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 OECD 422 study summarized elsewhere.  These results suggest a low risk for reproductive toxicity.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type
Type of information:
experimental study
Adequacy of study:
key study
Study period:
In-life phase: between 01 March 2012 (first day of treatment) and 28 June 2012 (final necropsy).
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 relevant results.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: 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).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for seven days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 196 to 236g, the females weighed 158 to 195g, and were approximately six weeks old.

Initially, all animals were housed in groups of up to five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. From Week 14 of the study, as a result of increased body weight, males were housed in groups of up to three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used. Certificates of analysis of the batches of diet used are given in Addendum 1. 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) except for mated females during gestation and lactation.

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 temperatures and humidities are included in the study records. Study Plan target ranges for temperature and relative humidity were 22 ± 3ºC and 50 ± 20% respectively; there were no deviations from these targets.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
For the purpose of this study the test item was prepared at the appropriate concentrations as an emulsion in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services as part of this study (see Appendix 33). Results show the formulations to be stable for at least twenty-one days. Formulations were therefore prepared twice monthly and stored at approximately +4ºC in the dark.

Representative samples of test item formulations were taken and analysed for concentration of 1,1,4,4-tetramethylbutane-1,4-diylbis(2-ethylperoxyhexanoate) (CAS 13052-09-0) at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Appendix 33. The results indicated that the analysed formulations were within 86-107% of the nominal concentrations and were considered to confirm the accuracy of the formulation procedure.
Details on mating procedure:
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Summary
The concentration of CAS 13052-09-0 in the test item formulations was determined by gas chromatography (GC) using an external standard technique.


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


Standards
Standard solutions of test item were prepared in methanol 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 50 ºC for 2 mins
rate 10 ºC/min
final 260 ºC for 10 mins
Injection temperature :250 ºC
Flame ionisation detector temperature 250 ºC
Injection volume: 1 µl
Retention time : Profile of peaks from ~ 3 to 17 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 approximately +4ºC in the dark for twenty one days.


Verification of Test Item Formulation Concentrations
The test item formulations were sampled and analysed within six days of preparation.
Duration of treatment / exposure:
Up to sixteen weeks (including an eleven week pre-pairing phase, pairing, gestation and early lactation for females),
Frequency of treatment:
Daily
Details on study schedule:
Chronological Sequence of Study
i) Prior to the start of the study, all animals from the control and high dose groups were subjected to ophthalmoscopic examination.

ii) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.

iii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.

iv) Three weeks prior to pairing, females were subjected to a daily vaginal smear for assessment of oestrous cyclicity.

v) On Day 78, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

vi) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.

vii) During Week 13, females from the control and high dose groups were subjected to ophthalmoscopic examinations.

viii) During Week 15, all selected males were evaluated for functional/sensory responses to various stimuli. Males from the control and hig
h dose groups were also subjected to ophthalmoscopic examinations.

ix) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.

x) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.

xi) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 110. The male dose groups were killed and examined macroscopically on Day 111.

xii) Blood samples were taken from females from each dose group at termination for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 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:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The animals were allocated to dose groups using a randomisation procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups. The animals were uniquely identified within the study, by an ear punching system routinely used in these laboratories.
Positive control:
Not applicable
Parental animals: Observations and examinations:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.


Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.



Behavioural Assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait
Hyper/Hypothermia
Tremors
Skin colour
Twitches
Respiration
Convulsions
Palpebral closure
Bizarre/Abnormal/Stereotypic behaviour
Urination
Salivation
Defecation
Pilo-erection
Transfer arousal
Exophthalmia
Tail elevation
Lachrymation

This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioural Assessments and Sensory Reactivity Tests.


Functional Performance Tests
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).

Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).


Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).

The following parameters were observed:
Grasp response
Touch escape
Vocalisation
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach


Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.


Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.


Water Consumption
Water intake was measured daily throughout the study (with the exception of the pairing phase).


Ophthalmoscopic Examination
The eyes of all control and high dose animals were examined pre-treatment and before termination of treatment (during Week 13 for females and Week 15 for males). Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex. Following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.


Reproduction Screening

Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.


Pregnancy and Parturition
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:

i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition


Laboratory Investigations
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 110 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.


Haematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:

Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed

Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).


Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Calcium (Ca++)
Glucose
Inorganic phosphorus (P)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
Albumin
Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphatase (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl-)
Total bilirubin (Bili)
Bile acids

Oestrous cyclicity (parental animals):
During Weeks 9 to 11 a vaginal smear was taken from each female rat daily and a sample was placed on a glass slide. The smears were allowed to dry and then stained using a diluted giemsa stain. The slides were examined microscopically and the stage of oestrous was recorded.
Sperm parameters (parental animals):
Semen Assessment
At necropsy the following procedures were performed on all males:

i) The left testis and epididymis were removed, dissected from connective tissue and weighed separately.

ii) For the testis, the tunica albuginea was removed and the testicular tissue stored frozen at approximately -20ºC. At an appropriate later date the tissues were thawed, re-weighed and homogenised in a suitable saline/detergent mixture. Samples of the homogenate were stained with a DNA specific fluorescent stain and a sub-sample was analysed for numbers of homogenisation resistant spermatids.

iii) For the epididymis the distal region was incised and a sample of the luminal fluid collected and transferred to a buffer solution for analysis of sperm motility and sperm morphology. A minimum of 200 individual sperm were assessed using an automated semen analyser to determine the number of motile, progressively motile and non-motile sperm. The characteristics of motile sperm were also identified using the computer assisted sperm analyser (Hamilton-Thorne TOX IVOS system).

iv) A sample of semen was preserved in formalin and then stained with eosin. A sub-sample was placed on a glass slide with a coverslip and a morphometric analysis of sampled semen was performed manually.

v) The cauda epididymis was separated from the body of the epididymis and then weighed. The cauda epididmis was then stored frozen at approximately -20ºC. At an appropriate later date the tissues were thawed, re-weighed and homogenised in an appropriate saline/detergent mixture. Samples of the homogenate were stained with eosin and a sub-sample was analysed for homogenisation resistant spermatids.

vi) For both ii) and iv) above, samples from Groups 1 and 4 were evaluated only as no significant effects were seen.
Litter observations:
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:

i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)


Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.
Postmortem examinations (parental animals):
Pathology
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 111. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.


Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals
Prostate
Brain
Seminal vesicles
Epididymides
Spleen
Heart
Testes
Kidneys
Thymus
Liver
Thyroid (weighed post-fixation with Parathyroid)
Ovaries
Uterus (weighed with Cervix)
3.3.9.2
Histopathology


Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
Pituitary
Bone & bone marrow (sternum)
Prostate
Brain (including cerebrum, cerebellum and pons)
Oesophagus
Caecum
Rectum
Coagulating gland
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides•
Skin (hind limb)
Eyes*
Spinal cord (cervical, mid-thoracic and lumbar)
Gross lesions
Heart
Spleen
Ileum (including peyer’s patches)
Stomach
Jejunum
Thyroid/parathyroid
Kidneys
Trachea
Liver
Testes•
Lungs (with bronchi) #
Thymus
Lymph nodes (mandibular and mesenteric)
Urinary bladder
Mammary gland
Uterus/Cervix
Muscle (skeletal)
Vagina


All tissues were despatched to the histology processing Test Site (Harlan Laboratories Ltd, Itingen, Switzerland) for processing. The tissues from five selected control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 1000 mg/kg bw/day animals were also processed. In addition, sections of testes and epididymides from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Since there were indications of treatment-related kidney changes in males, and liver and thyroid changes in animals of either sex, examination was subsequently extended to include similarly prepared sections of kidney from five males from the low and intermediate groups and similarly prepared sections of liver and thyroid from five animals per sex from the low and intermediate groups (or all ten animals per sex in the case of reproductive tissues).

