Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No effects on reproductive organs were found in the 90-day OECD 408 study summarized elsewhere. In addition, there were no developmental effects reported in an OECD 414 study.These results suggest a low risk for reproductive toxicity; therefore, we are waiving the requirement for a reproductive toxicity study.

Justification for selection of Effect on fertility via oral route:

A reproduction study is scientifically unjustified based on the lack of adverse findings on reproductive organs in a 90-day repeat dose study as well as in a developmental study.

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Quality of whole database:
No effects on reproductive organs were found in the 90-day OECD 408 study summarized elsewhere. In addition, there were no developmental effects reported in an OECD 414 study. These results suggest a low risk for reproductive toxicity; therefore, we are waiving the requirement for a reproductive toxicity study.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

No adverse developmental findings were reported in an OECD 414 study when tested up to 300 mg/kg/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Esperimental starting date: 2nd Octiber 2013 Experimental completion date: 20th February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance wiith agreed protocols, with no/minor deviations from standard test guidelines and/or minor methodological deficincies, which do not affect the quality of relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (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 Minitsry 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
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 was designated Day 0 of gestation. On arrival the females weighed 197 to 270g.

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. A certificate of analysis of the batch of diet used is given in Appendix 16. 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 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 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 computerized system, and print-outs of hourly mean temperatures and humidity were included in the study records. The Study Plan 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; see Deviations from Study Plan.

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.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
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 previous toxicity work. The stability and homogeneity of the test item formulations were previously determined as part of Harlan Laboratories Ltd. Study Number 41301877 and showed the formulations to be stable for at least eight days. Formulations were therefore prepared on three occasions and stored at approximately +4 °C in the dark.

Representative samples were taken of each test item formulation and were analyzed for concentration of Di-tert-butyl 1,1,4,4-tetramethyl tetramethylene diperoxide, CAS# 78-63-7 at Harlan Analytical Laboratory, Shardlow. The method used for analysis of formulations and the results obtained are given in Appendix 14 - please see attached. The results indicate that the prepared formulations were within ± 7% of the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by gas chromtaography (GC) using an external standard technique. The test item gave a chromtaographic profile consisting of a single peak.

The test item described in the main part of this study was also used as the analytical standard.

Preparation of Standard Solutions
Stock solutions of test item in methanol were prepared for external standard calibration. An aliquotm 100 mg of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with methanol to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in methanol with a concentration of 0.1 mg/mL. Standard solutions contained the equivalent amount of vehicle to that of the relevant samples.

On each occasion, standard solutions derived from two stock standard solutins were used for calculation.

Analysis of samples
The formulations received were extracted with methanol. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with methanol. This was then ultra-sonicated for 15 minutes and centrifuged at 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with methanol to achieve the working concentration.

Instrumental Setip
GC system: Agilent Technologies 5890, incorporating autosampler and workstation
Column: DB-1 (30 m x 0.25 mm id x 0.25 micro-metre film)
Oven temperature program: Oven: 100°C for 2 minutes, with 10°C/minute to 250°C for 5 minutes
Injection temperature: 150°C
Flame ionisation detector temperature: 150°C
Injection volume: 1micro-litre
Retention time: ~7 minutes
Details on mating procedure:
Not described in the study
Duration of treatment / exposure:
Day 5 to Day 19 of gestation
Frequency of treatment:
Daily
Duration of test:
20 days
Remarks:
Doses / Concentrations:
15 mg/kg bw/day (3.75 mg/mL0
Basis:
actual ingested
Remarks:
Doses / Concentrations:
60 mg/kg bw/day (15 mg/mL)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day (75 mg/mL)
Basis:
actual ingested
No. of animals per sex per dose:
24 mated females
Control animals:
yes, concurrent vehicle
Details on study design:
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.


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.
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 and on Days 5 (before the start of treatment), 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for surviving animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each surviving 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.

