Bis-sec-butyl peroxydicarbonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 17 April 2013 Experimental Completion Date: 14 January 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis-sec-butyl peroxydicarbonate

EC Number:

243-424-3

EC Name:

Bis-sec-butyl peroxydicarbonate

Cas Number:

19910-65-7

Molecular formula:

C10H18O6

IUPAC Name:

2-[({[(butan-2-yloxy)carbonyl]peroxy}carbonyl)oxy]butane

Test material form:

liquid

Specific details on test material used for the study:

Identification : Di-sec-butyl peroxydicarbonate (CAS# 19910-65-7)
Test Item Active Substance Content : 100.8 % w/w
Batch Number : 12121BO702
Storage Conditions : Stored at approximately -20 ºC in darkness, may be
formulated/used in light
Expiry Date : 01 September 2013
Physical State/Appearance : Clear colourless liquid
No correction for purity was made.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system 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 ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 311 to 359g, the females weighed 191 to 218g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of four 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. 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. Mated females were also given softwood flakes, as bedding, throughout 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 Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; there were no deviations from these targets.

The animals were randomly allocated to treatment groups using a stratified body weight randomisation procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in chilled corn oil. 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 four hours. The test item was administered within four hours of formulation. The test item was stored and handled on ice during formulation, and the dosing formulations were also stored on ice until use.

Samples of the test item formulation were taken and analysed for concentration of Di-sec-butyl peroxydicarbonate (CAS# 19910-65-7) at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 9% of the nominal concentration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item concentration in the test samples was determined by hihg performance liquid chromatography with UV detection (HPLC/UV) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

Preparation of standard solutions
Stock solutions of test item in acetonitrile were prepared for external standard calibration. An aliquot, 100 mg of test material, was exactly weighed into a 100 mL volumetric flask and brought to volume with acetonitrile to yield a solution with a concentration of 1 mg/mL.

On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Analysis of Samples
The formulations recived were extracted with acetonitrile. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with acetonitrile this was then ultra-sonicated for 15 minutes and centrifuged at 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with acetonitrile to achieve the working concentration.

Preparation of Accuracy Samples
Samples of corn oil were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis as per the above.

Preparation of linearity standards
A range of standard solutions were prepared in acetonitrile from a stock solution of 2 mg/mL by serial dilution covering the concentration range 0 to 2.088 mg/mL.

Instrumental Setup
HPLC: Agilent Technologies 1200, incorporating autosampler and workstation
Colummn: Luna C18 5µ (250 x 4.6 mm id)
Mobile phase: Methanol:water (70:30 v/v)
Flow rate: 1 mL/min
UV detector wavelegth: 210 nm
Injection volume: 25 µL
Vial compartment temp.: 4°C
Retention time: ~13.5 mins

Duration of treatment / exposure:

Up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

twelve male and twelve female

Control animals:

yes, concurrent vehicle

Details on study design:

The animals were randomly allocated to treatment groups using a stratified body weight randomisation procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one hour 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

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 on each occasion (at least two hours after dosing), 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. Body weights were also recorded at terminal kill.

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 during the pre-pairing phase of the study.

Reproductive Performance
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

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
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).

Sacrifice and pathology:

Pathology

Necropsy
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski, 1964). The corpora lutea were also counted. 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 were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group. Tissues shown in bold were weighed from all remaining animals:
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 five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown in bold were preserved from all remaining animals:
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
Pituitary
Bone & bone marrow (sternum)
Prostate
Brain (including cerebrum, cerebellum and pons)
Oesophagus
Caecum
Rectum
Coagulating gland
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides Skin (hind limb)
Eyes*
Spinal cord (cervical, mid-thoracic and lumbar)
Gross lesions
Heart
Spleen
Ileum (including peyer’s patches)
Stomach
Jejunum
Thyroid/parathyroid
Kidneys
Trachea
Liver
Testes
Lungs (with bronchi) #
Thymus
Lymph nodes (mandibular and mesenteric)
Urinary bladder
Mammary gland
Uterus/Cervix
Muscle (skeletal)
Vagina

Tissues were despatched to the Test Site (TUPI Manufacturing, PO Box 305, Diss,Norfolk, IP22 4XL, UK) for processing. The tissues from five selected control and 500 mg/kg bw/day dose group animals, and any animals which 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 500 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 500 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.

