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; therefore, we are waiving the requirement for a reproductive toxicity study.  A 90-day subchronic toxicity study and an OECD 414 developmental toxicity study are proposed on the read-across substance, CAS# 53220-22-7, and this proposed data waiver may be re-evaluated when the results of those studies are available.

Link to relevant study records

Referenceopen allclose all

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reproductive effects observed:
not specified
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 22 March 2012 and 14 October 2012. The in-life phase of the study was conducted between 04 April 2012 (first day of treatment) and 28 May 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
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: approximately twelve weeks old.
- Weight at study initiation: males weighed 322 to 367g, the females weighed 198 to 224g
- Fasting period before study:
- Housing: 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.
- Diet: ad libitum; A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK)
- Water: ad libitum: Mains drinking water was supplied from polycarbonate bottles attached to the cage.
Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for mated females during gestation and lactation.
- Acclimation period: The animals were acclimatised for six days during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK, Barrier Maintained Rodent Facility.
- Temperature (°C): The temperature controls were set to achieve target values of 21 ± 2°C. Short term deviations from these targets were considered not to affect the purpose or integrity of the study. The actual range of temperature was 19.91 - 22.21°C.
- Humidity (%): The relative humidity controls were set to achieve target values of 55 ± 15%. Short term deviations from these targets were considered not to affect the purpose or integrity of the study. The actual range of relative humidity controls were 44.47 - 72.55%.
- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES: 04 April 2012 (first day of treatment) and 28 May 2012 (final necropsy).
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 suspension in Arachis oil BP.
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. The volume of test and control item administered was based on the most recent scheduled body weight and was adjusted at regular intervals.

VEHICLE
- Amount of vehicle (if gavage): Control: 4 mL/kg; in doses: made up to 4 mL/kg.
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:
The stability and homogeneity of the test item formulations were determined. Results show the formulations to be stable for at least nineteen days. Formulations were therefore prepared weekly for the first four weeks and fortnightly thereafter and stored at approximately 4ºC in the dark.
Samples of each test item formulation were taken and analysed for concentration of Dihexadecyl Peroxodicarbonate (CAS 26322-14-5). The concentration of Dihexadecyl Peroxodicarbonate (CAS 26322-14-5) in the test item formulations was determined by gas chromatography (GC) using an external standard technique. The results indicate that the prepared formulations were within ±10% of the nominal concentration.
Duration of treatment / exposure:
Up to eight weeks.
Frequency of treatment:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe.
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 pre-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 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.
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Ten animals per sex, per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were chosen based on the results of previous toxicity work (14 day repeated dose oral range-finding study)
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
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.

BODY WEIGHT: Yes
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 AND COMPOUND INTAKE (if feeding study):
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:
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 AND COMPOUND INTAKE (if drinking water study): Yes
Water intake was measured daily throughout the study (with the exception of the pairing phase).

OPHTHALMOSCOPIC EXAMINATION: No

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: Yes
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).

CLINICAL CHEMISTRY: Yes
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) and Bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
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 and Lachrymation.
This test was developed from the methods used by Irwin (1968) and Moser et al (1988).

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 and Finger approach.
Oestrous cyclicity (parental animals):
During mating 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).
Sperm parameters (parental animals):
Parameters examined in all males: testis weight and epididymis weight.
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)

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.
Postmortem examinations (parental animals):
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. 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.

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

* = eyes fixed in Davidson’s fluid
• = preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately
forty-eight hours later
# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before
immersion in fixative

All tissues were despatched to the histology processing Test Site 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 indicated as + 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.
Postmortem examinations (offspring):
Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent.
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.

Statistics:
Evaluation of Data
Treatment of Data
Data were processed to give group mean values and standard deviations where appropriate.
For body weights and food consumptions during gestation, group mean values were calculated using data from females which were observed to give birth to offspring.
For body weights and food consumptions during lactation, group mean values were calculated using data from females with live young at Day 5 of lactation.

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Water Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed in "Any other information on materials and methods" below.
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:
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 implantati on sites/Number of corpora lutea) x100

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

Clinical signs:
no effects observed
Description (incidence and severity):
There were no toxicologically significant clinical signs detected in treated animals.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in body weight development.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in body weight development.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related microscopic findings were detected.
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on mating performance.
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Mortality
One female treated with 1000 mg/kg bw/day died during bleeding on Day 4 post partum. This death was considered procedure related and therefore is of no toxicological significance.
There were no further unscheduled deaths.

