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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:
7th January 2013 to 24 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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:
2013

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
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
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: di-tert-butyl peroxide, (CAS# 110-05-4)
Description: Clear colourless liquid
Batch: 1208250897
EC Number: 203-733-6
CAS Number: 110-05-4
Purity: >99%
Date Received: 09 October 2012
Expiry Date: 01 September 2020
Storage Conditions: Stored at approximately -20 °C; used/formulated at ambient temperature <30 °C

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, Margate, Kent. 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 190 to 261g.

The animals were housed individually in solid-floor polypropylene cages with stainless steel lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan UK, Oxon, UK) was used. Certificates of analysis of the batches of diet used are given in Appendix 17. 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 (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment were 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 Harlan Laboratories Ltd., Shardlow, UK 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 computerised system, and print-outs of hourly mean temperatures and humidity are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3ºC and 50 ± 20% respectively; there were no deviations from these target ranges.

The animals were randomly allocated to treatment groups using a randomisation 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:
For purpose of the study, the test item was prepared at the appropriate concentrations as a solution in Corn Oil (Corn oil had been successfully used in previous toxicity work). The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services as part of this study. Results are given in Appendix 14 and show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately -20 °C in the dark.

Representative samples were taken of test item formulation used on the study and were analysed for concentration of di-tert butyl peroxide (CAS# 110-05-4) at Harlan Analytical Laboratory, Shardlow. The method used for analysis of formulations and the results obtained are given in Appendix 14. The results indicate that the prepared formulations were within ± 6% of the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Summary
The concentration of Di-Tert-butyl peroxide (CAS#110-05-4) in the test item formulations was determined by gas chromatography (GC) using an external standard technique.

Samples
The test item formulations were diluted with tetrahydrofuran to give a final, theoretical test item concentration of approximately 0.1 mg/ml.

Standards
Standard solutions of test item were prepared in tetrahydrofuran at a nominal concentration of 0.1 mg/ml.

Procedure
The standard and sample solutions were analysed by GC using the following conditions:

GC system : Agilent Technologies 5890, incorporating autosampler and workstation
Column : DB-5 (30 m x 0.53 mm id x 5 µm film)
Oven temperature program : initial 40 ºC for 0 mins
rate 10 ºC/min
temp 140 ºC for 0 mins
rate 50 ºC/min
final 260 ºC for 5 mins
Injection temperature : 150 ºC
Flame ionisation detector temperature :250 ºC
Injection volume: 1 µl
Retention time : ~ 4.9 mins


Appendix 14 (continued) Analytical Investigations
1.5 Homogeneity Determinations
The test item formulations were mixed and assessed visually.

Stability Determinations
The test item formulations were sampled, analysed initially and then after storage at approximately +4ºC in the dark for fourteen days.

Verification of Test Item Formulation Concentrations
The test item formulations were sampled and analysed within two days of preparation.
Details on mating procedure:
Not described in this study
Duration of treatment / exposure:
16 days (Between days 3 and 19 of gestation)
Frequency of treatment:
Daily
Duration of test:
20 days
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
The animals were randomly allocated to treatment groups using a randomisation 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.


Justification
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Examinations

Maternal examinations:
Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioural 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. An additional observation was also performed five hours after dosing during the normal working week (see Deviations from Study Plan). 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.


All implantations and viable foetuses were numbered according to their intrauterine position as follows (as an example):

Left Horn Cervix Right Horn

L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16

V = viable foetus

Organ Weights
The liver and kidneys were removed from all animals that were killed at the end of the study and were dissected free from fat and weighed before fixation. These tissues were not processed any further and are retained with the raw data for the study.
Ovaries and uterine content:
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. 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) Foetal sex
iv) External foetal appearance
v) Foetal weight
vi) Placental weight
vii) Gravid uterus weight

The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium poly sulphide 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/foetal and placental tissue visible

Dead Foetus: A foetus that had died shortly before necropsy. These were included as late deaths for reporting purposes
Fetal examinations:
The foetuses were killed by subcutaneous injection of sodium pentobarbitone. Foetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate foetuses were identified using an indelible marker and placed in Bouin’s fixative. Foetuses were transferred to 90% industrial methylated spirits (IMS) in distilled water and examined for visceral anomalies under a low power binocular microscope. The remaining foetuses were identified using colour coded wires and placed in 70% IMS in distilled water. The foetuses were eviscerated, processed and the skeletons stained with alizarin red S. The foetuses were examined for skeletal development and anomalies. Following examination foetuses that were examined for skeletal development were placed in 100% 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 foetal 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 nonhomogeneous Kruskal-Wallis and, if significant, pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test was employed.

Foetal evaluation parameters, including skeletal or visceral findings were analyzed by Kruskal-Wallis and, if significant, 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:no effects

Details on maternal toxic effects:
Mortality
There were no unscheduled deaths.


Clinical Observations
A summary incidence of daily clinical observations is given in Table 2. Individual data is presented in Appendix 1.

At 1000 mg/kg bw/day, the majority of females showed isolated incidences of increased salivation between Day 13 and Day 17 of gestation. One female at this dosage also showed noisy respiration on Days 8 to 10 and Day 14. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and, in isolation, are considered to be of no toxicological importance.

There were no other signs considered to indicate an effect of treatment.

