O,O-tert-butyl isopropyl monoperoxycarbonate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 23 August 2016 Experimental Completion Date: 21 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

O,O-tert-butyl isopropyl monoperoxycarbonate

EC Number:

219-143-7

EC Name:

O,O-tert-butyl isopropyl monoperoxycarbonate

Cas Number:

2372-21-6

Molecular formula:

C8H16O4

IUPAC Name:

tert-butoxy propan-2-yl carbonate

additive 1

Reference substance name:

Hydrocarbons, C4, 1,3-butadiene-free, polymd., triisobutylene fraction, hydrogenated

EC Number:

297-629-8

EC Name:

Hydrocarbons, C4, 1,3-butadiene-free, polymd., triisobutylene fraction, hydrogenated

Cas Number:

93685-81-5

Molecular formula:

not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

isododecane

Test material form:

liquid

Details on test material:

Identification : 0,0-tert-butyl isopropyl monoperoxycarbonate (CAS# 2372-21-6)
Physical State/Appearance : Clear colorless liquid
Product Name : Trigonox BPIC-C75
Chemical Name : tert-Butylperoxy isopropyl carbonate
Purity : 74.6%
Batch Number : 1512442974
Label : Trigonox BPIC-C75 500g
Date Received : 25 May 2016
Storage Conditions : Stored cold at ≈ 5°C in the dark; formulated in the light at ambient temperature 10 to 25°C
Expiry/Retest Date : 04 May 2026

Specific details on test material used for the study:

Identification : 0,0-tert-butyl isopropyl monoperoxycarbonate (CAS# 2372-21-6)
Physical State/Appearance : Clear colorless liquid
Product Name : Trigonox BPIC-C75
Chemical Name : tert-Butylperoxy isopropyl carbonate
Purity : 74.6%
Batch Number : 1512442974
Label : Trigonox BPIC-C75 500g
Date Received : 25 May 2016
Storage Conditions : Stored cold at ≈ 5°C in the dark; formulated in the light at ambient temperature 10 to 25°C
Expiry/Retest Date : 04 May 2026

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for eight 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 305 to 341g, the females weighed 181 to 214g, and were approximately twelve weeks old.

Animal Care and Husbandry
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, Envigo RMS (UK) Limited, Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Annex 5. 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 paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly 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. Short term deviations from these targets (relative humidity only) were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.

The animals were randomly allocated to treatment groups using a stratified body weight randomization 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 randomized. 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:

arachis oil

Details on exposure:

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least twenty-two days. As full stability data was not available prior to the start of the study, formulations were prepared daily during the first week of the study, this was followed by two batches made up weekly and then followed by batches of the formulation that were made up every two weeks, and stored at approximately 4 ºC in the dark.
Samples of test item formulation were taken and analyzed for concentration of 0,0-tert-butyl isopropyl monoperoxycarbonate (CAS# 2372-21-6) at Envigo Research Limited, Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Annex 2. The results indicate that the prepared formulations were generally within ± 10% of the nominal concentration and were generally within acceptable ranges for the purpose of this study. This is with the exception of the final high dose formulation which was found to be 87% of nominal. However, this was the final formulation and was only being used to dose one remaining high dose female animal for three days and as such it was considered not to have affected the purpose of this study.

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 gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of two peaks.

Preparation of calibration standards
Stock solutions of test item in dilution solvent were prepared for external standard calibration. An aliquot, approximately 0.1 g of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with dilution solvent to yield a solution with a concentration of 1mgmL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration of 0.1 mg/mL.

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

Calibration solutions were injected into the instrument, at the beginning and end of each sample analysis sequence as a minimum.

To assess the calibration range of the method, a ange of standard solutions were prepared in dilution solvent from a stock solution of 1.185 mg/mL by serial dilution covering the concentration range 0.0593 mg/mL to 0.1580 mg/mL.

Preparation of test samples
The formulations recievd were extracted with extract solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume wuth extract solvent. This was then ultra-sonicated for 125 minutes and centrifuged at 4500 rom for 10 minutes. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentration.

Preparation of Accuracy and Precision Samples
Samples of Arachis Oil BP 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.

