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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:
2012-05-02 to 2012-06-05
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
tert-butyl 3,5,5-trimethylperoxyhexanoate
EC Number:
236-050-7
EC Name:
tert-butyl 3,5,5-trimethylperoxyhexanoate
Cas Number:
13122-18-4
Molecular formula:
C13H26O3
IUPAC Name:
tert-butyl 3,5,5-trimethylhexaneperoxoate

Test animals

Species:
rat
Strain:
other: Hsd.Brl.Han:WIST
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt., 1103, Budapest Cserkesz u. 90., Hungary
- Age at study initiation (start of the mating period):
Females: 10-11 weeks
Males: 27-28 weeks
- Weight at study initiation: 164 g - 246 g
- Fasting period before study: no
- Housing:
before mating: 1-3 females/cage and 1-2 males/cage
mating: 1 male and 1-3 females/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: 13 days for females and 118 days for males

ENVIRONMENTAL CONDITIONS
- Temperature : 20 - 22 °C
- Humidity: 38 - 46 %
- Air changes: 8-12 air exchanges/hour by central air-condition system.
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 2012-05-02 To: 2012-06-05

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Oleum helianthy /sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
25 mg/mL and 75 mg/mL. Formulations were prepared in the laboratory of Toxi-Coop Zrt. daily to three day intervals 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 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: 10, 25, 75 mg/mL
- Amount of vehicle: 2 mL/kg bw
- Lot/batch no: 19/4
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 of treatment. Results were expressed in terms of percentage of the nominal concentration. The deviation from the nominal concentration was within the range of -4 and +7 %.

Analytical method:
- HPLC-UV
- Detector: 210 nm
- Column: HyperPrep HS C 18, 250 x 4.6 mm, 8 µm
- Mobile Phase: Acetonitrile:Water = 9:1 (v:v)
- Flow: 1.2 mL/min
- Injection volume: 50 µL
- Temperature: 25°C
- Retention time: for TBPIN 6.0 min ± 5%

Result:
- The TBPIN concentrations in the samples varied in the range from 96 % to 107 % in comparison to the nominal values.
- Recovery of TBPIN from sunflower oil formulations at four concentration levels (~2, ~8,~10 and ~100 mg/mL) was 99-97-100-99 %
- Test item proved to be stable in sunflower oil formulations on 2 mg/mL, 8 mg/mL, 10 mg/ml and ~100 mg/mL concentration levels at least for 72 hours in refrigerator (5 ± 3°C).
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male: one to three female
- Length of cohabitation: in the morning 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
- Sperm positive females were seperated and caged in groups of 2 to 3 animals. Caging of the females individually was avoided if possible
Duration of treatment / exposure:
The sperm positive females were treated from gestational day 6 to 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. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology.
Duration of test:
GD 5 to GD 19
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
test item treated group: 24 sperm positive females
control group: 25 sperm positive females
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
The dose have been chosen by the Sponsor on the basis a previous study (GLP OECD 421 Reproduction/Developmental Toxicity Screening Study with TBPIN)
Randomization:
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
Treatment volume:
2 mL dose preparation/kg bw

Examinations

Maternal examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day
Individual observation included the check of behavior and general condition. Duration and severity of the clinical signs were recorded. Observations for signs of morbidity and mortality were made twice daily, at the beginning of the working period and in the afternoon.

BODY WEIGHT: Yes (but only for female animals)
- Time schedule for examinations: Body weight of the sperm positive females was measured on gestation days 0, 3, 6, 9, 12, 15, 18 and 20 (accuracy of 1 g). The corrected body weight was calculated for the 20th day of pregnancy (body weight on day 20 minus the weight of the gravid uterus).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was measured between gestation days 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 20 by re-weighing the non-consumed diet (accuracy: 1 g).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organ examined:
The uterus with cervix and the left ovary were removed and weighted. 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 sites, but not fetuses have benn 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 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 a 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 study report contains all data of these animals, too.

The retarded body weight was calculated by sex. A male/female fetus was considered as retarded in body weight, when its weight was below the average minus twofold standard deviation of all control male/female fetuses.
Indices:
- Number of corpora lutea
- Number of implantations
- Number and percent of live fetuses
- Pre-implantation loss
- Post-implantation loss
- Sex distribution
- Fetal body weight (by sex)
- Fetal body weight (with sexes combined)
- External abnormalities/litter
- Visceral abnormalities/litter
- Skeletal abnormalities/litter
Historical control data:
Background pregnancy and fetal data from developmental toxicity studies in the Hsd.Brl.Han:WIST Rat were used for the evaluation of the study results.

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
There were no clinical signs and necropsy findings recorded for the survived dams in the experimental groups.

BODY WEIGHT
There was no indication of an effect of the test item on body weight, corrected body weight and food consumption of the dams in the 20, 50, 150 mg/kg bw/day dose groups.

