Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 13 June 2013; Experimental Completion Date: 06 January 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidenlines and/or minor methodlogical deficiences, which do not affect the quality of relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147 (24 November 2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Identification : Tert-butyl perbenzoate CAS# 614-45-9
Description : Clear colorless liquid
Purity : 98.8%
Batch Number : 0906225400
EC-Number : 210-382-2
CAS Number : 614-45-9
Date Received : 30 April 2013
Storage Conditions: Approximately 4°C in the dark. Test Item was removed from storage and allowed to reach room temperature before use. Expiry Date : 01 July 2019

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited. Animals were received in two deliveries each containing two separate batches of females on either Day 0 or Day 1 of presumed gestation. The day that positive evidence of mating was observed at the supplier was designated Day 0 of gestation. On arrival the females weighed 194 to 262g.

The animals were housed individually in solid-floor polypropylene cages with stainless steel lids furnished with softwood flakes. The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidity were included in the study records. The target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; there were no deviations from the target range for temperature. Deviations from the target range for relative humidity were observed but were considered not to have affected the purpose or integrity of the study.

The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.






Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Test Item Formulation:
For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in Corn Oil. Corn oil was used as the vehicle for this study, as it had been successfully used in previously toxicity work. The stability and homogeneity of the test item formulations were determined as part of this study. Results showed the formulations to be stable for at least twenty one days. Bulk formulations were therefore prepared twice and divided into daily aliquots which were stored at approximately +4 °C in the dark.

Representative samples were taken of each bulk test item formulation and were analyzed for concentration of Tert-butyl perbenzoate CAS# 614-45-9.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Representative samples were taken of each bulk test item formulation and were analyzed for concentration of Tert-butyl perbenzoate CAS# 614-45-9. The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

The results indicate that the prepared formulations were within ± 5% of the nominal concentration.

Details on mating procedure:
Details on mating procedure not included in report. The test item was administered to mated female rats.
Duration of treatment / exposure:
Mated females were dosed once daily by gavage, from Day 3 (prior to implantation) to Day 19 of gestation (day prior to expected parturition).
Frequency of treatment:
Once daily.
Duration of test:
All females were terminated on Day 20 of gestation.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
24 females per dose group (including control).
Control animals:
yes, concurrent vehicle
Details on study design:
Four dose groups (control, low, intermediate and high) each comprising twenty four mated females (to ensure as far as possible twenty pregnant females per group) were used. Dose levels of 100, 300 and 1000 mg/kg bw/day were chosen in collaboration with the sponsor based on available toxicity data including a rat OECD 421 toxicity study. In the OECD 421 study a dosage of 1000 mg/kg bw/day was generally well tolerated by females, both prior to and during pregnancy.

As no effect of treatment was observed for pre-implantation loss in a previous rat OECD 421 toxicity study, mated females were dosed once daily by gavage, from Day 3 (prior to implantation) to Day 19 of gestation (day prior to expected parturition). Control animals were treated in an identical manner with the vehicle alone.

Examinations

Maternal examinations:
CLINICAL OBSERVATIONS:
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.

BODY WEIGHT:
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

FOOD CONSUMPTION:
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

WATER CONSUMPTION:
Water intake was observed daily by visual inspection of the water bottles for any overt changes.

POST MORTEM:
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.










Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes; The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight

The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.

Implantation types were divided into:

Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes

All implantations and viable fetuses were numbered according to their intrauterine position.







Fetal examinations:
The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were transferred to 90% industrial methylated spirits (IMS) in distilled water and examined for visceral anomalies under a low power binocular microscope. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed in 70% IMS in distilled water. The fetuses were eviscerated, processed and the skeletons stained with alizarin red S. The fetuses were examined for skeletal development and anomalies. Following examination fetuses that were examined for skeletal development were placed in 50% glycerol.
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:

Body weight and body weight change (including adjustment for the contribution of the gravid uterus), food consumption, gravid uterus weight, absolute and body weight relative organ weights, litter data and fetal litter and placental weights: Bartlett’s test for homogeneity of variance. Where the data were shown to be homogeneous one way analysis of variance and, if significant, Dunnett’s multiple comparison test was employed; where the data were found to be non-homogeneous Kruskal-Wallis and, if significant, pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test was employed.

Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.

Probability values (p) are presented as follows:

p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Body weight

Details on maternal toxic effects:
Adult Responses:

A summary of female performance is given in Table 1 (see attached background material).