The concentration of test item in the final solution was quantified by GC using FID detection as described in the instrument parameters below:

GC system: Agelient Technologies 6890, incorporating autosampler and workstation
Column: DB-17 (30 m x 0.53 mm id x 1 µm film)
Oven temperature programme:
Oven: 40°C for 2 minutes
with 10°C/minute to 120°C for 0 minutes
then 50°C/minute to 260°C for 5 minutes
Injection temperature: 150°C
Flame ionisation detector temperature: 300°C
Injection volume: 1 µL
Retention time: ~ 2.8 and 6.5 mins

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 estrus 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. Mated females were housed individually during the period of gestation and lactation.

Duration of treatment / exposure:

Males: approximately six weeks
Females: approximately eight weeks

Frequency of treatment:

Daily

Duration of test:

Males: approximately six weeks
Females: approximately eight weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Low dose level treatment group: 12.5 mg/mL

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Intermediate dose level treatment group - 37.5 mg/mL

Doses / concentrations 3

Dose / conc.:

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

Remarks:

High dose level treatment group - 112.5 mg/mL

No. of animals per sex per dose:

12 males/12 females

Control animals:

yes, concurrent vehicle

Details on study design:

The animals were randomly allocated to treatment groups using a stratified body weight randomization 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 randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Examinations

Maternal examinations:

General Observations/Measurements
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.

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 pairing. During the pairing phase females were weighed daily until mating was confirmed. 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.
Normal range data for body weight changes in pregnant and lactating females are shown in Annex 7.

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 during gestation and lactation.
Normal range data for pregnant and lactating females are presented in Annex 7.

Water Consumption
Water intake was measured daily during the pre-pairing phase of the study.

Specialist Evaluations
Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral 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.

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

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

Functional Performance Tests
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time 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
Vocalization
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach

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 estrus 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. Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible 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


In-Life Sampling and Analysis
Hematological 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.
The methods used for hematological and blood chemical investigations are presented in Annex 6 and normal ranges are shown in Annex 9.

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

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:

Urea
Inorganic phosphorus (P)
Glucose
Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.)
Alanine aminotransferase (ALAT)
Albumin
Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation)
Creatinine (Creat)
Sodium (Na+)
Total cholesterol (Chol)
Potassium (K+)
Total bilirubin (Bili)
Chloride (Cl-)
Bile acids
Calcium (Ca++)

Terminal Investigations
Necropsy
Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent. Any females which failed to achieve pregnancy 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
Pituitary (post-fixation)
Brain
Seminal Vesicles
Epididymides
Spleen
Heart
Kidneys
Thymus
Liver
Thyroid (weighed post-fixation with Parathyroid)
Ovaries
Uterus (weighed with Cervix)

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
Muscle (skeletal)
Aorta (thoracic)
Ovaries
Bone & bone marrow (femur including stifle joint)
Pancreas
Bone & bone marrow (sternum)
Pituitary
Brain (including cerebrum, cerebellum and pons)
Caecum
Rectum
Coagulating gland
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides ♦
Skin
Esophagus
Spinal cord (cervical, mid thoracic and lumbar)
Eyes *
Gross lesions
Spleen
Heart
Stomach
Ileum (including peyer’s patches)
Testes ♦
Jejunum
Thyroid/Parathyroid
Kidneys
Trachea
Liver
Thymus
Lungs (with bronchi)#
Urinary bladder
Lymph nodes (mandibular and mesenteric)
Uterus & Cervix
Mammary gland
Vagina

Tissues were dispatched to the Test Site (Envigo CRS Limited, Eye, Suffolk, IP23 7PX) for processing. The tissues from five selected control and 450 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 450 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 450 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 changes in the stomachs of males and females and in the kidneys of males only, examination was subsequently extended to include similarly prepared sections of stomach (both sexes) and kidneys (males only) from animals in the low and intermediate groups.

Pathology
Microscopic examination was conducted by the Study Pathologist. A peer review of the findings observed was conducted by V Mowat at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS, UK. A complete histopathology phase report is presented in Annex 1 and represents the consensus view of both pathologists.

Ovaries and uterine content:

Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible 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

Fetal examinations:

Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

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

Statistics:

Please refer to "Any other information on materials and methods"

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 from 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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Animals of either sex treated with 450 mg/kg bw/day showed incidences of increased salivation from Day 2 (males) and Day 5 (females) until Day 37. Occasional instances of increased salivation were also noted in three males and two females treated with 150 mg/kg bw/day from Days 14 to 30 (males) and on Day 18 (females). Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and are generally considered to be of no toxicological importance. One male animal from each treatment group exhibited signs of noisy respiration which persisted for one or two days throughout the treatment period only. However, these clinical signs were considered to reflect occasional difficulties with dosing these animals rather than any underlying toxicological effect of the test item.