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 8 onwards. One female treated with 300 mg/kg bw/day also showed increased salivation on Day 33. 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.
One control female had mis-aligned posture characterised by the head tilting to the right from Day 35 onwards. The eyes of this animal also focussed in different directions. In the absence of treatment this was considered to be an isolated finding.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no toxicologically significant effects detected in body weight development.
Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in body weight gain during Week 6. Body weight gain in the remaining treated groups during this week did not follow a true dose related response and in the absence of any significant effect on the percent of overall body weight gain, the intergroup difference was considered not to be of toxicological significance. Females treated with 1000 mg/kg bw/day showed a statistically significant reduction in body weight on Day 20 of gestation. Subsequent body weight gain during the final week of gestation was also statistically significantly reduced in these females. In isolation and in the absence of any effect on overall body weight development in females the intergroup differences were considered not to be of toxicological significance.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
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 and one female treated with 300 mg/kg bw/day did not achieve pregnancy following evidence of mating. One female treated with 30 mg/kg bw/day had one implantation site but failed to give birth to any offspring. No histopathological correlates were evident in the female reproductive organs. No histopathological correlates were evident in the reproductive organs of the male partners treated with 300 or 30 mg/kg bw/day. The male partner for the control female however showed bilateral minimal/sporadic spermatid retention. 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.

Gestation Length:
There were no differences in gestation lengths. The distribution for treated females was comparable to controls. All animals showed gestation lengths of 22 to 23½ days.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No toxicologically significant effects were detected in the organ weights measured.
Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in prostate weight, both absolute and relative to terminal body weight. Females treated with 300 mg/kg bw/day showed a statistically significant increase in absolute and relative kidney weight. In the absence of any histology correlates the intergroup differences were considered not to be of toxicological importance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No toxicologically significant macroscopic abnormalities were detected.
One female treated with 1000 mg/kg bw/day had an enlarged spleen at necropsy. In the absence of any histology correlates the intergroup difference was considered not to be of toxicological importance.
One control male, two control females and one female treated with 30 mg/kg bw/day had reddened lungs at necropsy. In the absence of similar findings in the 1000 mg/kg bw/day dose group and in view of this finding also being present in control animals the intergroup differences were considered not to be of toxicological significance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment related microscopic findings were detected.
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically significant effects.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically significant effects.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected
Mortality / viability:
no mortality observed
Description (incidence and severity):
No significant differences were detected for corpora lutea, implantation counts, litter size or litter viability for treated animals when compared to controls.
Body weight and weight changes:
not specified
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically significant effects were detected in the organ weights measured.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring.
Histopathological findings:
no effects observed
Description (incidence and severity):
No treatment related microscopic findings were detected.
LITTER RESPONSES:
In total nine females from the control, 30 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 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 RATION AND VIABILITY:
No significant differences were detected for corpora lutea, implantation counts, litter size or litter viability 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:
There were no toxicologically significant effects detected.
Statistical analysis of the litter, offspring weights or surface righting reflex 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, no milk in stomach, 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.


CLINICAL SIGNS (OFFSPRING)
There were no toxicologically significant clinical signs detected in treated animals.

ORGAN WEIGHTS (OFFSPRING)
No toxicologically significant effects were detected in the organ weights measured.

GROSS PATHOLOGY (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.

HISTOPATHOLOGY (OFFSPRING)
No treatment related microscopic findings were detected.


Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically significant effects.
Reproductive effects observed:
not specified
Conclusions:
The oral administration of Dihexadecyl Peroxodicarbonate (CAS 26322-14-5) to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 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 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 and 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 4 post 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 surviving females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results.

Adult Responses:

Mortality.One female treated with 1000 mg/kg bw/day died during bleeding on Day 4 post partum. There were no further unscheduled deaths. 

Clinical Observations.There were no toxicologically significant 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:

Mating.There 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 4 post partum were comparable to controls. Sex ratio was also comparable to controls.

Offspring Growth and Development.Offspring bodyweight gain and litter weights at birth and subsequently on Days 1 and 4 post partum were comparable to controls. Surface righting was also comparable to controls. No clinically observable signs of toxicity were detected in offspring.

Laboratory Investigations:

Haematology.No toxicologically significant effects were detected in the haematological parameters examined. 

Blood Chemistry.No toxicologically significant effects were detected in the blood chemical parameters examined. 

Pathology:

Necropsy.No toxicologically significant macroscopic abnormalities were detected.

Organ Weights.No toxicologically significant effects were detected in the organ weights measured.

Histopathology.No treatment related microscopic abnormalities were detected.