Mortality:
There were no unscheduled deaths.

Clinical Observations:
A summary incidence of daily clinical observations is given in Table 2 (see attached background material).

At 1000 mg/kg bw/day, the majority of females showed incidences of increased post-dosing salivation between Day 10 and Day 19 of gestation. At 300 mg/kg bw/day, increased post-dosing salivation was restricted to just one female on Day 10 and two females on Day 19 of gestation. Increased post-dosing salivation is commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and, in isolation, is considered to be of no toxicological importance.

There were no other clinical signs apparent during the study.

Body Weight:
Group mean body weights and standard deviations are given in Table 3 and presented graphically in Figure 1. Group mean body weight gains and adjusted body weights and standard deviations are given in Table 4 and Table 5 (see attached background material).

At 1000 mg/kg bw/day, body weight gain was generally lower than control throughout gestation, with differences frequently attaining statistical significance and this lower weight gain was still apparent after values were adjusted for the contribution of the gravid uterus.

Body weight gain during gestation, included where adjusted for the contribution of the gravid uterus, was considered to be unaffected by treatment at 100 and 300 mg/kg bw/day.

Food Consumption:
Group mean food consumptions are given in Table 6 and presented graphically in Figure 2 (see attached background material).

At 1000 mg/kg bw/day, food consumption was generally lower than control between Day 3 and Day 8 of gestation, with differences attaining statistical significance. Thereafter, although food intake remained lower than control, differences failed to attain statistical significance and the magnitude of the differences observed was such that they were considered to represent normal biological variation.

Food consumption appeared unaffected by treatment at 100 and 300 mg/kg bw/day.

Water Consumption:
Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Post Mortem Studies:
A summary incidence of female necropsy findings is given in Table 7 (see attached background material).

No macroscopic abnormalities were detected for adult animals at Day 20 of gestation.














Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Litter Responses:
Litter Data:
Summary fetal data is given in Table 8 (see attached background material).

There was no effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio and pre and post-implantation losses at 100, 300 and 1000 mg/kg bw/day.

At 300 and 1000 mg/kg bw/day, lower pre-implantation losses attained statistical significance when compared with control; these differences were considered to reflect normal biological variation particularly as an improvement in survival is unlikely to represent an adverse effect of treatment.

Litter Placental and Fetal Weights:
Group mean litter data values are given in Table 8 (see attached background material).

At 1000 mg/kg bw/day, mean fetal weight for both sexes was lower than control, resulting in lower mean litter weights; differences from control attaining statistical significance. There was considered to be no effect of treatment on placental weights.

There were no effects of maternal treatment on mean fetal, litter or placental weights at 100 or 300 mg/kg bw/day.

External Fetal Examinations:
Summary fetal external findings are given in Table 9 (see attached background material).

At 1000 mg/kg bw/day, there was a higher incidence of fetuses that were small in appearance compared with control; this finding was consistent with the lower fetal weight observed at this dosage.

At 100 and 300 mg/kg bw/day external fetal findings were unremarkable and did not indicate any effect of maternal treatment.

Detailed Visceral Fetal Examinations:
Summary fetal visceral findings are given in Table 10 (see attached background material).

At 1000 mg/kg bw/day, there was an increased incidence of fetuses/litters showing non-uniform patterning of the rugae, kinked/dilated ureter(s), increased renal pelvic cavitation, absent renal papila and partially undescended thymus lobe, with the percentage incidence of these findings frequently attaining statistical significance. The increased incidence of these findings was principally responsible for a statistically significant increased incidence in the overall number of fetuses showing visceral findings.

At 300 mg/kg bw/day, there was an increased incidence of fetuses/litters showing kinked/dilated ureter(s) compared with control; although the percentage incidence of these findings did not attain statistical significance when compared with control, it was higher than the recent background control incidence. The overall percentage incidence of visceral findings was not greatly increased in comparison to the control and failed to attain statistical significance.

At 100 mg/kg bw/day, the incidence and type of visceral findings observed did not indicate any effect of maternal treatment.

Detailed Skeletal Fetal Examinations:
Summary fetal skeletal findings are given in Table 11 (see attached background material).

