Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No effects on reproductive organs or sperm analysis were found in the OECD 408 study

An OECD 422 did not reveal any effects on fertility up to 1000 mg/kg bw/d. These results suggest a low risk for reproductive toxicity; therefore, we are waiving the requirement for a reproductive toxicity study.  

According to Annex IX 8.7.3 a reproductive study only needs to be conducted if there are adverse effects on reproductive organs or tissues.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
In-life phase: 23/05/12 to 16/07/12
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 five 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 310 to 363g, the females weighed 188 to 216g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of 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.

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 paired animals and 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. The temperatures and relative humidity controls were set to achieve target values of 21 ± 2°C and 55 ± 15% respectively. Short term deviations from these targets were considered not to affect the purpose or integrity of the study.

The animals were allocated to dose groups using a randomisation procedure based on stratified body weights, 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.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least nineteen days. Formulations were therefore prepared either weekly or fortnightly and stored at 4ºC in the dark.

Samples of each test item formulation were taken and analysed for concentration of 1,1,3,3-tetramethylbutyl 2-ethylperoxyhexanoate (CAS 22288-43-3) at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ±6% of the nominal concentration.
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 1,1,3,3-Tetramethylbutyl 2-Ethylperoxyhexanoate (CAS 22288-43-3) in the test item formulations was determined by gas chromatography (GC) using an external standard technique.


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


Standards
Standard solutions of test item were prepared in acetonitrile at a nominal concentration of 0.1 mg/ml. The standard solutions contained the equivalent amount of vehicle to that of the relevant samples.


Procedure
The standard and sample solutions were analysed by GC using the following conditions:
GC system : Agilent Technologies 5890, incorporating autosampler and workstation
Column : ZB-5 (30 m x 0.53 mm id x 5 µm film)
Oven temperature program :initial 50 ºC for 1 min
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 ~ 4 to 8 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 nineteen days.


Verification of Test Item Formulation Concentrations
The test item formulations were sampled and analysed within three days of preparation.

Duration of treatment / exposure:
Oral administration of the test substance to the rats for a period of up to fifty-four consecutive days.
(Male dose groups terminated Day 43, females - Day 5 post partum)

Frequency of treatment:
Daily
Details on study schedule:
Chronological Sequence of Study
i) 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.

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

iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

iv) 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.

v) On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.

vi) 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.

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

viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43.

ix) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all 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:
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 (including control)
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, 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 five selected 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).


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 42 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):
A vaginal smear was prepared for each female and the stage of the oestrous cycle was recorded.

Sperm parameters (parental animals):
Detailed qualitative examination of the testes was performed, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
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)
Postmortem examinations (parental animals):
Pathology
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. 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 a suitable barbiturate agent. 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)
Pituitary (post fixation)


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
Gross lesions lumbar)
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

The tissues from five selected control and 1000 mg/kg bw/day dose group animals 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. Detailed qualitative examination of the testes was performed, taking into account the tubular stages of the spermatogenic cycle.

Since there were indications of treatment-related liver, spleen, kidney and thyroid changes, examinations were subsequently extended to include similarly prepared sections of liver, spleen, kidney and thyroid from five animals per sex from the low and intermediate groups.


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:
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
Description (incidence and severity):
Please refer to the "Details on results" section below
Gross pathological findings:
no effects observed
Description (incidence and severity):
Observed effects were considered not to be of toxicological importance. Please refer to the "Details on results" section below
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Observed effects were considered not to be of toxicological importance. Please refer to the "Details on results" section below
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Please refer to the "Details on results" section below
Clinical Observations
There were no toxicologically significant clinical signs detected in treated animals.

Animals of either sex treated with 1000 mg/kg bw/day showed episodes of increased salivation from Day 3 (males) and Day 5 (females) to Day 34. Two males treated with 300 mg/kg bw/day also showed increased salivation on Day 15. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are considered not to be of toxicological importance.


Functional Observations

Behavioural Assessments
Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.

Functional Performance Tests
There were no toxicologically significant changes in functional performance.

Females treated with 1000 mg/kg bw/day showed a statistically significant increase in hind and fore limb grip strength. Females treated with 1000 and 300 mg/kg bw/day also showed a statistically significant reduction in the final 20% of mobility (considered to be the asymptotic period). The statistically significant differences in hind/fore limb grip strength were confined to one out of the three tests and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered to be of no toxicological significance.

Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.


Body Weight
There were no toxicologically significant effects detected in body weight development.