Microscopic examination was conducted by the Study Pathologist ( AnaPath GmbH, Oberbuchsiten, Switzerland).
Postmortem examinations (offspring):
All offspring, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
Due to the nature and quantity of this data please see section "any other information on material and methods including tables".
Reproductive indices:
Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:

i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices
For each group the following were calculated:

Mating Index (%) = (Number of animals mated / Number of animals paired) x 100

Pregnancy Index (%) = (number of pregnant females / Number of animals mated) x 100


Gestation and Parturition Data
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.

i) Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii) Parturition Index
The following was calculated for each group:

Parturition Index (%) = (Number of females delivering live offspring / number of pregnant females) x 100
Offspring viability indices:
Offspring viability indices
The standard unit of assessment was considered to be the litter, therefore, values were first calculated for each litter and the group mean was
calculated using the individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:

Pre–implantation loss = ((Number of corpora lutea - number of implantation sites) / Number of corpora lutea) x100

Post–implantation loss = ((Number of implantation sites - Total number of offspring born) / Number of implantation sites) x 100


ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:

Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) x 100

Viability Index (%) = (Number of offspring alive on Day 4 / Number of offpsring alive on Day 1) x 100


iii) Sex Ratio (% males)
Sex ratio was calculated for each litter on Days 1 and 4 post partum, using the following formula:

Sex Ratio = (Number of male offspring / Total number of offspring) x 100
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "Details on Results" section below
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Mortality
There was one unscheduled death on the study.

At 1000 mg/kg bw/day, female number 76 was found dead at the morning check on Day 98 of the study (Day 18 of gestation). No significant clinical signs had been observed for this animal prior to its death. Macroscopic necropsy revealed enlarged adrenals and a reddened left lung lobe but microscopic examination of the tissues for this animal did not reveal the aetiology of this death.


Clinical Observations
Clinical signs were mainly confined to increased post dosing salivation for both sexes at 1000 mg/kg bw/day, and to a lesser extent, males at 300 mg/kg bw/day. One female at 300 mg/kg bw/day also showed increased post dosing salivation on a single occasion.

One female at 1000 mg/kg bw/day showed piloerection, hunched posture and pallor of the extremities on Day 104 (around the time of parturition) but these findings were consistent with the physiological strains associated with littering and were considered to be unrelated to treatment.

The type, incidence and distribution of other clinical signs observed on the study did not indicate any obvious association with treatment.


Functional Observations

Behavioural Assessments
Assessment of the animals in a standard arena did not reveal any obvious effects of treatment at 30, 300 or 1000 mg/kg bw/day.

Functional Performance Tests
Assessment of grip strength or motor activity did not reveal any consistent pattern of results that indicated an adverse effect of treatment at 30, 300 or 1000 mg/kg bw/day.

During these assessments, reduced hind limb grip strength for males at all dosages at Test 1 and reduced fore limb grip strength for males at 30 and 300 mg/kg bw/day at Test 3 attained statistical significance compared to control. There was no dosage relationship and given the absence of statistically significant differences from control in the remaining tests of grip strength these findings were considered to be incidental and unrelated to treatment. For females at 1000 mg/kg bw/day, lower overall mobile score attained statistical significance compared to control, but in isolation this was considered to be of no toxicological significance.


Sensory Reactivity Assessments
Sensory reactivity to different stimuli did not reveal any adverse effect of treatment at 30, 300 or 1000 mg/kg bw/day.


Body Weight
There was no clear adverse effect of treatment on body weight performance of males at any of the dosages investigated. At 1000 mg/kg bw/day, occasionally lower body weight gain for males attained statistical significance when compared to control, but as overall gain was approximately 91% of control at the end of the study the differences observed were considered to reflect normal biological variation.

There was no clear adverse effect of treatment on body weight performance of females, including during gestation and early lactation, at any of the dosages investigated.


Food Consumption and Food Conversion Efficiency
There was no adverse effect of treatment on food consumption or food conversion efficiency of males during the study at any of the dosages investigated.

There was no adverse effect of treatment on food consumption or food conversion efficiency of females during the pre-pairing phase of the study and no subsequent adverse effect of treatment during gestation and early lactation, at any of the dosages investigated.


Water Consumption
For males at 1000 mg/kg bw/day, there was a tendency for slightly higher water intake compared to control throughout most of the study; water consumption for females at this dosage appeared unaffected.

At 30 and 300 mg/kg bw/day there was no clear effect of treatment on water intake for either sex.


Ophthalmoscopic Examination
No treatment-related effects were detected during ophthalmic examination of animals at 1000 mg/kg bw/day.


Reproductive Performance
Oestrus Cycle Assessments
Assessment of female oestrous cycles did not indicate any treatment-related effects at 30, 300 or 1000 mg/kg bw/day.

Mating
Mating performance was unaffected by treatment at 30, 300 or 1000 mg/kg bw/day.

Fertility
Fertility was unaffected by treatment at 30, 300 or 1000 mg/kg bw/day.

Gestation Length
Gestation length was unaffected by treatment at 30, 300 or 1000 mg/kg bw/day.


Laboratory Investigations
Haematology
For males at 1000 mg/kg bw/day, mean platelet count was statistically significantly higher than control with half of the individual values for treated animals exceeding the historical control range compared to only one in the control group.

Additionally, for males at 1000 mg/kg bw/day, prothrombin times were slightly faster than control with differences attaining statistical significance. All individual values were within the historical control range and, although these times may have been influenced to some extent by the higher mean platelet count at this dosage, it was considered that the differences in prothrombin times, particularly in the absence of any histopathological finding for the bone marrow, were incidental and of no toxicological significance.

At 1000 mg/kg bw/day, lower mean cell haemoglobin concentration for both sexes attained statistical significance when compared with control, however there were no other statistically significant differences from control apparent for other erythrocyte parameters for these animals and all individual values were within the historical control range. In isolation this finding was considered incidental and unrelated to treatment.

At 30 and 300 mg/kg bw/day there were no statistically significant differences from control observed for haematology parameters.


Blood Chemistry
No toxicologically significant effects were detected for males at 1000 mg/kg bw/day or either sex at 30 or 300 mg/kg bw/day.

For females at 1000 mg/kg bw/day, higher total protein and albumin levels attained statistical significance compared with control although no statistically significant change in albumin/globulin ratio was apparent. Although occasional control values for these parameters exceeded the historical control range, this range was exceeded for both parameters at a much higher incidence by the treated animals.

At 1000 mg/kg bw/day, lower bilirubin levels for both sexes attained statistical significance when compared with control. All individual values for treated animals were within the historical control.

