scientific literature umbilical hernia is a rather common finding in the rat strain used. There was no test item related effect indicated at skeletal examination of the fetuses in the 200 and 400 mg/kg bw/day dose group. The incidence of the fetuses with skeletal variations increased significantly (p<0.01) in the 1000 mg/kg bw/day dose group due to the higher incidence of the delayed ossification of skull and metacarpal/metatarsal. These findings might be attributed to the effects on body weight gain and food consumption of the dams observed in the 1000 mg/kg bw/day dose group. Based on these observations, and the assumptions in the international literature that retarded skull bone ossification is a relatively common observation and may not be a reliable indicator for developmental toxicity (Mylchreest et al. (2005)), the slight delay in ossification is considered to be non-adverse. Skeletal malformations were found only in the control and 1000 mg/kg bw/day dose group with an incidence of 2 and 1 respectively, thus the test item did not induced skeletal malformations.

 

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL maternal toxicity: 1000 mg/kg bw/day

NOAEL developmental toxicity: 1000 mg/kg bw/day