Conclusion.The oral administration of Dihexadecyl Peroxodicarbonate (CAS 26322-14-5) to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 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 fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
K1
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for selection of Effect on fertility via oral route:

No effects on reproductive organs were found in the OECD 422 study summarized elsewhere.  These results suggest a low risk for reproductive toxicity; therefore, we are waiving the requirement for a reproductive toxicity study.  A 90-day subchronic toxicity study and an OECD 414 developmental toxicity study are proposed on the read-across substance, CAS# 53220-22-7, and this proposed data waiver may be re-evaluated when the results of those studies are available.

Effects on developmental toxicity

Description of key information

No effects were found in the OECD 422 study summarized elsewhere.  These results suggest a low risk for developmental toxicity. A 90-day subchronic toxicity study and an OECD 414 developmental toxicity study are proposed on the read-across substance, CAS# 53220-22-7.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study planned (based on read-across)
Justification for type of information:
See attached read across document
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Species:
rat
Abnormalities:
not specified
Developmental effects observed:
not specified
Endpoint:
developmental toxicity
Remarks:
OECD 422
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 March 2012 and 14 October 2012
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
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: approximately twelve weeks old.
- Weight at study initiation: males weighed 322 to 367g, the females weighed 198 to 224g
- Fasting period before study:
- Housing: 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.
- Diet: ad libitum; A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK)
- Water: ad libitum: Mains drinking water was supplied from polycarbonate bottles attached to the cage.
Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for mated females during gestation and lactation.
- Acclimation period: The animals were acclimatised for six days during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK, Barrier Maintained Rodent Facility.
- Temperature (°C): The temperature controls were set to achieve target values of 21 ± 2°C. Short term deviations from these targets were considered not to affect the purpose or integrity of the study. The actual range of temperature was 19.91 - 22.21°C.
- Humidity (%): The relative humidity controls were set to achieve target values of 55 ± 15%. Short term deviations from these targets were considered not to affect the purpose or integrity of the study. The actual range of relative humidity controls were 44.47 - 72.55%.
- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES: 04 April 2012 (first day of treatment) and 28 May 2012 (final necropsy).
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 suspension in Arachis oil BP.
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. The volume of test and control item administered was based on the most recent scheduled body weight and was adjusted at regular intervals.

VEHICLE
- Amount of vehicle (if gavage): Control: 4 mL/kg; in doses: made up to 4 mL/kg.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item formulations were determined. Results show the formulations to be stable for at least nineteen days. Formulations were therefore prepared weekly for the first four weeks and fortnightly thereafter and stored at approximately 4ºC in the dark.
Samples of each test item formulation were taken and analysed for concentration of Dihexadecyl Peroxodicarbonate (CAS 26322-14-5). The concentration of Dihexadecyl Peroxodicarbonate (CAS 26322-14-5) in the test item formulations was determined by gas chromatography (GC) using an external standard technique. The results indicate that the prepared formulations were within ±10% 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.
Duration of treatment / exposure:
Animals were dosed daily through the eight weeks of the study.
Frequency of treatment:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe.
Duration of test:
Eight weeks.
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:
Ten animals per sex, per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were chosen based on the results of previous toxicity work (14 day repeated dose oral range-finding study)
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
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.

BODY WEIGHT: Yes
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 AND COMPOUND INTAKE (if feeding study):
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.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Water intake was measured daily throughout the study (with the exception of the pairing phase).

POST-MORTEM EXAMINATIONS: Yes
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. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.
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.

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

* = eyes fixed in Davidson’s fluid
• = preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later
# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

All tissues were despatched to the histology processing Test Site 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 indicated as + 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.

OTHER:
FOOD EFFICIENCY:
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.

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.

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.
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:
Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent.
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.
Statistics:
Evaluation of Data
Treatment of Data
Data were processed to give group mean values and standard deviations where appropriate.
For body weights and food consumptions during gestation, group mean values were calculated using data from females which were observed to give birth to offspring.
For body weights and food consumptions during lactation, group mean values were calculated using data from females with live young at Day 5 of lactation.

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Water Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed in "Any other information on materials and methods" below.
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

For Offspring viability indices, please see "any other information on materials and methods" below.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Mortality
One female treated with 1000 mg/kg bw/day died during bleeding on Day 4 post partum. This death was considered procedure related and therefore is of no toxicological significance.
There were no further unscheduled deaths.