No such effects were evident in female animals treated with 50 mg/kg bw/day.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was considered to be no adverse effect on body weight development for animals of either sex treated with 50, 150 or 450 mg/kg bw/day.
Male animals treated with 150 mg/kg bw/day showed a statistically significant increase (p<0.05) in body weight gain during the fourth week of treatment and subsequently a higher overall body weight gain. Body weight gains were generally similar to controls for all treated male animals throughout the treatment period.
Female animals treated with 450 mg/kg bw/day showed a slight reduction in body weight gain during the first week of treatment (without achieving statistical significance). Recovery was evident during the second week of treatment. During gestation and lactation, body weight gains were essentially similar to controls and resulted in overall body weight gains which were comparable to the control animals (at the end of maturation, gestation and lactation). No such effects were noted for female animals from the other two treatment groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of treatment with the test item up to a dose level of 450 mg/kg bw/day on dietary intake for animals of either sex throughout the treatment period. Minor fluctuations were noted in food conversion efficiency, however, these differences were deemed to be reflective of intergroup differences in body weights gains and/or dietary intake.

Food efficiency:

no effects observed

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

no effects observed

Description (incidence and severity):

Males treated with 450 mg/kg bw/day showed an increase in overall water consumption during the pre-pairing phase of the study. No such effects were detected in males treated with 150 or 50 mg/kg bw/day or in any treated females.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects detected in the hematological parameters examined.
Males treated with 150 mg/kg bw/day showed statistically significant increases (p<0.05) in lymphocytes and total leukocyte count with three and four of these values respectively being outside of the normal range data. However, as no such effects were detected in animals treated with 450 mg/kg bw/day this is likely to be incidental and unrelated to treatment.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no treatment-related changes in the behavioural parameters at 50, 150 or 450 mg/kg bw/day.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Females treated with 450 mg/kg bw/day and 150 mg/kg bw/day showed a statistically significant increase (p<0.05) in ovary weight both absolute and relative to terminal body weight. For each group one absolute weight and two relative ovary weights were higher than the normal data range.
As there were no histopathological correlates identified for the increases in liver and ovary weights and the fact that the majority were within the historical control data range, and ovary weights did not show a true dose related response, it is considered that these findings were not of any toxicological significance.
No treatment-related effects were detected in males treated with 150 mg/kg bw/day or in animals of either sex treated with 50 mg/kg bw/day.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Two control females, two males and one female treated with 50 mg/kg bw/day, one male and two females treated with 150 mg/kg bw/day and two females treated with 450 mg/kg bw/day had reddened lungs. One male treated with 450 mg/kg bw/day had discoloured lungs and one control male had dark lungs. One control male had a small right epididymis, seminal vesicle and testis. One male animal treated with 150 mg/kg bw/day that was paired with a female that failed to achieve pregnancy had small testes. One male treated with 50 mg/kg bw/day, one male treated with 150 mg/kg bw/day and one male treated with 450 mg/kg bw/day had an increased pelvic space in the right kidney. Two males treated with 450 mg/kg bw/day had an increased pelvic space in the kidneys which also showed a mottled appearance. One male treated with 150 mg/kg bw/day had an enlarged and dark spleen. In the absence of any histopathological correlates the intergroup differences were considered not to be of toxicological importance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following treatment-related microscopic abnormalities were detected:
Kidneys
An increase in hyaline droplets was present in all males examined which were treated with 450 mg/kg bw/day. There was an increase in incidence and severity of multifocal basophilic tubules in males treated with 450 mg/kg bw/day when compared to controls; 7/8 minimal (severity scale 4) or mild (severity scale 3) compared with 1/5 minimal in the control group. Proteinaceous casts were present in 4/8 males treated with 450 mg/kg bw/day. Increased hyaline droplets were present in 3/5 males treated with 150 mg/kg bw/day without any accompanying pathological changes. No changes were noted in animals treated with 50 mg/kg bw/day which could be attributed to treatment.
The pathological changes seen in the kidneys were confined to males treated with 450 mg/kg bw/day. The hyaline droplet accumulation within the tubules and associated pathology (increased tubular basophilia, proteinaceous casts) are consistent with the accumulation of α 2u globulin, a common finding in animals treated with various test items as well as untreated male rats (Khan KNM et al., 2002). Hyaline droplet accumulation is specific to the male rat, not seen in immature or female rats, and is not considered to be significant in man although due to the subsequent pathological changes it is considered to be adverse in terms of the affected animals in this study.
Accumulation of hyaline droplets only, seen in males treated with 150 mg/kg bw/day is considered to be non-adverse as no associated pathological changes were present.
Stomach
Minimal hyperplasia of the non-glandular stomach was present in 4/5 males and 3/5 females treated with 450 mg/kg bw/day. The minimal hyperplasia also included hyperkeratosis. No changes were apparent in any animals treated with 50 or 150 mg/kg bw/day.
The hyperplasia noted in the stomach of animals treated with 450 mg/kg bw/day is likely to be due to an irritant effect of the test item. Given the low severity and lack of other significant pathological changes (inflammation for example) this is considered to be an adaptive, non-adverse change within this study. This change in the rodent-specific non-glandular region is generally not considered to be significant to man as the corresponding anatomical area is not present.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Non-Productive Matings
The following pairings did not produce litters despite positive signs of mating.
Animals 64F and 52M (treated with 150 mg/kg bw/day) showed no histological changed to account for the lack of pregnancy. Animals 72F and 60M (treated with 150 mg/kg bw/day), the male of this pairing had marked tubular atrophy in the testes and subsequent aspermia in the epididymis and this is the reason for lack of pregnancy. This is not considered to be due to the test item.
Animals 89F and 77M (treated with 450 mg/kg bw/day) showed no histological changes to account for the lack of pregnancy.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Non-Productive Matings
The following pairings did not produce litters despite positive signs of mating.
Animals 64F and 52M (treated with 150 mg/kg bw/day) showed no histological changed to account for the lack of pregnancy. Animals 72F and 60M (treated with 150 mg/kg bw/day), the male of this pairing had marked tubular atrophy in the testes and subsequent aspermia in the epididymis and this is the reason for lack of pregnancy. This is not considered to be due to the test item.
Animals 89F and 77M (treated with 450 mg/kg bw/day) showed no histological changes to account for the lack of pregnancy.