At 1000 mg/kg bw/day, there was clear effect of treatment on a large number of ossification parameters affecting most regions of the skeleton, with the number of fetuses/litters affected being increased compared with control and differences frequently attaining statistical significance. These parameters included unossified hyoid and incomplete ossification of the nasal, frontal, parietal, interparietal, occipital, squamosal, premaxilla, presphenoid bones of the skull, incomplete ossification of the cervical arch, thoracic centrum and sacral arch, unossified thoracic centrum and sacral (neural) arch, less than 4 caudal vertebrae ossified, unossified/ incomplete ossification of the sternebra, incomplete ossification of the ischium, unossified/incomplete ossification of the pubis, unossified/incomplete ossification of the metacarpals and metatarsals and incomplete ossification of the humerus and femur. There was also a lower incidence of fetuses showing ossification of the ventral arch of the first cervical vertebra (vertebra 1).

At 100 and 300 mg/kg bw/day, the incidence and type of skeletal findings observed did not indicate any effect of maternal treatment. The percentage incidence of occasional skeletal parameters did attained statistical significance but these differences were considered to reflect normal biological variation.








Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Discussion

Treatment at 1000 mg/kg bw/day was associated with lower maternal body weight gain during gestation and an initial effect on food consumption. While part of the lower overall weight gain observed was attributable to lower litter weight due to reduced fetal weight, an underlying effect of the pregnant dam was still present when body weight gain was adjusted for the contribution of the gravid uterus.

In-utero survival of the developing conceptus appeared unaffected by maternal treatment at 1000 mg/kg bw/day with both pre and post-implantation losses being lower than control. This was despite a clear reduction in fetal weight which resulted in lower litter weight at this dosage. The lower foetal weight at 1000 mg/kg bw/day is suggestive of a retardation of fetal growth at this dosage and this was supported by subsequent findings observed at fetal examination. Externally many of the fetuses appeared small and there was a plethora of skeletal findings indicating incomplete ossification or no ossification for many regions of the skeleton. Visceral findings included non-uniform patterning of the rugae, kinked/dilated ureter(s), increased renal pelvic cavitation, absent renal papila and partially undescended thymus lobe. These visceral findings were also considered to indicate a retardation of fetal growth. 

While there is no doubt that there has been an effect of maternal treatment on the fetuses, there was no evidence of any structural defects that would indicate any adverse effect on development per se and fetal survival was clearly unaffected. The examination of the fetuses on Day 20 is a window in time and the fetuses will continue to grow and develop after this time. 

For females at 300 mg/kg bw/day, clinical signs, body weight performance, food consumption and macroscopic necropsy examinations did not indicate any obvious effect of treatment and this dosage is considered to represent a No Observed Effect Level (NOEL) for the pregnant female. Litter data, fetal, litter and placental weights, external fetal appearance and detailed skeletal fetal evaluation did not indicate any obvious effect of maternal treatment on the developing conceptus. However, visceral examination of the fetuses showed an increased incidence of fetuses/litters with kinked/dilated ureter(s) compared with control. Although the incidence was much lower than observed at 1000 mg/kg bw/day and failed to attain statistical significance, it was higher than the recent background control incidence and an association with maternal treatment cannot be discounted. These visceral findings may be the most sensitive indicator of the retardation of fetal growth demonstrated at the high dosage and therefore the classification of this dosage as a No Observed Effect Level (NOEL) for the developing conceptus is equivocal. However, in view of the findings apparent at 1000 mg/kg bw/day, these findings at 300 mg/kg bw/day are considered not to indicate any effect on the structural development of the fetuses at 300 mg/kg bw/day.

A dosage of 100 mg/kg bw/day is therefore considered to be a clear No Observed Effect Level (NOEL) for the developing conceptus.

Kinked/dilated uretus are considered considered reversible variations (Solecki, R. et al. Reproductive Toxicilogy 17 (2003) 635 -637) and therefore not considered adverse. The NOAEL was therefore 300 mg/kg bw/day.

Applicant's summary and conclusion

Conclusions:
Treatment at 1000 mg/kg bw/day was associated with lower maternal body weight gain during gestation and an initial effect on food consumption. No similar effects were apparent at 300 mg/kg bw/day and this dosage is considered to represent the No Observed Effect Level (NOEL) for the pregnant female. Kinked/dilated uretus are considered considered reversible variations (Solecki, R. et al. Reproductive Toxicilogy 17 (2003) 635 -637) and therefore not considered adverse. The NOAEL was therefore 300 mg/kg bw/day.

