Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The available data, including chronic oral exposure, provide no evidence of toxicity to reproductive organs. Moreover, the developmental study revealed no toxicological significant changes in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg/day. Therefore, a reproduction study is scientifically unjustified.

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:

Well conducted studies are available to assess effects to the reproductive system as well as to the developing fetus.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The available data, including chronic oral exposure, provide no evidence of toxicity to reproductive organs. Moreover, the developmental study revealed no toxicological significant changes in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg/day. Therefore, a reproduction study is scientifically unjustified.

Justification for selection of Effect on fertility via oral route:

The avaialble data, including chronic oral exposure, provide no evidence of toxicity to reproductive organs. Moreover, the developmental study revealed no toxicological significant changes in the offspring parameters measured.  The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg/day.  Therefore, a reproduction study is scientifically unjustified.

Effects on developmental toxicity

Description of key information

An OECD 414 Developmental Study did not reveal any significant effects to offspring when tested up to 1000 mg/kg/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 December 2012 to 07 June 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 guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, forestry abd Fisheries Testing guidelines for Toxicology Studies, 12 NohSan No 8147, (24 November 2000)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

A total of one hundred and twenty time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 212 to 292g.


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 22. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). There were no contaminants 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 were included in the study records. The temperature and relative humidity controls were set to achieve target values of 21 ± 2 ºC and 55 ± 15% respectively. Occasional deviations from the humidity target were considered not to have affected the purpose or integrity of the study.

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.

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

dried

Details on exposure:

For the purpose of the study, the test item was prepared at the appropriate concentrations as a solution in Dried Corn Oil. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services. Results are given in Appendix 15 and show the formulations to be stable for four hours. Formulations were therefore prepared daily.


Samples were taken of each test item formulation and were analysed for concentration of Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide (CAS# 6731-36-8) at Harlan Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within ± 4% 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 3,3,5-trimethylcyclohexylidene diperoxide (CAS# 6731-36-8) in the test item formulations was determined by gas chromatography (GC) using an external standard technique.

Samples
The test item formulations were extracted with acetonitrile to give a final, theoretical test item concentration of approximately 0.2 mg/ml.

Standards
Standard solutions of test item were prepared in acetonitrile at a nominal concentration of 0.2 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 50 ºC for 0 mins
rate 10 ºC/min
temp 130 ºC for 0 mins
rate 50 ºC/min
final 260 ºC for 10 mins
Injection temperature :150 ºC
Flame ionisation detector temperature :250 ºC
Injection volume: 2 µl
Retention time : ~ 6.5 mins

 
Homogeneity Determinations
The test item formulations were assessed visually.

Stability Determinations
The test item formulations were sampled and analysed initially and then after storage at ambient conditions for four hours.

Verification of Test Item Formulation Concentrations
The test item formulations were sampled and analysed on the day of preparation.

Details on mating procedure:

Not described in this study

Duration of treatment / exposure:

14 days (Between days 5 and 19 of gestation)

Frequency of treatment:

Daily

Duration of test:

20 days

Remarks:

Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

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.

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. All observations were recorded.


Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 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.


3.4.5 Post Mortem
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation.



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.


Histopathology
The liver and kidneys were removed from all parental animals and preserved in buffered 10% formalin. The tissues from all control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Haematoxylin and Eosin for subsequent microscopic examination.

Since there were indications of treatment-related liver changes, examination was subsequently extended to include similarly prepared sections of the liver from all animals from the low and intermediate groups.

Ovaries and uterine content:

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. 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:

Female body weight change, food consumption and gravid uterus weight: Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
All caesarean necropsy parameters and foetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.

Foetal 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:
Twenty-one females treated with 1000 mg/kg bw/day, seventeen females treated with 300 mg/kg bw/day and three females treated with 100 mg/kg bw/day had an enlarged liver at necropsy. One of the females treated with 1000 mg/kg bw/day also had discolouration on the right lobe of the liver.

No such effects were detected in females treated with 30 mg/kg bw/day.

One female treated with 1000 mg/kg bw/day had a mass on the mammary gland. This was confirmed as a malignant tumour adenocarcinoma following histopathological examination. In isolation, this was considered not to be related to treatment.



Females from all treatment groups showed an increase in liver weight both absolute and relative to terminal body weight. Statistical significant was achieved in 100, 300 and 1000 mg/kg bw/day females (p<0.05 - 0.001).

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

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No effects of any toxicological significance

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: embryotoxic / teratogenic effects

Abnormalities:

not specified

Developmental effects observed:

not specified

              

Mortality

There were no unscheduled deaths.

 

Clinical Observations 

Females treated with 1000 and 300 mg/kg bw/day showed incidences of increased salivation from Day 6 and 14, respectively. No such effects were detected in females treated with 100 or 30 mg/kg bw/day.

  

               

Body Weight

No adverse effect on body weight development was detected. Statistical analysis did not reveal any significant differences.

Food Consumption

No adverse effect on dietary intake was detected.

 

Females treated with 1000 mg/kg bw/day showed a statistically significant reduction (p<0.001) in food consumption between Days 5 and 8. Recovery was evident thereafter, with values for these treated females actually being higher than control values therefore in isolation the intergroup difference was considered not to be of toxicological significance. 

 

               

Water Consumption

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

 

No adverse effect on water consumption was detected.

Post Mortem Studies

Twenty-one females treated with 1000 mg/kg bw/day, seventeen females treated with 300 mg/kg bw/day and three females treated with 100 mg/kg bw/day had an enlarged liver at necropsy. One of the females treated with 1000 mg/kg bw/day also had discolouration on the right lobe of the liver.  

 

No such effects were detected in females treated with 30 mg/kg bw/day.