Organ Weights
The liver was removed from all surviving females at termination and dissected free from fat and weighed before fixation.
Ovaries and uterine content:
3.4.5 Post Mortem
All surviving 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) 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 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 fetus

Fetal examinations:
The fetuses were killed by subcutaneous injection of a suitable barbiturate agent. 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 subsequently transferred to water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed in 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.
Indices:
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 and gravid uterus weight, absolute and body weight relative liver 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)
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
At 300 mg/kg bw/day there was an increase in absolute and body weight relative liver weights, compared with control, for females at Day 20 of gestation, with differences attaining statistical significance.
Dose descriptor:
NOEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
300 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: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No effects of toxicological significance
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
other: developmental toxicity
Remarks:
The No Observed Effect Level (NOEL) for the in-utero survival, growth and development of the offspring was considered to be 300 mg/kg bw/day.
Abnormalities:
not specified
Developmental effects observed:
not specified

1.1.1            Adult Responses

1.1.1.1      Mortality

There were no unscheduled deaths during the study that were considered to be attributable to treatment with Di-tert-butyl 1,1,4,4-tetramethyl tetramethylene diperoxide, CAS# 78-63-7 at 15, 60 or 300 mg/kg bw/day.

 

At 60 mg/kg bw/day, female number 49 was found dead at the initial check on Day 8 of gestation; no clinical signs had been apparent for this animal prior to this event. Necropsy revealed a tear in the esophagus; this was considered to have arisen from accidental trauma during the dosing procedure and to be the underlying reason for the death of the animal. As such, this death was considered to be incidental and unrelated to treatment.

 

Clinical Observations

Neither the type or incidence of clinical signs observed during the study indicated any obvious effect of treatment at 15, 60 or 300 mg/kg bw/day.

 

At 300 mg/kg bw/day, three females showed increased post-dosing salivation on the last day of treatment (Day 19 of gestation) but this was considered to reflect a slightly unpalatable test item formulation and, in isolation, was considered to be of no toxicological importance.

 

Body Weight

Body weight gain during pregnancy, including when adjusted for the contribution of the gravid uterus, was unaffected by treatment at 15, 60 or 300 mg/kg bw/day. Intergroup differences observed in body weight performance were considered to reflect normal biological variation.

  

Food Consumption

Food consumption during pregnancy was unaffected by treatment at 15, 60 or 300 mg/kg bw/day.

  

Water Consumption

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

 

Post Mortem Studies

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

  

Organ Weights

At 300 mg/kg bw/day there was an increase in absolute and body weight relative liver weights, compared with control, for females at Day 20 of gestation, with differences attaining statistical significance.

 

Absolute and body weight relative liver weights at 15 and 60 mg/kg bw/day appeared unaffected by treatment.

 

Litter Responses

Litter Data and Litter Placental and Fetal Weights

There was no effect of maternal treatment on litter data as assessed by numbers of implantations,in-uterooffspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio and pre and post-implantation losses at 15, 60 or 300 mg/kg bw/day.

 

There were no effects of maternal treatment on mean fetal, litter or placental weights at 15, 60 or 300 mg/kg bw/day.

 

Fetal Examination

External Examinations

External fetal findings were unremarkable and did not indicate any effect of maternal treatment on fetal development at 15, 60 or 300 mg/kg bw/day. 

 

Detailed Visceral Examinations

The incidence, type or distribution of visceral findings observed did not indicate any effect of maternal treatment on fetal development at 15, 60 or 300 mg/kg bw/day.

 

Detailed Skeletal Examinations

The incidence, type or distribution of skeletal findings observed did not indicate any effect of maternal treatment on fetal development at 15, 60 or 300 mg/kg bw/day.

Conclusions:
Treatment of pregnant females with Di-Tert-Butyl 1,1,4,4-Tetramethyl Tetramethylene Diperoxide (CAS# 78-63-7) at dosages of up to 300 mg/kg bw/day was not associated with any obvious treatment-related effects on clinical signs, body weight performance or food consumption. As anticipated, necropsy revealed an increased in absolute and body weight relative liver weights but there were no obvious macroscopic abnormalities apparent. Increased liver weights were also apparent for both sexes at 150 mg/kg bw/day in a Ninety Day Toxicity Study in the Rat (Harlan Laboratories Ltd Study Number 41301877) with this test item; this finding was not accompanied by any evidence of microscopic change and was considered to be adaptive in nature. The increased liver weights at 300 mg/kg bw/day are considered most likely to reflect the same adaptive process and, as such, are considered not to represent an adverse effect of treatment. A dosage of 300 mg/kg bw/day is therefore considered to represent the No Observed Adverse Effect Level (NOAEL) for the pregnant females with the No Observed Effect Level (NOEL) being 60 mg/kg bw/day.
 