In-utero survival of the developing conceptus was unaffected by maternal treatment at 1000 mg/g bw/day although reduced fetal weight and external, visceral and skeletal findings indicated an adverse effect on fetal growth. The absence any structural defects indicated that development per se was unaffected at this dosage. Only an equivocal increase in the incidence of fetuses/litter showing kinked/dilated ureter(s) prevented 300 mg/kg bw/day being classified as a fetal No Observed Effect Level and a dosage of 100 mg/kg bw/day is therefore considered to be a clear No Observed Effect Level (NOEL) for the developing conceptus.
Executive summary:

Introduction

The study was designed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female from Day 3 to Day 19 of gestation (and including the period of organogenesis). The results of the study are believed to be of value in predicting the toxicity of the test item during pregnancy, and the estimation of both a maternal and embryofetal ‘No Observed Effect Level’ (NOEL).

 

The study was designed to comply with the following guidelines:

- US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

- Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

- OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

Methods

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD (SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels 100, 300, and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Corn Oil) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study. 

 

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were preserved in 70% Industrial Methylated Spirit (IMS) and then following examination for skeletal development transferred into 50% glycerol. The remaining half were preserved in Bouin’s solution and transferred to 90% IMS in distilled water and the viscera were examined.

Results…….

Mortality

There were no unscheduled deaths.

 

Clinical Observations

At 1000 mg/kg bw/day, the majority of females showed incidences of increased post-dosing salivation between Day 10 and Day 19 of gestation. At 300 mg/kg bw/day, increased post-dosing salivation was restricted to just three females on isolated days on the study.

 

Body Weight

At 1000 mg/kg bw/day, body weight gain was lower than control throughout gestation and this lower weight gain was still apparent after values were adjusted for the contribution of the gravid uterus.

 

Food Consumption

At 1000 mg/kg bw/day, food consumption was lower than control between Day 3 and Day 8 of gestation.

 

Water Consumption

No treatment related effects were apparent during the study.

 

Post Mortem Studies

No macroscopic abnormalities were detected during adult necropsy.

 

Litter Responses

Litter Data

There was no effect of maternal treatment on litter data at dosages up to 1000 mg/kg bw/day.

 

Litter Placental and Fetal Weights

At 1000 mg/kg bw/day, mean fetal weights for both sexes was lower than control, resulting in lower mean litter weights; placental weights were unaffected.

 

Fetal Examination

External Examinations

At 1000 mg/kg bw/day, there was a higher incidence of fetuses that were small in appearance compared with control.

 

Detailed Visceral Examinations

At 1000 mg/kg bw/day, there was an increased incidence of fetuses/litters showing non-uniform patterning of the rugae, kinked/dilated ureter(s), increased renal pelvic cavitation, absent renal papila and partially undescended thymus lobe, leading to an increased incidence in the overall number of fetuses showing visceral findings.

 

At 300 mg/kg bw/day, there was an equivocal increased incidence of fetuses/litters showing kinked/dilated ureter(s) compared with control.    

 

Detailed Skeletal Examinations

At 1000 mg/kg bw/day, there was clear effect of treatment on a large number of ossification parameters affecting most regions of the skeleton, with the number of fetuses/litters affected being increased compared with control.

 

Conclusion

Treatment at 1000 mg/kg bw/day was associated with lower maternal body weight gain during gestation and an initial effect on food consumption. No similar effects were apparent at 300 mg/kg bw/day and this dosage is considered to represent the No Observed Effect Level (NOEL) for the pregnant female.    

 

In-utero survival of the developing conceptus was unaffected by maternal treatment at 1000 mg/kg bw/day although reduced fetal weight and external, visceral and skeletal findings indicated an adverse effect on fetal growth. The absence any structural defects indicated that development per se was unaffected at this dosage. Only an equivocal increase in the incidence of fetuses/litter showing kinked/dilated ureter(s) prevented 300 mg/kg bw/day being classified as a fetal No Observed Effect Level and a dosage of 100 mg/kg bw/day is therefore considered to be a clear No Observed Effect Level (NOEL) for the developing conceptus.

Kinked/dilated uretus are considered considered reversible variations (Solecki, R. et al. Reproductive Toxicilogy 17 (2003) 635 -637) and therefore not considered adverse. The NOAEL was therefore 300 mg/kg bw/day.