Males treated with 30 mg/kg bw/day showed a statistically significant increase in body weight gain during the first two weeks of treatment. An increase in body weight gain is considered not to represent an adverse effect of treatment and in the absence of a true dose related response the intergroup difference was considered not to be of toxicological importance.


Food Consumption
No adverse effect on food consumption or food efficiency was detected in treated animals when compared to control animals.

Females treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in food consumption during the final week of gestation.

An increase in food consumption is considered not to represent an adverse effect of treatment therefore the intergroup differences were considered not to be of toxicological importance.


5.1.6 Water Consumption
No toxicologically significant effect on water consumption was detected.

Females treated with 1000 mg/kg bw/day showed an increase in water consumption throughout maturation, gestation and lactation. Statistically significant increases were evident throughout gestation and lactation. Females treated with 300 mg/kg bw/day also showed a statistically significant increase in water consumption during the final week of gestation and during lactation. Males from all treatment groups showed an increase in overall water consumption throughout the treatment period however a true dose related response was not evident at 300 and 30 mg/kg bw/day. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in the absence of any associated microscopic gastric changes the intergroup differences were considered not to be of toxicological importance.


Reproductive Performance

Mating
There were no treatment-related effects on mating performance.


Fertility
No treatment-related effects on fertility were detected for treated animals, when compared to controls.

One control female, one female treated with 300 mg/kg bw/day and two females treated with 1000 mg/kg bw/day did not achieve pregnancy following evidence of mating. No histopathological correlates were evident in the female reproductive organs. Microscopic examination including qualitative examination of the testes in the male partners from the control and 1000 mg/kg bw/day dose groups revealed testicular degeneration. This was considered to be the reason for the non pregnancies. This lesion is a naturally occurring background change in rats and due to its presence in a control animal was considered not to be of toxicological significance. No histopathological correlates were evident in the reproductive organs of the male partner treated with 300 mg/kg bw/day.

There were no differences in gestation lengths. The distribution for treated females was comparable to controls. All animals showed gestation lengths of 22 to 24½ days.


Laboratory Investigations

Haematology
Males treated with 1000 mg/kg bw/day showed an increase in platelet count. Females treated with 1000 mg/kg bw/day showed a statistically significant increase in neutrophil (P<0.01) and monocyte (P<0.05) count. Females treated with 300 mg/kg bw/day also showed a statistically significant increase in neutrophil (P<0.01) count. The majority of the individual values for these parameters were outside of the historical control ranges for rats of the strain and aged used.

No toxicologically significant effects were detected in males treated with 300 mg/kg bw/day or animals of either sex treated with 30 mg/kg bw/day.

Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in mean corpuscular haemoglobin concentration whilst females from this treatment group showed a statistically significant reduction in haemoglobin and erythrocyte count. Females treated with 300 mg/kg bw/day also showed a statistically significant reduction in haemoglobin. Males from all treatment groups showed a statistically significant increase in total leucocyte count and males treated with 30 mg/kg bw/day also showed a statistically significant increase in neutrophil count. For all parameters listed in this paragraph the majority of individual values were within normal ranges for rats of the strain and age used, and in the absence of true dose related responses the intergroup differences were considered not to be of toxicological importance.


Blood Chemistry
Males treated with 1000 mg/kg bw/day showed a statistically significant increase in albumin/globulin ratio (P<0.01) and alanine aminotransferase (P<0.01). Females from this treatment group showed a statistically significant increase in albumin (P<0.01) and total protein (P<0.01). Females from all treatment groups also showed a statistically significant increase in cholesterol levels (P<0.05 - P<0.01). The majority of the individual values for these parameters were outside of the historical control ranges for rats of the strain and aged used.

No toxicologically significant effects were detected in males treated with 300 or 30 mg/kg bw/day.

Males from all treatment groups showed statistically significant increases in creatinine and albumin (P<0.05 - P<0.01). Females from all treatment groups showed a statistically significant increase in urea (P<0.05 - P<0.01). Males treated with 1000 mg/kg bw/day also showed a statistically significant increase in sodium concentration (P<0.05). For all parameters listed in this paragraph all of the individual values were within normal ranges for rats of the strain and age used, and in the absence of true dose related responses the intergroup differences were considered not to be of toxicological importance.



Pathology

Necropsy

Adults
At 1000 mg/kg bw/day macroscopic examination revealed enlarged, mottled and pale kidneys in five males, enlarged kidneys in one male and enlarged and mottled kidneys in a further male. Three males treated with 300 mg/kg bw/day had pale and mottled kidneys, one male had pale and enlarged kidneys and a further two males had either pale or mottled kidneys at necropsy.