At 300 mg/kg bw/day, one female treated showed chromodacryorrhoea on Days 4 to 5 and another female at 100 mg/kg bw/day showed this clinical sign on Day 10. Additionally at 100 mg/kg bw/day, a further female showed increased salivation and decreased respiratory rate on Day 3 with noisy respiration on Days 3 and 5 and one female had fur loss from Day 14 onwards. In isolation, these low incidence findings were considered to incidental and unrelated to treatment.


Body Weight
Group mean body weights and standard deviations are given in Table 3. Group mean body weight gains and adjusted body weights and standard deviations are given in Table 4 and Table 5 and cumulative body weight change is presented graphically in Figure 1. Individual data are given in Appendix 2 to Appendix 4.

Body weights and body weight gain, including values adjusted for the contribution of the gravid uterus, were unaffected by treatment at 100, 300 and 1000 mg/kg bw/day.


Food Consumption
Group mean food consumptions are given in Table 6 and presented graphically in Figure 2. Individual data are given in Appendix 5.

There were no effects of treatment on food intake during gestation at 100, 300 or 1000 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. Individual data are given in Appendix 6.

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


Organ Weights
Group mean absolute and body weight-relative values and standard deviations for test and control animals are presented in Table 8. Individual data are given in Appendix 7.

At 1000 mg/kg bw/day mean absolute and body weight-relative liver weight were higher than control with differences for relative weights attaining statistical significance. Absolute and body weight-relative kidney weights at this dosage were considered to be unaffected by treatment and there were no statistically significant differences in kidney weights compared with control.

At 100 and 300 mg/kg bw/day there were no statistically significant differences in absolute and body weight-relative liver and kidney weight compared with control.

Effect levels (maternal animals)

open allclose all
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
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Litter Data and Litter Placental and Foetal Weights
Group Mean Litter data is given in Table 9. Individual data are given in Appendix 8 and Appendix 9.

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

There were no effects of treatment on mean foetal, litter or placental weights at 100, 300 or 1000 mg/kg bw/day.

At 300 mg/kg bw/day, one female showed total resorption (total litter loss in utero). In the absence of any observed increase in mean post-implantation loss for other litters at this dosage, or at 1000 mg/kg bw/day, this finding was considered incidental and unrelated to treatment.


Foetal Examination
Summary foetal external findings, visceral findings, skeletal findings and skeletal development are given in Table 10 to Table 13. Individual data are given in Appendix 10 to Appendix 13.

The type, incidence or distribution of findings observed externally at necropsy examination and subsequently during detailed visceral and skeletal assessment did not indicated any effect of treatment on foetal growth or development.

Females from all treatment groups showed a statistically significant increase in the number of foetuses presenting with one thoracic vertebral centra semi-bipartite. Additionally females treated with 1000 mg/kg bw/day showed a statistically significant reduction in the number of foetuses presenting with incomplete ossification of more than one cranial bone variant. The observation of one variant at a higher or lower incidence when compared to controls is not significant when evaluated in isolation and, due to the number of parameters evaluated, it is highly likely that at least one finding will be more/less frequently observed in dosed groups in comparison to controls. A true developmental effect is represented by effects observed in a number of variants or a syndrome of variance and therefore the intergroup differences observed in this study were considered incidental and unrelated to treatment.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
A dosage of 1000 mg/kg bw/day was considered to represent a No Observed Adverse Effect Level (NOAEL) for the pregnant female, with the No Observed Effect Level (NOEL) being 300 mg/kg bw/day. The NOEL for the survival, growth and morphological development of the conceptus was considered to be 1000 mg/kg bw/day.
Executive summary:

Introduction

The study was performed according to the study plan presented in Appendix15and was designed to investigate the effects of the test item on embryonic and foetal development following repeated administration by gavage to the pregnant female (from Day 3 to Day 19 of gestation) (and including 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 embryofoetal ‘No Observed Effect Level’ (NOEL).

 

This study was designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:

 

·     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)

·     Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

 

 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 and food and water consumptions were monitored during the study. Liver and kidney weights were recorded for all females at termination.

 

All surviving 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, foetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were preserved in 70% Industrial Methylated Spirit (IMS) in distilled water and then following examination for skeletal development transferred into 100% 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

There were no toxicological significant clinical signs observed at 100, 300 or 1000 mg/kg bw/day.

 

Body Weight

There were no effects of treatment on body weight or body weight gain during gestation at 100, 300 or 1000 mg/kg bw/day.

 

Food Consumption

There were no effects of treatment on food intake during gestation at 100, 300 or 1000 mg/kg bw/day.

 

Water Consumption

No effect on water consumption during gestation was detected at 100, 300 or 1000 mg/kg bw/day.

 

Post Mortem Studies

No macroscopic abnormalities were detected for adult animals.

 

Organ Weights

At 1000 mg/kg bw/day, liver weights were considered to be increased in comparison to control.

 

Litter Data and Litter Placental and Foetal Weights

There were no effects of maternal treatment onin uterolitter data or on litter, placental or foetal weights at 100, 300 or 1000 mg/kg bw/day.

 

Foetal Examination

Detailed examination of foetal morphology did not indicate any effect of maternal treatment on embryofoetal development at 100, 300 or 1000 mg/kg bw/day.

 

Conclusion

A dosage of 1000 mg/kg bw/day was considered to represent a No Observed Adverse Effect Level (NOAEL) for the pregnant female, with the No Observed Effect Level (NOEL) being 300 mg/kg bw/day. The NOEL for the survival, growth and morphological development of the conceptus was considered to be 1000 mg/kg bw/day.