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

Other examinations:

Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:

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

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

Statistics:

Due to the quantity of data, please see below re "Any other information"

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Animals of either sex treated with 500 and 150 mg/kg bw/day showed episodes of increased salivation from Day 5 and Day 16 (respectively) onwards. One male treated with 150 mg/kg bw/day also showed noisy respiration on Day 40 only.

No such effects were detected in animals of either sex treated with 50 mg/kg bw/day.

One female treated with 150 mg/kg bw/day showed ptosis, pilo-erection, lethargy, hunched posture and diarrhoea between Days 37 and 40. This was the littering phase for this female and observations of this nature are not uncommon during this period. The observations were therefore not considered to be related to test item toxicity.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males treated with 500 mg/kg bw/day showed a reduction in body weight gain throughout the treatment period. Statistical significance was achieved during Weeks 1 (p<0.05), 5 (p<0.001) and 6 (p<0.05). Overall body weight gain was subsequently reduced in these animals throughout the study.

No such effects were detected in females treated with 500 or 150 mg/kg bw/day or animals of either sex treated with 50 mg/kg bw/day.

Males treated with 150 mg/kg bw/day showed a statistically significant reduction in body weight gain during the final week of treatment. Body weight gain during all the previous weeks were
comparable to controls. Therefore the intergroup difference was considered not to be of toxicological significance.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Males treated with 500 mg/kg bw/day showed a reduction in overall food consumption (-12%) when compared to control males. Food efficiency was also reduced in males treated with 500 mg/kg bw/day.

No such effects were detected in females treated with 500 mg/kg bw/day or animals of either sex treated with 150 or 50 mg/kg bw/day.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Males treated with 500 mg/kg bw/day showed a reduction in overall food consumption (-12%) when compared to control males. Food efficiency was also reduced in males treated with 500 mg/kg bw/day.

No such effects were detected in females treated with 500 mg/kg bw/day or animals of either sex treated with 150 or 50 mg/kg bw/day.

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

effects observed, non-treatment-related

Description (incidence and severity):

Males treated with 500 mg/kg bw/day showed an increase in overall water consumption (+36%) during the maturation phase when compared to control males.

No such effects were detected in females treated with 500 mg/kg bw/day or animals of either sex treated with 150 or 50 mg/kg bw/day.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Males treated with 500 mg/kg bw/day showed a statistically significant reduction in mean corpuscular haemoglobin concentration when compared to controls. The majority of individual values were within normal ranges for rats of the strain and age used, therefore the intergroup difference was considered not to be of toxicological importance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Males treated with 500 mg/kg bw/day showed statistically significant reductions in total protein and alkaline phosphatase and a statistically significant increase in bile acids. Males treated with 500 and 150 mg/kg bw/day showed a statistically significant increase in albumin/globulin ratio. The majority of individual values were within the normal ranges for rats of the strain and age used and in the absence of any associated histology correlates the intergroup differences were considered not to be of toxicological importance. Females treated with 500 mg/kg bw/day showed a statistically significant reduction in alkaline phosphatase. The level of statistical significance was minimal (p<0.01) and in the absence of any associated histopathology correlates, the intergroup difference was considered of no toxicological importance.

Males treated with 500 and 150 mg/kg bw/day showed a statistically significant reduction in bilirubin levels. The majority of individual values for treated animals were within the normal ranges for rats of the strain and age used. However control values were all higher than the expected normal ranges. The intergroup differences were therefore considered to be due to the higher control values rather than test item toxicity.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant changes in functional performance.
Males from all treatment groups showed a statistically significant reduction (p<0.05) in mean forelimb grip strength whilst females treated with 150 or 50 mg/kg bw/day showed a statistically significant (p<0.05) reduction in hindlimb grip strength. The intergroup differences were confined to one out of the three tests for either sex and in the absence of a dose related response was considered not to be of toxicological importance.