No toxicologically significant macroscopic abnormalities were detected in females treated with 1000 or 300 mg/kg bw/day or in animals of either sex treated with 30 mg/kg bw/day.

Two females treated with 1000 mg/kg bw/day and one female treated with 300 mg/kg bw/day had reddened lungs at necropsy. In the absence of any histology correlates the intergroup differences were considered not to be of toxicological importance. One female treated with 1000 mg/kg bw/day showed increased renal pelvic space. This finding is considered to be a congenital abnormality and unrelated to treatment. One male treated with 30 mg/kg bw/day had an enlarged spleen. This finding was associated with the microscopic finding identified as increased hemopoiesis in the spleen. At this dose level, the increased hemopoiesis was considered to be incidental and is therefore, of no toxicological importance. One control female had an enlarged left adrenal and a small right adrenal. In the absence of treatment, this was considered to be an incidental finding. One control male and one male treated with 1000 mg/kg bw/day had small epididymides and small and flaccid testes. A further male treated with 1000 mg/kg bw/day had small epididymides and testes. These findings correlated histopathologically with testicular tubular degeneration and were considered to be within the range of normal background alterations and were therefore considered not to be of toxicological importance. One male treated with 1000 mg/kg bw/day also showed small seminal vesicles and prostate. In the absence of any histology correlates the intergroup differences were considered not to be of toxicological importance.


Organ Weights
Males treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in kidney, liver and spleen weight both absolute and relative to terminal body weight. Females treated with 1000 mg/kg bw/day showed a statistically significant increase in absolute and relative liver and spleen weight. Females treated with 300 mg/kg bw/day also showed a statistically significant increase in liver weight both absolute and relative to terminal body weight.

No such effects were detected in animals of either sex treated with 30 mg/kg bw/day.

Males treated with 1000 mg/kg bw/day showed a statistically significant increase in adrenal weight both absolute and relative to terminal body weight. Females treated with 1000 mg/kg bw/day showed a statistically significant increase in absolute and relative kidney weight. In the absence of any associated histopathological changes the intergroup differences were considered not to be of toxicological importance. Females treated with 300 mg/kg bw/day showed a statistically significant reduction in thyroid weight both absolute and relative to terminal body weight. In the absence of a similar effect at 1000 mg/kg bw/day or any associated histopathological changes the intergroup difference was considered not to be of toxicological importance.


Histopathology: Please refer to the section below "Any other information on results"
Key result
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on a reduced live birth index and a reduction in offspring body weights in litters from females treated with 1000 mg/kg bw/day.
Key result
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'
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Please refer to the "Details on results" section below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Please refer to the "Details on results" section below
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
In total, all females from the 30 mg/kg bw/day dose group, nine females from the control and 300 mg/kg bw/day dose groups and eight females from the 1000 mg/kg bw/day dose group gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.


Offspring Litter Size, Sex Ratio and Viability
A statistically significant reduction in live birth index was evident in litters from females treated with 1000 mg/kg bw/day. Litter viability at Day 4, however, was comparable to control females. No significant differences were detected for corpora lutea, implantation counts or litter size for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.


Offspring Growth and Development
Offspring body weights in litters from females treated with 1000 mg/kg bw/day were reduced on Day 1. Statistical significance was, however, only achieved for males. A slight reduction in offspring body weight was also evident at this dose level on Day 4 post partum and subsequent offspring body weight gain was slightly reduced. Statistical analysis of the data did not however reveal and significant intergroup differences.

A statistically significant reduction in surface righting was also evident in litters from females treated with 1000 mg/kg bw/day.

Statistical analysis of the litter weights data did not reveal any significant intergroup differences.

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, reddened/swollen hind limbs, found dead or missing, were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.


Necropsy

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 toxicity of the test item.
Key result
Dose descriptor:
NOEL
Remarks:
Reproductive toxicity
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on a reduced live birth index and a reduction in offspring body weights in litters from females treated with 1000 mg/kg bw/day.
Reproductive effects observed:
not specified

Histopathology

The following treatment related microscopic findings were detected:

Liver: Centrilobular to diffuse hepatocellular hypertrophy was evident in animals of either sex treated with 1000 and 300 mg/kg bw/day and in males treated with 30 mg/kg bw/day.