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 8 onwards. One female treated with 300 mg/kg bw/day also showed increased salivation on Day 33. 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.
One control female had mis-aligned posture characterised by the head tilting to the right from Day 35 onwards. The eyes of this animal also focussed in different directions. In the absence of treatment this was considered to be an isolated finding.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no toxicologically significant effects detected in body weight development.
Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in body weight gain during Week 6. Body weight gain in the remaining treated groups during this week did not follow a true dose related response and in the absence of any significant effect on the percent of overall body weight gain, the intergroup difference was considered not to be of toxicological significance. Females treated with 1000 mg/kg bw/day showed a statistically significant reduction in body weight on Day 20 of gestation. Subsequent body weight gain during the final week of gestation was also statistically significantly reduced in these females. In isolation and in the absence of any effect on overall body weight development in females the intergroup differences were considered not to be of toxicological significance.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
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 and one female treated with 300 mg/kg bw/day did not achieve pregnancy following evidence of mating. One female treated with 30 mg/kg bw/day had one implantation site but failed to give birth to any offspring. No histopathological correlates were evident in the female reproductive organs. No histopathological correlates were evident in the reproductive organs of the male partners treated with 300 or 30 mg/kg bw/day. The male partner for the control female however showed bilateral minimal/sporadic spermatid retention. 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.

Gestation Length:
There were no differences in gestation lengths. The distribution for treated females was comparable to controls. All animals showed gestation lengths of 22 to 23½ days.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No toxicologically significant effects were detected in the organ weights measured.
Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in prostate weight, both absolute and relative to terminal body weight. Females treated with 300 mg/kg bw/day showed a statistically significant increase in absolute and relative kidney weight. In the absence of any histology correlates the intergroup differences were considered not to be of toxicological importance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No toxicologically significant macroscopic abnormalities were detected.
One female treated with 1000 mg/kg bw/day had an enlarged spleen at necropsy. In the absence of any histology correlates the intergroup difference was considered not to be of toxicological importance.
One control male, two control females and one female treated with 30 mg/kg bw/day had reddened lungs at necropsy. In the absence of similar findings in the 1000 mg/kg bw/day dose group and in view of this finding also being present in control animals the intergroup differences were considered not to be of toxicological significance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment related microscopic findings were detected.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:not examined

Details on embryotoxic / teratogenic effects:
LITTER RESPONSES:
In total nine females from the control, 30 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 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 RATION AND VIABILITY:
No significant differences were detected for corpora lutea, implantation counts, litter size or litter viability 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:
There were no toxicologically significant effects detected.
Statistical analysis of the litter, offspring weights or surface righting reflex 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, no milk in stomach, 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.

CLINICAL SIGNS (OFFSPRING)
There were no toxicologically significant clinical signs detected in treated animals.

ORGAN WEIGHTS (OFFSPRING)
No toxicologically significant effects were detected in the organ weights measured.

GROSS PATHOLOGY (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.

HISTOPATHOLOGY (OFFSPRING)
No treatment related microscopic findings were detected.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The oral administration of Dihexadecyl Peroxodicarbonate (CAS 26322-14-5) to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 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 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 and 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 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 and histopathological evaluation of selected tissues was performed.

Results.

Adult Responses:

Mortality.One female treated with 1000 mg/kg bw/day died during bleeding on Day 4post partum. There were no further unscheduled deaths. 

Clinical Observations.There were no toxicologically significant 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:

Mating.There 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. Sex ratio was also comparable to controls.

Offspring Growth and Development.Offspring bodyweight gain and litter weights at birth and subsequently on Days 1 and 4post partumwere comparable to controls. Surface righting was also comparable to controls. No clinically observable signs of toxicity were detected in offspring.

Laboratory Investigations:

Haematology.No toxicologically significant effects were detected in the haematological parameters examined. 

Blood Chemistry.No toxicologically significant effects were detected in the blood chemical parameters examined. 

Pathology:

Necropsy.No toxicologically significant macroscopic abnormalities were detected.

Organ Weights.No toxicologically significant effects were detected in the organ weights measured.

Histopathology.No treatment related microscopic abnormalities were detected.

Conclusion.The oral administration of Dihexadecyl Peroxodicarbonate (CAS 26322-14-5) to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 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
Quality of whole database:
K1
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

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

No effects were found in the OECD 422 study summarized elsewhere.  These results suggest a low risk for developmental toxicity. A 90-day subchronic toxicity study and an OECD 414 developmental toxicity study are proposed on the read-across substance, CAS# 53220-22-7.

Justification for classification or non-classification

No effects were found in the OECD 422 study summarized elsewhere.  These results suggest a low risk for developmental toxicity. A 90-day subchronic toxicity study and an OECD 414 developmental toxicity study are proposed on the read-across substance, CAS# 53220-22-7, and this proposed reproductive data waiver may be re-evaluated when the results of those studies are available.