For males at 1000 mg/kg bw/day, higher urea levels attained statistical significance compared with control, but only two values for treated animals exceeded the historical control range. Additionally levels of potassium, calcium and phosphorus were also statistically significantly higher than control but, with the exception of two individual values for potassium, individual values for treated animals were within the historical control range.

At 30 and 300 mg/kg bw/day there were no statistically significant differences for blood chemistry parameters compared with controls.



Pathology
Necropsy

Adults
At 1000 mg/kg bw/day, two males showed mottled kidneys and this finding may be associated with the treatment related histopathological renal changes observed for males at this dosage.

Additionally another male at 1000 mg/kg bw/day showed a small and malformed left kidney but this finding was considered likely to be congenital in nature and unrelated to treatment.

Remaining necropsy observations observed on the study were unremarkable and did not indicate any adverse effect of treatment at 30, 300 or 1000 mg/kg bw/day.

Sperm Analysis
There were no toxicologically significant effects detected in sperm motility values, morphological assessments or in homogenisation-resistant spermatid counts for males receiving 1000 mg/kg bw/day.


Organ Weights
At 1000 mg/kg bw/day, absolute and body weight relative kidney weights for both sexes were higher than control with differences attaining statistical significance. A statistically significant increase in kidney weights was also apparent for males receiving 300 mg/kg bw/day.

For males at 1000 mg/kg bw/day, increased absolute and body weight relative liver weights also attained statistical significance compared with control.

For males at all dosages, higher absolute heart weights attained statistical significance compared with control, although no individual values for treated animals exceeded the historical control. When adjusted for body weight, mean male heart weights at all dosages were statistically significantly lower than control but showed no dosage relationship. For females at all dosages, absolute heart weights were statistically significantly lower than control but showed no dosage-relationship and only one individual value in each treatment group was lower than the historical control range. When adjusted for body weight, mean female heart weights at all dosages were statistically significantly lower than control. In the absence of any histopathological change these differences in heart weight were considered incidental and unrelated to treatment.

For males at all dosages, lower absolute and body weight relative thymus weights attained statistical significance compared with control, however there was no dosage relationship and all individual values for treated animals were within the historical control range. In the absence of any supporting histopathological change this finding was considered incidental and unrelated to treatment.

For females at 300 and 1000 mg/kg bw/day, lower absolute and body weight relative brain weights attained statistical significance compared with control, but there was no dosage relationship. In the absence of any supporting histopathological change this finding was considered incidental and unrelated to treatment.


Histopathology
At 300 and 1000 mg/kg bw/day the livers of several males showed centrilobular hepatocellular hypertrophy; since this finding was not accompanied by degenerative or inflammatory lesions it was considered to be reversible and to represent an adaptive effect.

Males at 300 and 1000 mg/kg bw/day also showed hyaline droplet nephropathy of the kidney, consisting of increased incidence and severity of hyaline droplets, tubular degeneration, granulated tubular casts and interstitial inflammatory infiltrate. This nephropathy was deemed to be related to treatment. Hyaline droplets were also present for males at 30 mg/kg bw/day but these occurred in the absence of any degenerative changes.

For females at 30 mg/kg bw/day and both sexes at 300 and 1000 mg/kg bw/day increased incidence and severity of follicular hypertrophy was recorded in the thyroid. Follicular hypertrophy of the thyroids is deemed to be associated with the increased hepatic metabolisation of thyroid hormones (T3/T4) due to hepatocellular hypertrophy in the respective groups. The reason for hypertrophic changes in the thyroids of females was not clear but it was assumed that the mechanism of increased hepatic metabolisation and subsequent thyroidal changes also applies for females, however with no overt morphological hepatic correlate. Therefore, this finding is deemed to represent a secondary effect.

There were no abnormal lesions encountered during sperm staging regarding completeness of stages and maturation of cell populations for males at 1000 mg/kg bw/day. Individual lesions recorded were within the range of background alterations that may be recorded in this type of study, in rats of this strain and age.

The remainder of histopathological findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Remarks:
Reproductive toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Enhanced evaluation of reproduction for this study did not indicate any effect of treatment on reproduction including litter size and offspring survival, growth and development at dosages up to 1000 mg/kg bw/day.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Litter Responses
As previously discussed one female at 1000 mg/kg bw/day was found dead during late gestation; additionally at this dosage one female was found to be non-pregnant. For the non-pregnant animal, histopathological examination revealed a vaginal retention cyst which was deemed to be the underlying cause for the unsuccessful mating. Two control litters showed post partum total litter loss but there were no similar losses for treated groups.
The following assessment is generally based on the 8, 10, 10 and 8 litters successfully reared to Day 5 of age for the control, 30, 300 and 1000 mg/kg bw/day dosage groups respectively.


Offspring Litter Size, Sex Ratio and Viability
There was no adverse effect of treatment on corpora lutea and implantation counts, or on subsequent post-natal litter size, sex ratio or offspring survival at 30, 300 and 1000 mg/kg bw/day.


Offspring Growth and Development
Litter weight and offspring body weight on Day 1 and subsequent offspring growth to Day 4 of age was unaffected by treatment at 30, 300 and 1000 mg/kg bw/day.

Assessment of surfacing righting ability on Day 1 of age was unaffected by treatment at 30, 300 and 1000 mg/kg bw/day. The clinical signs observed for the offspring on this study were typical for the age observed and neither the incidence or distribution of these signs indicated any underlying adverse effect on offspring development.

Offspring
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to maternal treatment with the test item.

Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Enhanced evaluation of reproduction for this study did not indicate any effect of treatment on reproduction including litter size and offspring survival, growth and development at dosages up to 1000 mg/kg bw/day.
Reproductive effects observed:
not specified
Conclusions:
The oral administration of 1,1,4,4-tetramethylbutane-1,4-diylbis(2-ethylperoxyhexanoate) (CAS 13052-09-0) to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day, resulted in treatment related findings in animals of either sex treated with 30, 300 and 1000 mg/kg bw/day. The effects detected in females were mainly confined to adaptive microscopic thyroid changes and there were no findings observed that were considered to represent an adverse effect of treatment. The ‘No Observed Adverse Effect Level' (NOAEL) for females was considered to be 1000 mg/kg bw/day.

Kidney effects detected in males at 300 and 1000 mg/kg bw/day consisted of increased incidence and severity of hyaline droplets, tubular degeneration, granulated tubular casts and interstitial inflammatory infiltrate. This nephropathy was deemed to be related to treatment and to represent an adverse effect of treatment to the rat. Hyaline droplets were also present for males at 30 mg/kg bw/day but, occurred in the absence of any degenerative changes and the ‘No Observed Adverse Effect Level' (NOAEL) for males was therefore considered to be 30 mg/kg bw/day. However, the kidney changes of hyaline droplets were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. This effect is, therefore, not indicative of a hazard to human health. In the context of this study, the remaining kidney findings, consisting of tubular and/or degeneration, tubular dilation/vacuolation, granulated tubular casts and interstitial inflammatory infiltrate detected in males are more likely to be correlated to the same condition as hyaline droplet accumulation and are, therefore, considered to represent limited relevance to humans.