Incidence and Mean Severity of Main Findings in Liver

Finding / Groups

1

2

3

4

Total Affected /
Mean Severity

(5) M

(5) F

(5) M

(5) F

(5) M

(5) F

(5) M

(5) F

Hepatocellular hypertrophy

-

-

5

1.2

-

5

1.8

3

1.0

5

2.2

4

1.3

Kidneys: Nephropathy consisting of increased incidence and severity of hyaline droplets, tubular degeneration, tubular dilation/vacuolation, granulated tubular casts and interstitial inflammatory infiltrate was evident in males treated with 1000, 300 and 30 mg/kg bw/day.


Incidence and Mean Severity of Main Findings inKidneys

Finding / Groups

1

2

3

4

Total Affected /
Mean Severity

(5) M

(5) F

(5) M

(5) F

(5) M

(5) F

(5) M

(5) F

Hyaline droplets

5

1.0

-

5

2.2

-

8

3.1

-

10

3.0

-

Granular tubular casts

-

-

3

2.0

-

5

3.0

-

8

2.8

-

Tubular degeneration

-

-

5

1.4

-

8

2.8

-

10

3.2

-

Interstitial inflammatory infiltrate

1

1.0

-

2

1.0

-

4

1.0

-

7

1.1

-

Tubular dilation/vacuolation

-

-

-

-

-

-

5

2.0

-

Spleen: Slightly increased incidence and/or severity of splenic extramedullary hematopoiesis was evident in animals of either sex treated with 1000 mg/kg bw/day.

Incidence and Mean Severity of Main Findings in Spleen

Finding / Groups

1

2

3

4

Total Affected /
Mean Severity

(5) M

(5) F

(5) M

(5) F

(5) M

(5) F

(5) M

(5) F

Increased hematopoiesis

-

5

1.8

1

1.0

3

1.3

1

1.0

4

1.8

3

1.0

5

2.6

Thyroids: Increased incidence and severity of follicular hypertrophy was evident in animals of either sex treated with 1000 and 300 mg/kg bw/day and in males treated with 30 mg/kg bw/day.

Incidence and Mean Severity of Main Findings in Thyroids

Finding / Groups

1

2

3

4

Total Affected /
Mean Severity

(5) M

(5) F

(5) M

(5) F

(5) M

(5) F

(5) M

(5) F

Follicular Hypertrophy

4

1.0

3

1.0

5

1.6

1

1.0

5

2.0

4

1.3

5

2.0

4

1.8

 


Conclusions:
The oral administration of 1,1,3,3-tetramethylbutyl 2-ethylperoxyhexanoate (CAS 22288-43-3) 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 from all treatment groups. A 'No Observed Effect Level' (NOEL) for either sex was not achieved. The effects detected in females however were confined to increases in neutrophil and monocyte counts, increased total protein, albumin and cholesterol and adaptive microscopic liver, thyroid and spleen changes. These were considered not to represent an adverse effect of treatment therefore the 'No Observed Adverse Effect Level' (NOAEL) was considered to be 1000 mg/kg bw/day for females.

The kidney effects detected in males from all treatment groups were considered to represent an adverse effect of the test item, therefore, a 'No Observed Adverse Effect Level' (NOAEL) for males has not been established. 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 droplets and are, therefore, considered to represent limited relevance to humans.

The effects excluding the kidney changes detected in males were confined to increases in platelet count, albumin/globulin ratio and alanine aminotransferase and adaptive microscopic liver, thyroid and spleen 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.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 300 mg/kg bw/day based on a reduced live birth index and a reduction in offspring body weights in litters from females treated with 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 eight weeks (including a two 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 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 15 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 litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4post partumHaematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. 

Adult males were terminated on Day 43, followed by the termination of all 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 and histopathological evaluation of selected tissues was performed.

Results.

Adult Responses:

Mortality.There were no unscheduled deaths.

Clinical Observations.There were no toxicologically relevant clinical signs detected in treated animals.

Behavioural Assessment.There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests.There were no toxicologically significant changes in functional performance.

Sensory Reactivity Assessments.There were no treatment-related changes in sensory reactivity.

Body Weight.There were no toxicologically significant effects detected in body weight development.

Food Consumption.No adverse effect on food consumption or food efficiency was detected in treated animals.

Water Consumption.No toxicologically significant effect on water consumption was detected in treated animals.

Reproductive Performance:

MatingThere were no treatment-related effects on mating for treated animals.

Fertility.There were no treatment-related effects in conception rates for treated animals.

Gestation Lengths.There were no differences in gestation lengths. The distribution for treated females was comparable to controls.


Litter Responses:

Offspring Litter Size, Sex Ratio and Viability.Of the litters born, litter size at birth and subsequently on Days 1 and 4post partumwere comparable to controls. A reduction in live birth index was evident in litters from females treated with 1000 mg/kg bw/day. Litter viability at Day 4 however was comparable to control females. There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls.