The No Observed Effect Level (NOEL) for thein-uterosurvival, growth and development of the offspring was considered to be 300 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 fetal development following repeated administration by gavage to the pregnant female from Day 5 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)

·        Commission Regulation (EC) No 440/2008 of 30 May 2008 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 5 and 19 of gestation inclusive at dose levels 15, 60, and 300 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. Liver weights were recorded for all surviving 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, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for skeletal development. The remaining half were microdissected and the viscera were examined.

 

 

Results…….

Mortality

There were no unscheduled deaths on the study that were considered to be attributable to treatment at 15, 60 or 300 mg/kg bw/day.

  

Clinical Observations

Clinical signs did not indicate any effect of treatment at 15, 60 or 300 mg/kg bw/day.

 

 Body Weight

Body weight and body weight gain, including adjustment for the contribution of the gravid uterus, was unaffected by treatment at 15, 60 or 300 mg/kg bw/day.

 

 Food Consumption

Food consumption was unaffected by treatment at 15, 60 or 300 mg/kg bw/day.

 

 Water Consumption

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

 

 Post Mortem Studies

No macroscopic abnormalities were detected for females at Day 20 of gestation at 15, 60 or 300 mg/kg bw/day.

 

 Organ Weights

At 300 mg/kg bw/day, there was an increase in maternal absolute and body weight relative liver weights on Day 20 of gestation. No similar increase was observed for pregnant females at 15 or 60 mg/kg bw/day.

 

 Litter Data

The number of implantations, subsequent embryofetal survival and litter size, sex ratio and mean fetal, litter and placental weights on Day 20 of gestation were unaffected by maternal treatment at 15, 60 or 300 mg/kg bw/day.

 

 Litter, Placental and Fetal Weights

There were no effects of maternal treatment on mean fetal, litter or placental weights at 15, 60 or 300 mg/kg bw/day. There was no effect of maternal treatment on morphological development of the fetuses at 15, 60 or 300 mg/kg bw/day.

 

 Fetal Examination

There was no effect of maternal treatment on morphological development of the fetuses at 15, 60 or 300 mg/kg bw/day.

 

 Conclusion

Within this study, only an increase in maternal absolute and body weight relative liver weights precluded a dosage of 300 mg/kg bw/day from being the No Observed Effect Level (NOEL) for the pregnant females and this dosage probably represents the No Observed Adverse Effect Level (NOAEL). The clear No Observed Effect Level (NOEL) for the pregnant females was considered to be 60 mg/kg bw/day.

 

The No Observed Effect Level (NOEL) for thein-uterosurvival, growth and development of the offspring was considered to be 300 mg/kg bw/day.

Endpoint:
developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Treatment of pregnant females with Di-Tert-Butyl 1,1,4,4-Tetramethyl Tetramethylene Diperoxide (CAS# 78-63-7) at dosages of up to 300 mg/kg bw/day was not associated with any obvious treatment-related effects on clinical signs, body weight performance or food consumption. As anticipated, necropsy revealed an increased in absolute and body weight relative liver weights but there were no obvious macroscopic abnormalities apparent. Increased liver weights were also apparent for both sexes at 150 mg/kg bw/day in a Ninety Day Toxicity Study in the Rat (Harlan Laboratories Ltd Study Number 41301877) with this test item; this finding was not accompanied by any evidence of microscopic change and was considered to be adaptive in nature. The increased liver weights at 300 mg/kg bw/day are considered most likely to reflect the same adaptive process and, as such, are considered not to represent an adverse effect of treatment. A dosage of 300 mg/kg bw/day is therefore considered to represent the No Observed Adverse Effect Level (NOAEL) for the pregnant females with the No Observed Effect Level (NOEL) being 60 mg/kg bw/day.   The No Observed Effect Level (NOEL) for the in-utero survival, growth and development of the offspring was considered to be 300 mg/kg bw/day.

Justification for selection of Effect on developmental toxicity: via oral route:

Apparently well conducted study GLP.

Justification for classification or non-classification

There were no adverse effects, on reproductive organs, in a 90-day repeat dose study and no adverse developmental effects reported in an OECD 414 study.