Effects excluding the kidney changes detected in males were confined to adaptive microscopic liver and thyroid changes. These were considered not to represent an adverse effect of treatment. In terms of extrapolation to man and risk assessment calculations whereby effects relating to male rat renal changes are species and sex specific and therefore are not relevant, a NOAEL for males can be established at 1000 mg/kg bw/day.
Executive summary:

Introduction.The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to be compatible with the 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 ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to sixteen weeks (including an eleven week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 30, 300 and 1000 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioural assessments, body weight change, food and water consumption, oestrous cycles and opthalmic change were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 78 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with assessments of litter size, offspring weights and surface righting reflex.

Extensive functional observations were performed on males from each dose group during Week 15 and for parental females from each dose group on Day 4post partumHaematology and blood chemistry were evaluated prior to termination on males (Day 110) and females (Day 4 of lactation) from each dose group. An opthalmic examination was made for control and high dose animals prior to treatment and during Week 13 for females and Week 15 for males. 

Adult males were terminated on Day 112, followed by the termination of all surviving females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on or after Day 25post coitum. All animals were subjected to a gross necropsy examination with recording of selected organ weight and additionally sperm analysis for males. Histopathological evaluation of selected tissues was performed, with enhanced assessment of testes for males.

Results.

Adult Responses:

Mortality.At 1000 mg/kg bw/day, one female was found dead on Study Day 98 (Day 18 of gestation) without any significant clinical signs being seen prior to this event. Enlarged adrenals and a reddened left lung lobe were apparent at necropsy but microscopic examination did not reveal the aetiology of this death and this death was considered to be unrelated to treatment.

Clinical Observations.Clinical signs associated with treatment were confined to increased post dosing salivation for both sexes at 1000 mg/kg bw/day, and males and one female at 300 mg/kg bw/day.

Behavioural Assessment.No obvious neurological effects of treatment at 30, 300 or 1000 mg/kg bw/day were apparent.

Functional Performance Tests.There was no consistent pattern of results that indicated an effect of treatment at 30, 300 or 1000 mg/kg bw/day.

Sensory Reactivity Assessments.Sensory reactivity assessments to different stimuli did not reveal any effect of treatment at 30, 300 or 1000 mg/kg bw/day.

Body Weight.There was no adverse effect of treatment on body weight performance for males or females, including during gestation and lactation phases at 30, 300 or 1000 mg/kg bw/day.

Food Consumption and Food Conversion Effeciency.There was no adverse effect of treatment on food consumption and food conversion efficiency for males or females, including during gestation and lactation phases, at 30, 300 or 1000 mg/kg bw/day.

Water Consumption.At 1000 mg/kg bw/day, water consumption for males was slightly higher than control throughout most of the study.

Ophthalmoscopy.No treatment-related effects were detected for animals at 1000 mg/kg bw/day.

Reproductive Performance:

Oestrous Cycle.There was no adverse effect of treatment on female oestrous cycles at 30, 300 or 1000 mg/kg bw/day.

Mating.Mating performance was unaffected by treatment at 30, 300 or 1000 mg/kg bw/day.

Fertility.Fertility was unaffected by treatment at 30, 300 or 1000 mg/kg bw/day.

Gestation Lengths.Gestation length was unaffected by treatment at 30, 300 or 1000 mg/kg bw/day.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability.Corpora lutea and implantation counts and subsequent post-natal litter size, sex ratio and offspring survival were unaffected by treatment at 30, 300 and 1000 mg/kg bw/day.

Offspring Growth and Development.Offspring body weight, litter weight and surface righting ability on Day 1 and subsequent clinical signs and offspring growth to Day 4 of age were unaffected by treatment at 30, 300 and 1000 mg/kg bw/day.

Laboratory Investigations:

Haematology.Males treated with 1000 mg/kg bw/day showed increased platelet count compared with control. No toxicologically significant effects were detected for females at this dosage or either sex at 30 or 300 mg/kg bw/day.

Blood Chemistry.Females at 1000 mg/kg bw/day showed increased total protein and albumin levels compared to control. No similar effects were apparent for males at 1000 mg/kg bw/day or either sex at 30 or 300 mg/kg bw/day.

Pathology:

Necropsy.At 1000 mg/kg bw/day, two adult males showed mottled kidneys at necropsy examination. There were no other necropsy findings for adult animals or their offspring that were considered to be associated with treatment at 30, 300 or 1000 mg/kg bw/day.

Organ Weights.For both sexes at 1000 mg/kg bw/day and males at 300 mg/kg bw/day, absolute and body weight relative kidney weights were statistically significantly higher than control.

For males at 1000 mg/kg bw/day, increased absolute and body weight relative liver weights also attained statistical significance compared with control.

Sperm Analysis.Assessment of sperm motility values, morphological assessments and homogenisation-resistant spermatid counts for males at 1000 mg/kg bw/day did not indicate any effects of treatment.

Histopathology.At 300 and 1000 mg/kg bw/day males showed adaptive liver changes consisting of centrilobular hepatocellular hypertrophy; there were no degenerative or inflammatory lesions.

At 300 and 1000 mg/kg bw/day males also showed treatment related hyaline droplet nephropathy of the kidney, consisting of increased incidence and severity of hyaline droplets, tubular degeneration, granulated tubular casts and interstitial inflammatory infiltrate.

For females at 30 mg/kg bw/day and both sexes at 300 and 1000 mg/kg bw/day increased incidence and severity of follicular hypertrophy was recorded in the thyroid. This finding was considered to be a secondary effect of increased hepatic metabolism.

There were no abnormal lesions encountered during testicular spermatogenesis staging for males at 1000 mg/kg bw/day.

Conclusion.The oral administration of 1,1,4,4-tetramethylbutane-1,4-diylbis(2-ethylperoxyhexanoate) (CAS 13052-09-0) to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day, resulted in treatment related findings in animals of either sex treated with 30, 300 and 1000 mg/kg bw/day. The effects detected in females were mainly confined to adaptive microscopic thyroid changes and there were no findings observed that were considered to represent an adverse effect of treatment. The ‘No Observed Adverse Effect Level' (NOAEL) for females was considered to be 1000 mg/kg bw/day.

Kidney effects detected in males at 300 and 1000 mg/kg bw/day consisted of increased incidence and severity of hyaline droplets, tubular degeneration, granulated tubular casts and interstitial inflammatory infiltrate. This nephropathy was deemed to be related to treatment and to represent an adverse effect of treatment to the rat. Hyaline droplets were also present for males at 30 mg/kg bw/day but, occurred in the absence of any degenerative changes and the ‘No Observed Adverse Effect Level' (NOAEL) for males was therefore considered to be 30 mg/kg bw/day. However, the kidney changes of hyaline droplets were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. This effect is, therefore, not indicative of a hazard to human health. In the context of this study, the remaining kidney findings, consisting of tubular and/or degeneration, tubular dilation/vacuolation, granulated tubular casts and interstitial inflammatory infiltrate detected in males are more likely to be correlated to the same condition as hyaline droplet accumulation and are, therefore, considered to represent limited relevance to humans.