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

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

Males treated with 500 mg/kg bw/day showed a statistically significant reduction in prostate weight both absolute and relative to terminal body weight. Males from all treatment groups also
showed a statistically significant reduction in thymus weight both absolute and relative to terminal body weight. The majority of individual values were within normal range for rats of the strain and age used and in the absence of any histology correlates or a true dose related response, the intergroup differences were considered not to be of toxicological importance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant macroscopic abnormalities were detected.

One control male had increased pelvic space in the right kidney and the right kidney was also fluid filled. One control female had reddened lungs at necropsy. In the absence of treatment these were considered to be incidental findings.

One female treated with 150 mg/kg bw/day had reddened lungs at necropsy.

In the absence of a similar effect at 500 mg/kg bw/day or any histology correlates, the intergroup difference was considered to be of no toxicological importance.

One male treated with 50 mg/kg bw/day had small testes and epididymides. The testes were also flaccid. In the absence of a similar effect at 500 mg/kg bw/day, the intergroup difference was
considered to be of no toxicological importance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Epithelial hyperplasia in the forestomach was evident in animals of either sex treated with 500 mg/kg bw/day and in two males treated with 150 mg/kg bw/day. One male treated with 500 mg/kg bw/day also showed edema and a further male also showed erosion and vesiculation in the forestomach. Edema and erosion of the forestomach was also evident in one male treated with 150 mg/kg bw/day. No such effects were detected in females treated with 150 mg/kg bw/day or animals of either sex treated with 50 mg/kg bw/day.

These findings detected in the forestomach were consistent with local irritation.

Three males treated with 500 mg/kg bw/day showed squamous metaplasia in the trachea. Out of these three males, one also showed cellular exudate and one also showed cellular exudate and
erosion. The remaining male also showed inhalation pneumonia in the lungs. These findings in the trachea and lungs were consistent with local irritation following inhalation of the test
compound. The inhalation of test item droplets may have occurred as a consequence of the dosing procedure when the cannula is removed from the animal.

Histopathological findings: neoplastic:

not examined

Details on results:

The oral administration of Di-sec-butyl peroxydicarbonate (CAS# 19910-65-7) to rats for a period of up to eight weeks (including two weeks pre-pairing, gestation and early lactation for females) at dose levels of up to 500 mg/kg bw/day, resulted in treatment-related effects detected in animals of either sex treated with 500 mg/kg bw/day and males treated with 150 mg/kg bw/day.

Clinical signs were detected in animals of either sex treated with 500 mg/kg bw/day during the study. Incidents of increased salivation were evident throughout the treatment period and an isolated incident of noisy respiration was evident in one male treated with 150 mg/kg bw/day.

The physical condition of males treated with 500 mg/kg bw/day was also affected with reductions in body weight development throughout the treatment period and subsequently a reduction in overall body weight gain for these males was detected throughout the study period. A reduction in food consumption and food efficiency during the treatment period was evident in males treated with 500 mg/kg bw/day and an increase in water consumption during maturation was also evident. Observations of this nature are often reported when a test item formulation is unpalatable or irritating. This was supported microscopically, with stomach changes identified as epithelial hyperplasia in animals of either sex treated with 500 mg/kg bw/day and in males treated with 150 mg/kg bw/day and edema; erosion and vesiculation was observed in males treated with 500 and 150 mg/kg bw/day. While these findings probably represent an adverse effect of treatment they are considered to reflect local irritation rather than any adverse systemic toxicity of the test item.

There were no toxicologically significant effects observed during the weekly open field arena observations or in the haematological/blood chemical parameters measured.

There were no treatment-related effects detected in the reproductive parameters observed.

Effect levels

1

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

The stomach changes identified together with the associated reductions in body weight and food consumption in 500 mg/kg bw/day males may be considered to be an adverse effect of treatment, however they are also considered to be a result of local irritation of the test item rather than a true effect of systemic toxicity. In terms of risk assessment, the findings observed on this study would suggest that a NOAEL can be established at 500 mg/kg bw/day for females and 150 mg/kg bw/day for males because the findings were not evidence of true systemic toxicity.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 500 mg/kg bw/day.

