Di-tert-butyl 1,1,4,4-tetramethylbut-2-yn-1,4-ylene diperoxide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Minitsry of Agriculture, Forestry and Fishseries Testing Guideline for Toxicology Studies, 12 NohSan 8147, (24 November 2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide

EC Number:

201-128-1

EC Name:

Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide

Cas Number:

78-63-7

Molecular formula:

C16H34O4

IUPAC Name:

2,5-bis(tert-butylperoxy)-2,5-dimethylhexane

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

A total of ninety-six time female Spargue-Daley Crl:CD (DS) 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 270 g.
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 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 °C 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.

Administration / exposure

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 toxic 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 analysed for concentration of Di-tert-butyl1,1,4,4-tetramethyl tetramethylene diperoxide, CAS' 78-63-7 at Harlan Analytical Laboratory, Shardlow. 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 sample was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic 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 the test item in methanol were prepared for external standard calibration. An aliquot 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 standards 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 Setup:
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: 1 micro-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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

60 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

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.

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 and on day 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:

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 diet shortly before necropsy. These were included as late deaths for reporting purposes.
Allimplantations 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 fetus 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 analysed 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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

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

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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.

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The number of implantations on Day 20 of gestation were unaffected by maternal treatment at 15, 60 or 300 mg/kg bw/day.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

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.

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

not examined

Other effects:

not examined

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.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOEL

Effect level:

60 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

maternal abnormalities

2

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

maternal abnormalities

3

Key result

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects

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

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

external malformations

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Adult Responses
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.

Applicant's summary and conclusion

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.