Effects excluding the kidney changes detected in males were confined to adaptive microscopic liver and thyroid changes. These were considered not to represent an adverse effect of treatment. In terms of extrapolation to man and risk assessment calculations whereby effects relating to male rat renal changes are species and sex specific and therefore are not relevant, a NOAEL for males can be established at 1000 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
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

The oral administration of 1,1,4,4-tetramethylbutane-1,4-diylbis(2-ethylperoxyhexanoate) (CAS 13052-09-0)  to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day, resulted in treatment related findings in animals of either sex treated with 30, 300 and 1000 mg/kg bw/day.  The effects detected in females were mainly confined to adaptive microscopic thyroid changes and there were no findings observed that were considered to represent an adverse effect of treatment.  The ‘No Observed Adverse Effect Level' (NOAEL) for females was considered to be 1000 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
In-life phase: 01 March 2012 (first day of treatment) and 28 June 2012 (final necropsy)
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.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, No.422: “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).
Deviations:
no
Qualifier:
according to
Guideline:
other: Compatible with Commission Regulation (EC) No. 440/2008 of 30/05/08 laying down test methods pursuant to Reg'n (EC) No.1907/2006 of the European Parliament and the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for seven days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 196 to 236g, the females weighed 158 to 195g, and were approximately six weeks old.

Initially, all animals were housed in groups of up to five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. From Week 14 of the study, as a result of increased body weight, males were housed in groups of up to three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used. Certificates of analysis of the batches of diet used are given in Addendum 1. 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) except for mated females during gestation and lactation.

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 temperatures and humidities are included in the study records. Study Plan target ranges for temperature and relative humidity were 22 ± 3ºC and 50 ± 20% respectively; there were no deviations from these targets.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
For the purpose of this study the test item was prepared at the appropriate concentrations as an emulsion in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services as part of this study (see Appendix 33). Results show the formulations to be stable for at least twenty-one days. Formulations were therefore prepared twice monthly and stored at approximately +4ºC in the dark.

Representative samples of test item formulations were taken and analysed for concentration of 1,1,4,4-tetramethylbutane-1,4-diylbis(2-ethylperoxyhexanoate) (CAS 13052-09-0) at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Appendix 33. The results indicated that the analysed formulations were within 86-107% of the nominal concentrations and were considered to confirm the accuracy of the formulation procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Summary
The concentration of CAS 13052-09-0 in the test item formulations was determined by gas chromatography (GC) using an external standard technique.


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


Standards
Standard solutions of test item were prepared in methanol 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 50 ºC for 2 mins
rate 10 ºC/min
final 260 ºC for 10 mins
Injection temperature :250 ºC
Flame ionisation detector temperature 250 ºC
Injection volume: 1 µl
Retention time : Profile of peaks from ~ 3 to 17 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 approximately +4ºC in the dark for twenty one days.


Verification of Test Item Formulation Concentrations
The test item formulations were sampled and analysed within six days of preparation.
Details on mating procedure:
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.
Duration of treatment / exposure:
Up to sixteen weeks (including an eleven week pre-pairing phase, pairing, gestation and early lactation), at dose levels of 30, 300 and 1000 mg/kg bw/day.
Frequency of treatment:
Daily
Duration of test:
Up to sixteen weeks (including an eleven week pre-pairing phase, pairing, gestation and early lactation), at dose levels of 30, 300 and 1000 mg/kg bw/day.
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 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:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The animals were allocated to dose groups using a randomisation procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups. The animals were uniquely identified within the study, by an ear punching system routinely used in these laboratories.


Chronological Sequence of Study
i) Prior to the start of the study, all animals from the control and high dose groups were subjected to ophthalmoscopic examination.

ii) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.

iii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.

iv) Three weeks prior to pairing, females were subjected to a daily vaginal smear for assessment of oestrous cyclicity.

v) On Day 78, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

vi) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.

vii) During Week 13, females from the control and high dose groups were subjected to ophthalmoscopic examinations.

viii) During Week 15, all selected males were evaluated for functional/sensory responses to various stimuli. Males from the control and high dose groups were also subjected to ophthalmoscopic examinations.

ix) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.

x) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.

xi) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 110. The male dose groups were killed and examined macroscopically on Day 111.

xii) Blood samples were taken from females from each dose group at termination for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.
Maternal examinations:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.


Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.


Behavioural Assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:

Gait
Hyper/Hypothermia
Tremors
Skin colour
Twitches
Respiration
Convulsions
Palpebral closure
Bizarre/Abnormal/Stereotypic behaviour
Urination
Salivation
Defecation
Pilo-erection
Transfer arousal
Exophthalmia
Tail elevation
Lachrymation

This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioural Assessments and Sensory Reactivity Tests.


Functional Performance Tests
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).

Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).


Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).

The following parameters were observed:
Grasp response
Touch escape
Vocalisation
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach


Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.
Normal range data for body weight changes in pregnant and lactating females are presented in Addendum 3.


Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase.
For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.


Water Consumption
Water intake was measured daily throughout the study (with the exception of the pairing phase).


Ophthalmoscopic Examination
The eyes of all control and high dose animals were examined pre-treatment and before termination of treatment (during Week 13 for females and Week 15 for males). Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex. Following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.


Reproduction Screening

Oestrus Cycle
During Weeks 9 to 11 a vaginal smear was taken from each female rat daily and a sample was placed on a glass slide. The smears were allowed to dry and then stained using a diluted giemsa stain. The slides were examined microscopically and the stage of oestrous was recorded.

Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.


Pregnancy and Parturition
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:

i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition


Laboratory Investigations
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 110 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.


Haematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).


Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Calcium (Ca++)
Glucose
Inorganic phosphorus (P)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
Albumin
Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphatase (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl-)
Total bilirubin (Bili)
Bile acids


Pathology
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 111. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).

All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.


Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals
Prostate
Brain
Seminal vesicles
Epididymides
Spleen
Heart
Testes
Kidneys
Thymus
Liver
Thyroid (weighed post-fixation with Parathyroid)
Ovaries
Uterus (weighed with Cervix)


Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
Pituitary
Bone & bone marrow (sternum)
Prostate
Brain (including cerebrum, cerebellum and pons)
Oesophagus
Caecum
Rectum
Coagulating gland
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides•
Skin (hind limb)
Eyes*
Spinal cord (cervical, mid-thoracic and lumbar)
Gross lesions
Heart
Spleen
Ileum (including peyer’s patches)
Stomach
Jejunum
Thyroid/parathyroid
Kidneys
Trachea
Liver
Testes•
Lungs (with bronchi) #
Thymus
Lymph nodes (mandibular and mesenteric)
Urinary bladder
Mammary gland
Uterus/Cervix
Muscle (skeletal)
Vagina

All tissues were despatched to the histology processing Test Site (Harlan Laboratories Ltd, Itingen, Switzerland) for processing. The tissues from five selected control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 1000 mg/kg bw/day animals were also processed. In addition, sections of testes and epididymides from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Since there were indications of treatment-related kidney changes in males, and liver and thyroid changes in animals of either sex, examination was subsequently extended to include similarly prepared sections of kidney from five males from the low and intermediate groups and similarly prepared sections of liver and thyroid from five animals per sex from the low and intermediate groups (or all ten animals per sex in the case of reproductive tissues).