 

One female treated with 1000 mg/kg bw/day had a mass on the mammary gland. This was confirmed as a malignant tumour adenocarcinoma following histopathological examination. In isolation, this was considered not to be related to treatment.

 

   

Organ Weights

Females from all treatment groups showed an increase in liver weight both absolute and relative to terminal body weight. Statistical significant was achieved in 100, 300 and 1000 mg/kg bw/day females (p<0.05 - 0.001).

 

         

Histopathology

Liver:Minimal to moderate centrilobular hypertrophy of hepatocytes was evident in females from all treatment groups.

 

Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of associated inflammatory or degenerative changes is generally considered to be adaptive in nature and does not represent an adverse health effect.

 

Litter Responses

 

Litter Data and Litter Placental and Foetal Weights

There was no adverse effect onin-uterooffspring survival, as assessed by the mean numbers of early or late resorptions, live litter size and post-implantation losses. There were also no adverse effects on pre-implantation losses or sex ratio.

 

Females treated with 300 mg/kg bw/day showed an increase in male and female foetal weights. Females treated with 30 mg/kg bw/day also showed an increase in male foetal weights. An increase in body weight is considered not to be of toxicological significance.

 

 Foetal Examination

For all dose groups, there were no significant treatment-related trends in the proportion of foetuses (or litters) with evidence of visceral or skeletal anomalies. The type of visceral and skeletal anomalies were those commonly observed for this type of study.

 

Females treated with 300 mg/kg bw/day showed a statistically significant increase in the number of foetuses showing greater than six ossified metacarpals. Subsequently these females also showed a statistically significant reduction in the number of foetuses showing six ossified metacarpals. The standard number of ossified metacarpals in Day 20 foetuses is greater than six. Therefore the intergroup differences indicate a higher number of foetuses showing a normal number of ossified metacarpals compared to controls and in the absence of a true dose related response as such, are considered of no toxicological importance.

 

Females treated with 1000 mg/kg/day showed a statistically significant increase in the percentage of foetuses showing one lumbar vertebral centre semi-bipartite. Females treated with 100 mg/kg bw/day showed a statistically significant increase in the percentage of foetuses showing one thoracic vertebral centre bipartite. The observation of one variant at a higher incidence compared with controls is not significant when evaluated in isolation. The number of parameters evaluated makes it highly likely that one finding will be seen at a higher level in dosed groups compared with controls. A true developmental effect is only seen when a number of variants or a syndrome of variance is observed. Therefore these intergroup differences are of no biological significance.

 

        

Conclusions:

The oral administration of Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide (CAS# 6731-36-8) to pregnant rats by oral gavage during organogenesis at dose levels of 30, 100, 300 and 1000 mg/kg/day resulted in adaptive liver effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 1000 mg/kg/day.

No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg/day.

Executive summary:

SUMMARY

Introduction

The study was performed according to the study plan presented in Appendix16and was designed to investigate the effects of the test item on embryonic and foetal development following repeated administration by gavage to the pregnant female during the period of organogenesis. 

 

The study was designed to comply with the following guidelines:

* US EPA Health Effects Test Guidelines OPPTS 870.3700, 'Prenatal Developmental Toxicity Stidy' (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 (ES) 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 four 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 30, 100, 300 and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Dried Corn oil) to serve as a control.

 

Clinical signs, body weight change, food and water consumptions were monitored during the study. Liver and kidney weights were recorded for all females at termination and histopathological evaluation of the liver and kidneys was performed.

 

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, 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 examined viscerally.

  

Results…….

  

Mortality

There were no unscheduled deaths.

 

 Clinical Observations

Females treated with 1000 and 300 mg/kg bw/day showed incidences of increased salivation. No such effects were detected in females treated with 100 or 30 mg/kg bw/day.

 

 Body Weight

No treatment related effects in body weight development were detected.

 

 Food Consumption

No toxicologically significant effects were detected in food consumption.

 

 Water Consumption

No adverse effect on water consumption was detected.

  

Post Mortem Studies

Females treated with 1000, 300 and 100 mg/kg bw/day had an enlarged liver at necropsy. One female treated with 1000 mg/kg bw/day also had discolouration on the right lobe of the liver. No such effects were detected in females treated with 30 mg/kg bw/day.

 

 Organ Weights

Females from all treatment groups showed an increase in liver weight both absolute and relative to terminal body weight.

 

Histopathology

The following treatment related microscopic effects were detected:

 

Liver:Minimal to moderate centrilobular hypertrophy of hepatocytes was evident in females from all treatment groups.

 

Litter Data and Litter Placental and Foetal Weights

No treatment-related effects were detected in the uterine parameters examined, in foetal viability or in growth and development.

 

Foetal Examination

No treatment-related effects were detected on skeletal development or in the type and incidence of skeletal or visceral findings.

 

Conclusion

The oral administration of Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide (CAS# 6731-36-8) to pregnant rats by oral gavage during organogenesis at dose levels of 30, 100, 300 and 1000 mg/kg/day resulted in adaptive liver effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 1000 mg/kg/day. 

 

No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg/day.

Reference 2

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Qualifier:

no guideline required

Principles of method if other than guideline:

Waiver

Species:

rabbit

Strain:

Angora

Abnormalities:

not specified

Developmental effects observed:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

A well conducted GLP study is available to assess the effects to the developing fetus.

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

The oral administration of Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide (CAS# 6731-36-8) to pregnant rats by oral gavage during organogenesis at dose levels of 30, 100, 300 and 1000 mg/kg/day resulted in adaptive liver effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 1000 mg/kg/day. No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg/day.

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

Apparently well conducted GLP study.

Justification for classification or non-classification

Repeat dose studies did not reveal adverse effects to the reproductive system. Nor where any effects noted to developing offspring in an OECD 414 study. Therefore, the registered substance does not meet the criteria for classification.