INTRAUTERINE MORTALITY OF THE CONCEPTUSES, NUMBER OF IMPLANTIONS
There was no effect indicated related to the administration of TBPIN in the intrauterine mortality of the conceptuses and the number of implantations.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: (no adverse effect)

Details on embryotoxic / teratogenic effects:
VIABILITY OF FETUSES AND SEX DISTRIBUTION
There was no effect indicated related to the administration of TBPIN in the viability of fetuses and their sex distribution.

BODY WEIGHT OF FETUSES
The mean fetal weight, was similar in the control and 20 mg/kg bw/day groups. The slight but statistically significant reduction in the bodyweight of the fetuses in the 50 mg/kg bw/day and 150 mg/kg bw/day dose groups might be attributed to an effect of the test item. Since fetal weights were still within the range of the laboratory´s historical control data the weight reduction was considered as non-adverse. Relative placental weight was similar in all experimental groups.

EXTERNAL EXAMINATION
The increased incidence of external variations i.e. growth retarded fetuses in the 150 mg/kg bw/day dose group was within the laboratory´s historical control range and thus, considered as non-adverse. One control fetus had agnathy, microphthalmy and a celft palate and one female of the high dose group had agnathy, an absent oral orifice, one single nasal orifice, open eye and exophthalmy. The presence of the fetal malformations found was considered to be incidental.

VISCERAL EXAMINATION
The multiply malformed control and high dose fetus found at external examination were proven to be multiply malformed also at visceral examination. The control fetus had microphthalmy, agnathy, and dilated lateral brain ventricle, the fetus in the high dose group had exophthalmy, open eye, narrow and peaky head and nose, absent nasal cavity; absent nasal conchae, agnathy, absent palatine rouge, dilated lateral brain ventricles. Other visceral malformations such as a dilated IIIrd brain ventricle in one fetus and microphthalmy in another fetus in the 20 mg/kg bw/day dose group and situs inversus totalis in two control fetuses were found. The visceral malformations were considered to be incidental and were not attributed to the administration of the test item to the dams. The incidence of visceral variations were similar to the vehicle control level.

SKELETAL EXAMINATION
Skeletal malformations in two fetuses of the low dose group and in one fetus of the control group were without a relationship to the test item. Statistically significant skeletal variations were seen in the 20 and in the 150 mg/kg bw/day dose group when the ossification of sternum (3 or less ossified sternebra) was evaluated. Considering that there was no dose response indicated between the low and mid dose group and that the mean percent of the high dose group was slightly above the laboratory´s historical control level, this variation was judged to be in the biological variations.
There was a slightly but statistically significant (p<0.05) increase in the incidence of fetuses with markedly incomplete ossification of the skull-bones in the 50 mg/kg bw/day dose group and in the incidence of incomplete ossification of the skull in the 150 mg/kg bw/day dose group.
Incomplete ossification of the skull bones correlated with the slightly lower mean fetal weight observed at 50 and 150 mg/kg bw/day dose groups. The statistical significance might be partially attributed to the low control value, which was below the mean value seen in the laboratory´s historical control data. However, the data pool of laboratory´s historical control data comprises only a few studies and thus, it is not considered that the whole range of this rat strain is reflected. In general, retarded skull bone ossification is a relatively common observation and may not be a reliable indicator for developmental toxicity (Mylchreest et al., 2005). The maximum incidence of incomplete ossification of 8% observed in this study is within the normal range reported for rats (e.g. Mylchreest et al., 2005, RMS France, 2007). Moreover, since the incomplete ossification was observed in skull bones and not in bones that are normally well ossified the effect is classified as a low significant finding which may not be adverse according to Carney and Kimmel (2007). Overall, incomplete ossification of the skull bones (marked and less marked) seen at 50 mg/kg bw/day and at 150 mg/kg bw/day is considered to be non adverse.


REFERENCES
- Carney, E.W. and C.A. Kimmel (2007): Interpretation of skeletal variations for human risk assessment: delayed ossification and wavy ribs. Birth Defects Res., 80:473-496

- Mylchreest, E.et al. (2005): Evaluation of the developmental toxicity of 8-2 Telomer B Alcohol. Drug and Chemical Toxicology., 28:315-328

- Rapportour Member State (RMS) France (2007): Draft Assessment Report, Initial risk assessment provided by the rapporteur Member State France for the existing active substance Fluazifop-P-Butyl of the third stage (part A) of the review programme referred to the Article 8(2) of Council Directive 91/414/EEC. Volume 3, Annex B, part 4, B.8

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fetuses with incomplete ossification of the skull-bones, reduced fetal body weight at 50 and 150 mg/kg bw dose group.
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
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

Any other information on results incl. tables

LITTERS EVALUATED

In total, there were 19, 20, 21 and 22 evaluated litters in the control, 20, 50 and 150 mg/kg groups, respectively.

MORTALITY

Two pregnant females died in the course of the study, one female in the 50 mg/kg bw/day group on gestational day 17 and one in the 150 mg/kg bw/day dose group on gestational day 9. The death and clinical signs such as reduced body tone and reduced activity shortly before death of the female in the mid dose group and the death and necropsy findings (brownish-yellowish discolouration in the whole lung tissue and dark areas in the liver) of the dam at 150 mg/kg bw/day were considered to be likely without a relationship with an effect of the test item.