Microscopic examination was conducted by AnaPath GmbH, Oberbuchsiten, Switzerland.
Ovaries and uterine content:
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).
Fetal examinations:
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)


Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.


Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using the individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:

Pre–implantation loss = (Number of corpora lutea - number of implantation sites / number of corpora lutea) x100

Post–implantation loss = (Number of iomplantation sites - Total number of offpsring born / Number of implantation sites) x 100


ii) Live Birth and Viability Indices

The following indices were calculated for each litter as follows:

Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offpsring born) x 100

Viability Index (%) = (Number of offspring alive on Day 4 / Number of ofpsring alive on Day 1) x 100


iii) Sex Ratio (% males)

Sex ratio was calculated for each litter on Days 1 and 4 post partum, using the following formula:

(Number of male offspring / Total number of offpsring) x 100

Statistics:
Due to the nature and quantity of this data please see Section "any other information on materials and methods incl. tables"
Indices:
The following parameters were calculated from the individual data during the mating period of the parental generation:

i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices

For each group the following were calculated:
Mating Index (%) = (Number of animals mated/number if animals paired) x 100

Pregnancy Index (%) = (number of pregnant females / number of animals mated) x 100


Gestation and Parturition Data
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.

i) Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii) Parturition Index
The following was calculated for each group:

Parturition Index (%) = (number of females delivering live offspring / number of pregnant females) x 100
Historical control data:
Not applicable
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality
There was one unscheduled death on the study.

At 1000 mg/kg bw/day, female number 76 was found dead at the morning check on Day 98 of the study (Day 18 of gestation). No significant clinical signs had been observed for this animal prior to its death. Macroscopic necropsy revealed enlarged adrenals and a reddened left lung lobe but microscopic examination of the tissues for this animal did not reveal the aetiology of this death.


Clinical Observations
Clinical signs were mainly confined to increased post dosing salivation for both sexes at 1000 mg/kg bw/day, and to a lesser extent, males at 300 mg/kg bw/day. One female at 300 mg/kg bw/day also showed increased post dosing salivation on a single occasion.

One female at 1000 mg/kg bw/day showed piloerection, hunched posture and pallor of the extremities on Day 104 (around the time of parturition) but these findings were consistent with the physiological strains associated with littering and were considered to be unrelated to treatment.

The type, incidence and distribution of other clinical signs observed on the study did not indicate any obvious association with treatment.


Functional Observations

Behavioural Assessments
Assessment of the animals in a standard arena did not reveal any obvious effects of treatment at 30, 300 or 1000 mg/kg bw/day.

Functional Performance Tests
Assessment of grip strength or motor activity did not reveal any consistent pattern of results that indicated an adverse effect of treatment at 30, 300 or 1000 mg/kg bw/day.

During these assessments, reduced hind limb grip strength for males at all dosages at Test 1 and reduced fore limb grip strength for males at 30 and 300 mg/kg bw/day at Test 3 attained statistical significance compared to control. There was no dosage relationship and given the absence of statistically significant differences from control in the remaining tests of grip strength these findings were considered to be incidental and unrelated to treatment. For females at 1000 mg/kg bw/day, lower overall mobile score attained statistical significance compared to control, but in isolation this was considered to be of no toxicological significance.

Sensory Reactivity Assessments
Sensory reactivity to different stimuli did not reveal any adverse effect of treatment at 30, 300 or 1000 mg/kg bw/day.


Body Weight
There was no clear adverse effect of treatment on body weight performance of males at any of the dosages investigated. At 1000 mg/kg bw/day, occasionally lower body weight gain for males attained statistical significance when compared to control, but as overall gain was approximately 91% of control at the end of the study the differences observed were considered to reflect normal biological variation.

There was no clear adverse effect of treatment on body weight performance of females, including during gestation and early lactation, at any of the dosages investigated.


Food Consumption and Food Conversion Efficiency
There was no adverse effect of treatment on food consumption or food conversion efficiency of males during the study at any of the dosages investigated.

There was no adverse effect of treatment on food consumption or food conversion efficiency of females during the pre-pairing phase of the study and no subsequent adverse effect of treatment during gestation and early lactation, at any of the dosages investigated.


Water Consumption
Group mean water consumptions are given in Table 11. Individual and group mean daily water consumptions are presented in Appendix 11 and 12.
For males at 1000 mg/kg bw/day, there was a tendency for slightly higher water intake compared to control throughout most of the study; water consumption for females at this dosage appeared unaffected.

At 30 and 300 mg/kg bw/day there was no clear effect of treatment on water intake for either sex.


Ophthalmoscopic Examination
No treatment-related effects were detected during ophthalmic examination of animals at 1000 mg/kg bw/day.


Reproductive Performance

Oestrus Cycle Assessments
Assessment of female oestrous cycles did not indicate any treatment-related effects at 30, 300 or 1000 mg/kg bw/day.


Mating
Mating performance was unaffected by treatment at 30, 300 or 1000 mg/kg bw/day.

Fertility
Fertility was unaffected by treatment at 30, 300 or 1000 mg/kg bw/day.

Gestation Length
Gestation length was unaffected by treatment at 30, 300 or 1000 mg/kg bw/day.


Laboratory Investigations

Haematology
For males at 1000 mg/kg bw/day, mean platelet count was statistically significantly higher than control with half of the individual values for treated animals exceeding the historical control range compared to only one in the control group.

Additionally, for males at 1000 mg/kg bw/day, prothrombin times were slightly faster than control with differences attaining statistical significance. All individual values were within the historical control range and, although these times may have been influenced to some extent by the higher mean platelet count at this dosage, it was considered that the differences in prothrombin times, particularly in the absence of any histopathological finding for the bone marrow, were incidental and of no toxicological significance.

At 1000 mg/kg bw/day, lower mean cell haemoglobin concentration for both sexes attained statistical significance when compared with control, however there were no other statistically significant differences from control apparent for other erythrocyte parameters for these animals and all individual values were within the historical control range. In isolation this finding was considered incidental and unrelated to treatment.

At 30 and 300 mg/kg bw/day there were no statistically significant differences from control observed for haematology parameters.


Blood Chemistry
No toxicologically significant effects were detected for males at 1000 mg/kg bw/day or either sex at 30 or 300 mg/kg bw/day.

For females at 1000 mg/kg bw/day, higher total protein and albumin levels attained statistical significance compared with control although no statistically significant change in albumin/globulin ratio was apparent. Although occasional control values for these parameters exceeded the historical control range, this range was exceeded for both parameters at a much higher incidence by the treated animals.

At 1000 mg/kg bw/day, lower bilirubin levels for both sexes attained statistical significance when compared with control. All individual values for treated animals were within the historical control.

For males at 1000 mg/kg bw/day, higher urea levels attained statistical significance compared with control, but only two values for treated animals exceeded the historical control range. Additionally levels of potassium, calcium and phosphorus were also statistically significantly higher than control but, with the exception of two individual values for potassium, individual values for treated animals were within the historical control range.
At 30 and 300 mg/kg bw/day there were no statistically significant differences for blood chemistry parameters compared with controls.