Offspring Growth and Development.Offspring body weights in litters from females treated with 1000 mg/kg bw/day were reduced on Day 1. A slight reduction in offspring body weight was also evident at this dose level on Day 4post partumand subsequent offspring body weight gain was slightly reduced. Surface righting was also reduced in litters from females treated with 1000 mg/kg bw/day. No clinically observable signs of toxicity were detected in offspring.

Laboratory Investigations:

Haematology.Males treated with 1000 mg/kg bw/day showed an increase in platelet count. Females treated with 1000 mg/kg bw/day showed an increase in neutrophil and monocyte count. Females treated with 300 mg/kg bw/day also showed an increase in neutrophil count. No toxicologically significant effects were detected in males treated with 300 mg/kg bw/day or in animals of either sex treated with 30 mg/kg bw/day.

Blood Chemistry.Males treated with 1000 mg/kg bw/day showed increases in albumin/globulin ratio and alanine aminotransferase whilst females from this treatment group showed increases in total protein and albumin. Females from all treatment groups showed an increase in cholesterol. No such effects were detected in males treated with 300 or 30 mg/kg bw/day.

Pathology:

Necropsy.Enlarged, mottled and pale kidneys were evident in a number of males treated with 1000 mg/kg bw/day and to a lesser extent in males treated with 300 mg/kg bw/day.

Organ Weights.Males treated with 1000 and 300 mg/kg bw/day showed increases in kidney, liver and spleen weight both absolute and relative to terminal body weight. Females treated with 1000 mg/kg bw/day also showed increases in absolute and relative liver and spleen weight. Absolute and relative liver weights were also increased in females treated with 300 mg/kg bw/day. No such effects were detected in animals of either sex treated with 30 mg/kg bw/day.

Histopathology.The following treatment related microscopic findings were detected:

Liver: Centrilobular to diffuse hepatocellular hypertrophy was evident in animals of either sex treated with 1000 and 300 mg/kg bw/day and in males treated with 30 mg/kg bw/day.

Kidneys: Nephropathy consisting of increased incidence and severity of hyaline droplets, tubular degeneration, tubular dilation/vacuolation, granulated tubular casts and interstitial inflammatory infiltrate was evident in males treated with 1000, 300 and 30 mg/kg bw/day.

Spleen: Slightly increased incidence and/or severity of splenic extramedullary hematopoiesis was evident in animals of either sex treated with 1000 mg/kg bw/day.

Thyroids: Increased incidence and severity of follicular hypertrophy was evident in animals of either sex treated with 1000 and 300 mg/kg bw/day and in males treated with 30 mg/kg bw/day.

Conclusion.The oral administration of 1,1,3,3-tetramethylbutyl 2-ethylperoxyhexanoate (CAS 22288-43-3) 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 from all treatment groups. A 'No Observed Effect Level' (NOEL) for either sex was not achieved. The effects detected in females however were confined to increases in neutrophil and monocyte counts, increased total protein, albumin and cholesterol and adaptive microscopic liver, thyroid and spleen changes. These were considered not to represent an adverse effect of treatment therefore the 'No Observed Adverse Effect Level' (NOAEL) was considered to be 1000 mg/kg bw/day for females.

The kidney effects detected in males from all treatment groups were considered to represent an adverse effect of the test item, therefore, a 'No Observed Adverse Effect Level' (NOAEL) for males has not been established. 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 droplets and are, therefore, considered to represent limited relevance to humans.

The effects excluding the kidney changes detected in males were confined to increases in platelet count, albumin/globulin ratio and alanine aminotransferase and adaptive microscopic liver, thyroid and spleen 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.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 300 mg/kg bw/day based on a reduced live birth index and a reduction in offspring body weights in litters from females treated with 1000 mg/kg bw/day.

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no study available (further information necessary)
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

An OECD 414 study was conducted on the registered substance.  There were no adverse developmental effects when tested up to 1000 mg/kg/day. Therefore , further investigation of this endpoint in a seconf species is not required.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
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

No effects on reproductive organs or sperm analysis were found in the OECD 408 study. An OECD 422 did not reveal any effects on fertility up to 1000 mg/kg bw/d.

An OECD 414 developmental toxicity study did not reveal any adverse developmental effects when tested up to 1000 mg/kg/day.

 

Therefore, based on the data from an OECD 408, OECD 422 and an OECD 414 study the criteria for classification are not met.