Applicant's summary and conclusion

Conclusions:
In conclusion, under the conditions of this study, the no observed effect level (NOEL) for tert-Butylperoxy-3,5,5-trimethylhexanoat for maternal toxicity was 150 mg/kg bw /day and for developmental toxicity was 20 mg/kg bw/day for male and female rats. The no observed adverse effect level (NOAEL) for maternal and developmental toxicity was 150 mg/kg bw/day for male and female rats.

Executive summary:

Groups of 24 sperm-positive female Hsd. Brl. Han: WISTAR rats were treated with TBPIN by oral administration daily at three dose levels of 20, 50 and 150 mg/kg bw/day from day 6 up to and including day 19 post coitum. A control group of 25 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 19, 20, 21 and 22 evaluated litters in the control, 20, 50 and 150 mg/kg groups, respectively. Two pregnant females died in the course of the study, one female in the 50 mg/kg bw/day group on gestational day 17 and one in the 150 mg/kg bw/day dose group on gestational day 9. The death and clinical signs such as reduced body tone and reduced activity shortly before death of the female in the mid dose group and the death and necropsy findings (brownish-yellowish discolouration in the whole lung tissue and dark areas in the liver) of the dam at 150 mg/kg bw/day were considered to be likely without a relationship with an effect of the test item. There were no clinical signs and necropsy findings recorded for the survived dams in the experimental groups. There was no indication of an effect of the test item on body weight, corrected body weight and food consumption of the dams in the 20, 50, 150 mg/kg bw/day dose groups. There was no effect indicated related to the administration of TBPIN in the intrauterine mortality of the conceptuses, the number of implantations, viable fetuses and their sex distribution. The mean fetal weight, was similar in the control and 20 mg/kg bw/day groups. The slight but statistically significant reduction in the bodyweight of the fetuses in the 50 mg/kg bw/day and 150 mg/kg bw/day dose groups might be attributed to an effect of the test item. Since fetal weights were still within the range of the laboratory´s historical control data the weight reduction was considered as non-adverse. Relative placental weight was similar in all experimental groups.

The increased incidence of external variations i.e. growth retarded fetuses in the 150 mg/kg bw/day dose group was within the laboratory´s historical control range and thus, considered as non-adverse.

The presence of the fetal malformations found at external examination (one in the control and one in the high dose group) was considered to be incidental.

The visceral malformations were considered to be incidental and were not attributed to the administration of the test item to the dams. The incidence of visceral variations were similar to the vehicle control level.

Skeletal malformations in two fetuses of the low dose group and in one fetus of the control group were without a relationship to the test item. Statistically significant skeletal variations were seen in the 20 and in the 150 mg/kg bw/day dose group when the ossification of sternum (3 or less ossified sternebra) was evaluated. Considering that there was no dose response indicated between the low and mid dose group and that the mean percent of the high dose group was slightly above the laboratory´s historical control level, this variation was judged to be in the biological variations.

Incomplete ossification of the skull-bones (marked and less marked) was evaluated as variation during skeletal examination. Incomplete ossification of the skull bones correlated with the slightly lower mean fetal weight observed at 50 and 150 mg/kg bw/day dose groups, which was below the mean value seen in the laboratory´s historical control data. However, the data pool of laboratory´s historical control data comprises only a few studies and thus, it is not considered that the whole range of this rat strain is reflected. In general, retarded skull bone ossification is a relatively common observation and may not be a reliable indicator for developmental toxicity (Mylchreest et al., 2005). The maximum incidence of incomplete ossification of 8% observed in this study is within the normal range reported for rats (e.g. Mylchreest et al., 2005, RMS France, 2007). Moreover, since the incomplete ossification was observed in skull bones and not in bones that are normally well ossified the effect is classified as a low significant finding which may not be adverse according to Carney and Kimmel (2007). Overall, incomplete ossification of the skull bones (marked and less marked) seen at 50 mg/kg bw/day and at 150 mg/kg bw/day is considered to be non adverse.

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

NOELmaternal toxicity: 150 mg/kg bw/day

NOELdevelopmental toxicity: 20 mg/kg bw/day

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

NOAELmaternal toxicity: 150 mg/kg bw/day

NOAELdevelopmental toxicity: 150 mg/kg bw/day

REFERENCES

- Carney, E.W. and C.A. Kimmel (2007): Interpretation of skeletal variations for human risk assessment: delayed ossification and wavy ribs. Birth Defects Res., 80:473-496

- Mylchreest, E.et al. (2005): Evaluation of the developmental toxicity of 8-2 Telomer B Alcohol. Drug and Chemical Toxicology., 28:315-328

- Rapportour Member State (RMS) France (2007): Draft Assessment Report, Initial risk assessment provided by the rapporteur Member State France for the existing active substance Fluazifop-P-Butyl of the third stage (part A) of the review programme referred to the Article 8(2) of Council Directive 91/414/EEC. Volume 3, Annex B, part 4, B.8