Pathology

Necropsy

Adults
At 1000 mg/kg bw/day, two males showed mottled kidneys and this finding may be associated with the treatment related histopathological renal changes observed for males at this dosage.

Additionally another male at 1000 mg/kg bw/day showed a small and malformed left kidney but this finding was considered likely to be congenital in nature and unrelated to treatment.

Remaining necropsy observations observed on the study were unremarkable and did not indicate any adverse effect of treatment at 30, 300 or 1000 mg/kg bw/day.


Organ Weights
At 1000 mg/kg bw/day, absolute and body weight relative kidney weights for both sexes were higher than control with differences attaining statistical significance. A statistically significant increase in kidney weights was also apparent for males receiving 300 mg/kg bw/day.

For males at 1000 mg/kg bw/day, increased absolute and body weight relative liver weights also attained statistical significance compared with control.

For males at all dosages, higher absolute heart weights attained statistical significance compared with control, although no individual values for treated animals exceeded the historical control. When adjusted for body weight, mean male heart weights at all dosages were statistically significantly lower than control but showed no dosage relationship. For females at all dosages, absolute heart weights were statistically significantly lower than control but showed no dosage-relationship and only one individual value in each treatment group was lower than the historical control range. When adjusted for body weight, mean female heart weights at all dosages were statistically significantly lower than control. In the absence of any histopathological change these differences in heart weight were considered incidental and unrelated to treatment.

For males at all dosages, lower absolute and body weight relative thymus weights attained statistical significance compared with control, however there was no dosage relationship and all individual values for treated animals were within the historical control range. In the absence of any supporting histopathological change this finding was considered incidental and unrelated to treatment.

For females at 300 and 1000 mg/kg bw/day, lower absolute and body weight relative brain weights attained statistical significance compared with control, but there was no dosage relationship. In the absence of any supporting histopathological change this finding was considered incidental and unrelated to treatment.


Histopathology
At 300 and 1000 mg/kg bw/day the livers of several males showed centrilobular hepatocellular hypertrophy; since this finding was not accompanied by degenerative or inflammatory lesions it was considered to be reversible and to represent an adaptive effect.

Males at 300 and 1000 mg/kg bw/day also showed hyaline droplet nephropathy of the kidney, consisting of increased incidence and severity of hyaline droplets, tubular degeneration, granulated tubular casts and interstitial inflammatory infiltrate. This nephropathy was deemed to be related to treatment. Hyaline droplets were also present for males at 30 mg/kg bw/day but these occurred in the absence of any degenerative changes.

For females at 30 mg/kg bw/day and both sexes at 300 and 1000 mg/kg bw/day increased incidence and severity of follicular hypertrophy was recorded in the thyroid. Follicular hypertrophy of the thyroids is deemed to be associated with the increased hepatic metabolisation of thyroid hormones (T3/T4) due to hepatocellular hypertrophy in the respective groups. The reason for hypertrophic changes in the thyroids of females was not clear but it was assumed that the mechanism of increased hepatic metabolisation and subsequent thyroidal changes also applies for females, however with no overt morphological hepatic correlate. Therefore, this finding is deemed to represent a secondary effect.

There were no abnormal lesions encountered during sperm staging regarding completeness of stages and maturation of cell populations for males at 1000 mg/kg bw/day. Individual lesions recorded were within the range of background alterations that may be recorded in this type of study, in rats of this strain and age.

The remainder of histopathological findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

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:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: see Remarks:
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
5.3 Litter Responses
As previously discussed one female at 1000 mg/kg bw/day was found dead during late gestation; additionally at this dosage one female was found to be non-pregnant. For the non-pregnant animal, histopathological examination revealed a vaginal retention cyst which was deemed to be the underlying cause for the unsuccessful mating. Two control litters showed post partum total litter loss but there were no similar losses for treated groups. The following assessment is generally based on the 8, 10, 10 and 8 litters successfully reared to Day 5 of age for the control, 30, 300 and 1000 mg/kg bw/day dosage groups respectively.
5.3.1 Offspring Litter Size, Sex Ratio and Viability
Group mean corpora lutea and implantation counts, litter size, implantation losses, survival indices and sex ratio are given in Tables 14 to 16. Individual data are given in Appendices 15 to 17.
There was no adverse effect of treatment on corpora lutea and implantation counts, or on subsequent post-natal litter size, sex ratio or offspring survival at 30, 300 and 1000 mg/kg bw/day.
5.3.2 Offspring Growth and Development
Group values for total litter weights, offspring body weight and body weight change, surface righting reflex and a summary incidence of clinical signs are given in Tables 14, 17 and 18. Individual values and observations are given in Appendices 15, 16 and 19.
Litter weight and offspring body weight on Day 1 and subsequent offspring growth to Day 4 of age was unaffected by treatment at 30, 300 and 1000 mg/kg bw/day.
Assessment of surfacing righting ability on Day 1 of age was unaffected by treatment at 30, 300 and 1000 mg/kg bw/day. The clinical signs observed for the offspring on this study were typical for the age observed and neither the incidence or distribution of these signs indicated any underlying adverse effect on offspring development.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see Remarks
Abnormalities:
not specified
Developmental effects observed:
not specified

Sperm Analysis

There were no toxicologically significant effects detected in sperm motility values, morphological assessments or in homogenisation-resistant spermatid counts for males receiving 1000 mg/kg bw/day.

Conclusions:
The oral administration of 1,1,4,4-tetramethylbutane-1,4-diylbis(2-ethylperoxyhexanoate) (CAS 13052-09-0) to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day, resulted in treatment related findings in animals of either sex treated with 30, 300 and 1000 mg/kg bw/day. The effects detected in females were mainly confined to adaptive microscopic thyroid changes and there were no findings observed that were considered to represent an adverse effect of treatment. The ‘No Observed Adverse Effect Level' (NOAEL) for females was considered to be 1000 mg/kg bw/day.

The effects detected in males were mainly confined to adaptive microscopic liver and thyroid changes and hyaline droplet nephropathy at 300 and 1000 mg/kg bw/day. The hyaline droplet nephropathy of the kidney consisted of increased incidence and severity of hyaline droplets, tubular degeneration, granulated tubular casts and interstitial inflammatory infiltrate. This nephropathy was deemed to be related to treatment and to represent an adverse effect of treatment to the rat. Hyaline droplets were also present for males at 30 mg/kg bw/day but these occurred in the absence of any degenerative changes and the No Observed Adverse Effect Level' (NOAEL) for males was therefore considered to be 30 mg/kg bw/day. However, the hyaline droplets nephropathy at higher dosages were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. This effect is, therefore, not indicative of a hazard to human health. In the context of this study, the remaining kidney findings, consisting of tubular and/or degeneration, tubular dilation/vacuolation, granulated tubular casts and interstitial inflammatory infiltrate detected in males are more likely to be correlated to the same condition as hyaline droplet accumulation and are, therefore, considered to represent limited relevance to humans.

Enhanced evaluation of reproduction for this study did not indicate any effect of treatment on reproduction including litter size and offspring survival, growth and development at dosages up to 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.
Executive summary:

Introduction.The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to be compatible with the 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 ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to sixteen weeks (including an eleven week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 30, 300 and 1000 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioural assessments, body weight change, food and water consumption, oestrous cycles and opthalmic change were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 78 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with assessments of litter size, offspring weights and surface righting reflex.