Other effects:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

gross pathology

Remarks on result:

other: Systemic toxicity

Maternal abnormalities

Abnormalities:

effects observed, non-treatment-related

Localisation:

other: stomach

Description (incidence and severity):

The microscopic stomach changes identified in either sex at 450 mg/kg bw/day were considered to be the result of gastric irritancy rather than attributable to true systemic toxicity.

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed on F1 generation

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Any other information on results incl. tables

The microscopic stomach changes identified in either sex at 450 mg/kg bw/day were considered to be the result of gastric irritancy rather than attributable to true systemic toxicity. Kidney findings of tubular basophilia and proteinaceous casts in male kidneys were considered to be associated with alpha 2u-globulin and formation of hyaline droplets, an effect recognized as being both species and sex specific and not relevant for humans. In terms of risk assessment, these findings observed on this study would suggest that a ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 450 mg/kg bw/day for either sex because the findings do not reflect true systemic toxicity.

Applicant's summary and conclusion

Conclusions:

The oral administration of 0,0-tert-butyl isopropyl monoperoxycarbonate (CAS# 2372-21-6) to the Wistar Han™:RccHan™:WIST strain rat by gavage, at dose levels of 50, 150 and 450 mg/kg bw/day, resulted in treatment-related microscopic kidney changes in males treated with 450 and 150 mg/kg bw/day and microscopic stomach changes in animals of either sex treated with 450 mg/kg bw/day. A ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 150 mg/kg bw/day for females and 50 mg/kg bw/day for males.
The microscopic stomach changes identified in either sex at 450 mg/kg bw/day were considered to be the result of gastric irritancy rather than attributable to true systemic toxicity. Kidney findings of tubular basophilia and proteinaceous casts in male kidneys were considered to be associated with alpha 2u-globulin and formation of hyaline droplets, an effect recognized as being both species and sex specific and not relevant for humans. In terms of risk assessment, these findings observed on this study would suggest that a ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 450 mg/kg bw/day for either sex because the findings do not reflect true systemic toxicity.
Within the confines of this study, the ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was considered to be 450 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

0,0-tert-butyl isopropyl monoperoxycarbonate (CAS# 2372-21-6) 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 450 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioral 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 partum. Hematology 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 or Day 44, followed by the termination of all females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on or after Day 25post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results…….