Applicant's summary and conclusion

Conclusions:

The oral administration of di-sec-butyl peroxydicarbonate (CAS# 19910-65-7), to rats by gavage, at dose levels of 50, 150 and 500 mg/kg bw/day, resulted in treatment-related reduced body weight gain observed in males treated with 500 mg/kg bw/day, increased salivation in animals of either sex treated with 500 and 150 mg/kg bw/day and microscopic changes in the stomach of animals of either sex treated with 500 mg/kg bw/day and in males treated with 150 mg/kg bw/day. The No Observed Effect Level (NOEL) was therefore considered to be 150 mg/kg bw/day for females and 50 mg/kg bw/day for males. The stomach changes identified together with the associated reductions in body weight and food consumption in 500 mg/kg bw/day males may be considered to be an adverse effect of treatment, however they are also considered to be a result of local irritation of the test item rather than a true effect of systemic toxicity. In terms of risk assessment, the findings observed on this study would suggest that a NOAEL can be established at 500 mg/kg bw/day for females and 150 mg/kg bw/day for males because the findings were not evidence of true systemic toxicity.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 500 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 designed to be 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 twelve male and twelve 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 50, 150 and 500 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (corn oil).

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 partum. Haematology 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 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

There were no unscheduled deaths.

Clinical Observations

Animals of either sex treated with 500 and 150 mg/kg bw/day showed episodes of increased salivation throughout the treatment period. No adverse clinical signs were detected in animals of either sex treated with 50 mg/kg bw/day.

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

Males treated with 500 mg/kg bw/day showed a reduction in body weight gain throughout thetreatment period. No changes in overall body weight gain were detected in females treated with 500 mg/kg bw/day or animals of either sex treated with 150 or 50 mg/kg bw/day.

Food Consumption

Overall food consumption and food efficiency was reduced in males treated with 500 mg/kg bw/day when compared to controls. No such effects were detected in females treated with 500 mg/kg bw/day or animals of either sex treated with 150 or 50 mg/kg bw/day.

Water Consumption

Males treated with 500 mg/kg bw/day showed an increase in overall water consumption during the maturation phase when compared to control males. No such effects were detected in females treated with 500 mg/kg bw/day or animals of either sex treated with 150 or 50 mg/kg bw/day.

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 of gestation lengths for treatedfemales 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 werecomparable to controls. Sex ratio was also comparable to controls.

Offspring Growth and Development

Offspring body weight on Days 1 and 4 post partum were comparable to controls. Surfacerighting was also comparable to controls.

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

Epithelial hyperplasia in the forestomach was evident in animals of either sex treated with500 mg/kg bw/day and in two males treated with 150 mg/kg bw/day. One male treated with 500 mg/kg bw/day also showed edema and a further male also showed erosion and vesiculation in the forestomach. Edema and erosion of the forestomach was also evident in one male treated with 150 mg/kg bw/day. No such effects were detected in females treated with 150 mg/kg bw/day or animals of either sex treated with 50 mg/kg bw/day.

Conclusion

The oral administration of di-sec-butyl peroxydicarbonate (CAS# 19910-65-7), to rats bygavage, at dose levels of 50, 150 and 500 mg/kg bw/day, resulted in treatment-related reduced body weight gain observed in males treated with 500 mg/kg bw/day, increased salivation in animals of either sex treated with 500 and 150 mg/kg bw/day and microscopic changes in the stomach of animals of either sex treated with 500 mg/kg bw/day and in males treated with 150 mg/kg bw/day. The No Observed Effect Level (NOEL) was therefore considered to be 150 mg/kg bw/day for females and 50 mg/kg bw/day for males. The stomach changes identified together with the associated reductions in body weight and food consumption in 500 mg/kg bw/day males may be considered to be an adverse effect of treatment, however they are also

considered to be a result of local irritation of the test item rather than a true effect of systemic toxicity. In terms of risk assessment, the findings observed on this study would suggest that a NOAEL can be established at 500 mg/kg bw/day for females and 150 mg/kg bw/day for males because the findings were not evidence of true systemic toxicity.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 500 mg/kg bw/day.