Extensive functional observations were performed on males from each dose group during Week 15 and for parental females from each dose group on Day 4post partumHaematology and blood chemistry were evaluated prior to termination on males (Day 110) and females (Day 4 of lactation) from each dose group. An opthalmic examination was made for control and high dose animals prior to treatment and during Week 13 for females and Week 15 for males. 

Adult males were terminated on Day 112, followed by the termination of all surviving females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on or after Day 25post coitum. All animals were subjected to a gross necropsy examination with recording of selected organ weight and additionally sperm analysis for males. Histopathological evaluation of selected tissues was performed, with enhanced assessment of testes for males.

Results.

Adult Responses:

Mortality.At 1000 mg/kg bw/day, one female was found dead on Study Day 98 (Day 18 of gestation) without any significant clinical signs being seen prior to this event. Enlarged adrenals and a reddened left lung lobe were apparent at necropsy but microscopic examination did not reveal the aetiology of this death and this death was considered to be unrelated to treatment.

Clinical Observations.Clinical signs associated with treatment were confined to increased post dosing salivation for both sexes at 1000 mg/kg bw/day, and males and one female at 300 mg/kg bw/day.

Behavioural Assessment.No obvious neurological effects of treatment at 30, 300 or 1000 mg/kg bw/day were apparent.

Functional Performance Tests.There was no consistent pattern of results that indicated an effect of treatment at 30, 300 or 1000 mg/kg bw/day.

Sensory Reactivity Assessments.Sensory reactivity assessments to different stimuli did not reveal any effect of treatment at 30, 300 or 1000 mg/kg bw/day.

Body Weight.There was no adverse effect of treatment on body weight performance for males or females, including during gestation and lactation phases at 30, 300 or 1000 mg/kg bw/day.

Food Consumption and Food Conversion Effeciency.There was no adverse effect of treatment on food consumption and food conversion efficiency for males or females, including during gestation and lactation phases, at 30, 300 or 1000 mg/kg bw/day.

Water Consumption.At 1000 mg/kg bw/day, water consumption for males was slightly higher than control throughout most of the study.

Ophthalmoscopy.No treatment-related effects were detected for animals at 1000 mg/kg bw/day.

Reproductive Performance:

Oestrous Cycle.There was no adverse effect of treatment on female oestrous cycles at 30, 300 or 1000 mg/kg bw/day.

Mating.Mating performance was unaffected by treatment at 30, 300 or 1000 mg/kg bw/day.

Fertility.Fertility was unaffected by treatment at 30, 300 or 1000 mg/kg bw/day.

Gestation Lengths.Gestation length was unaffected by treatment at 30, 300 or 1000 mg/kg bw/day.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability.Corpora lutea and implantation counts and subsequent post-natal litter size, sex ratio and offspring survival were unaffected by treatment at 30, 300 and 1000 mg/kg bw/day.

Offspring Growth and Development.Offspring body weight, litter weight and surface righting ability on Day 1 and subsequent clinical signs and offspring growth to Day 4 of age were unaffected by treatment at 30, 300 and 1000 mg/kg bw/day.

Laboratory Investigations:

Haematology.Males treated with 1000 mg/kg bw/day showed increased platelet count compared with control. No toxicologically significant effects were detected for females at this dosage or either sex at 30 or 300 mg/kg bw/day.

Blood Chemistry.Females at 1000 mg/kg bw/day showed increased total protein and albumin levels compared to control. No similar effects were apparent for males at 1000 mg/kg bw/day or either sex at 30 or 300 mg/kg bw/day.

Pathology:

Necropsy.At 1000 mg/kg bw/day, two adult males showed mottled kidneys at necropsy examination. There were no other necropsy findings for adult animals or their offspring that were considered to be associated with treatment at 30, 300 or 1000 mg/kg bw/day.

Organ Weights.For both sexes at 1000 mg/kg bw/day and males at 300 mg/kg bw/day, absolute and body weight relative kidney weights were statistically significantly higher than control.

For males at 1000 mg/kg bw/day, increased absolute and body weight relative liver weights also attained statistical significance compared with control.

Sperm Analysis.Assessment of sperm motility values, morphological assessments and homogenisation-resistant spermatid counts for males at 1000 mg/kg bw/day did not indicate any effects of treatment.

Histopathology.At 300 and 1000 mg/kg bw/day males showed adaptive liver changes consisting of centrilobular hepatocellular hypertrophy; there were no degenerative or inflammatory lesions.

At 300 and 1000 mg/kg bw/day males also showed treatment related hyaline droplet nephropathy of the kidney, consisting of increased incidence and severity of hyaline droplets, tubular degeneration, granulated tubular casts and interstitial inflammatory infiltrate.

For females at 30 mg/kg bw/day and both sexes at 300 and 1000 mg/kg bw/day increased incidence and severity of follicular hypertrophy was recorded in the thyroid. This finding was considered to be a secondary effect of increased hepatic metabolism.

There were no abnormal lesions encountered during testicular spermatogenesis staging for males at 1000 mg/kg bw/day.

Conclusion.The oral administration of 1,1,4,4-tetramethylbutane-1,4-diylbis(2-ethylperoxyhexanoate) (CAS 13052-09-0) to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day, resulted in treatment related findings in animals of either sex treated with 30, 300 and 1000 mg/kg bw/day. The effects detected in females were mainly confined to adaptive microscopic thyroid changes and there were no findings observed that were considered to represent an adverse effect of treatment. The ‘No Observed Adverse Effect Level' (NOAEL) for females was considered to be 1000 mg/kg bw/day.

Kidney effects detected in males at 300 and 1000 mg/kg bw/day consisted of increased incidence and severity of hyaline droplets, tubular degeneration, granulated tubular casts and interstitial inflammatory infiltrate. This nephropathy was deemed to be related to treatment and to represent an adverse effect of treatment to the rat. Hyaline droplets were also present for males at 30 mg/kg bw/day but, occurred in the absence of any degenerative changes and the ‘No Observed Adverse Effect Level' (NOAEL) for males was therefore considered to be 30 mg/kg bw/day. However, the kidney changes of hyaline droplets were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. This effect is, therefore, not indicative of a hazard to human health. In the context of this study, the remaining kidney findings, consisting of tubular and/or degeneration, tubular dilation/vacuolation, granulated tubular casts and interstitial inflammatory infiltrate detected in males are more likely to be correlated to the same condition as hyaline droplet accumulation and are, therefore, considered to represent limited relevance to humans.

Effects excluding the kidney changes detected in males were confined to adaptive microscopic liver and thyroid changes. These were considered not to represent an adverse effect of treatment. In terms of extrapolation to man and risk assessment calculations whereby effects relating to male rat renal changes are species and sex specific and therefore are not relevant, a NOAEL for males can be established at 1000 mg/kg bw/day.

Enhanced evaluation of reproduction for this study did not indicate any effect of treatment on reproduction including litter size and offspring survival, growth and development at dosages up to 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
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

Justification for classification or non-classification

Data lacking