Adult Responses

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

There were no clinical signs of toxicity for males or females treated with 50, 150 or 450 mg/kg bw/day.

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters at 50, 150 or 450 mg/kg bw/day.

Functional Performance Tests

There were no changes in functional performance considered to be related to treatment at 50, 150 or 450 mg/kg bw/day.

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 50, 150 or 450 mg/kg bw/day.

Body Weight

There was considered to be no adverse effect on body weight development for animals of either sex treated with 50, 150 or 450 mg/kg bw/day.

Food Consumption

There was no effect of treatment with the test item up to a dose level of 450 mg/kg bw/day on dietary intake for animals of either sex throughout the treatment period.

Water Consumption

Only males treated with 450 mg/kg bw/day showed an increase in overall water consumption during the pre-pairing phase of the study. 

Reproductive Performance

Mating

No treatment-related effects were detected in mating performance.

Fertility

No treatment-related effects were detected in fertility.

Gestation Lengths

Gestation lengths were between 22 and 23½ days, the distribution of gestation lengths for treated females was essentially similar to control.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Of the litters born, litter size at birth and subsequently on Days 1 and 4post partum, sex ratio and viability were comparable to controls. 

Offspring Growth and Development

In general, offspring body weight gain and litter weights on Days 1 and 4post partumwere comparable to control litters. Surface righting was also comparable to controls.

Laboratory Investigations

Hematology

There were no treatment-related effects detected in the hematological parameters examined.

Blood Chemistry

There were no treatment-related effects detected in the blood chemical parameters examined.

Pathology

Necropsy

No toxicologically significant macroscopic effects were detected in animals of either sex treated with 50, 150 and 450 mg/kg bw/day.

Organ Weights

Males treated with 450 mg/kg bw/day showed a statistically significant increase in kidney weight both absolute and relative to terminal body weight. No such effects were detected in females treated with 450 mg/kg bw/day or in animals of either sex treated with 150 or 50 mg/kg bw/day.

Histopathology

The following treatment-related microscopic abnormalities were detected:

Kidneys

An increase in hyaline droplets was present in all males examined which were treated with 450 mg/kg bw/day. There was an increase in incidence and severity of multifocal basophilic tubules in males treated with 450 mg/kg bw/day when compared to controls. Proteinaceous casts were present in half of the males treated with 450 mg/kg bw/day. Increased hyaline droplets were also present in three males treated with 150 mg/kg bw/day without any accompanying pathological changes. No changes were noted in males treated with 50 mg/kg bw/day or any treated females.

Stomach

Minimal hyperplasia of the non-glandular stomach was present in 4/5 males and 3/5 females treated with 450 mg/kg bw/day. The minimal hyperplasia also included hyperkeratosis. No changes were apparent in any animals treated with 50 or 150 mg/kg bw/day.

Conclusion

The oral administration of 0,0-tert-butyl isopropyl monoperoxycarbonate (CAS# 2372-21-6) to the Wistar Han™:RccHan™:WIST strain rat by gavage, at dose levels of 50, 150 and 450 mg/kg bw/day, resulted in treatment-related microscopic kidney changes in males treated with 450 and 150 mg/kg bw/day and microscopic stomach changes in animals of either sex treated with 450 mg/kg bw/day. A ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 150 mg/kg bw/day for females and 50 mg/kg bw/day for males.

The microscopic stomach changes identified in either sex at 450 mg/kg bw/day were considered to be the result of gastric irritancy rather than attributable to true systemic toxicity. Kidney findings of tubular basophilia and proteinaceous casts in male kidneys were considered to be associated with alpha 2u-globulin and formation of hyaline droplets, an effect recognized as being both species and sex specific and not relevant for humans. In terms of risk assessment, these findings observed on this study would suggest that a ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 450 mg/kg bw/day for either sex because the findings do not reflect true systemic toxicity.

Within the confines of this study, the ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was considered to be 450 mg/kg bw/day.