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EC number: 219-143-7 | CAS number: 2372-21-6
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
No test substance-related adverse effects were observed in reproductive performance, mating, fertility, gestation lengths, litter responses (offspring litter size, sex ratio and viability) or offspring growth and development. Within the confines of the OECD 422 study in rats, the ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was considered to be 450 mg/kg bw/day. No classification was needed for this substance.
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Remarks:
- Combined with repeat dose 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
- 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
- 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 - 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:
- 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. - 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. - 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.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The test item concentration in the tst 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 - Duration of treatment / exposure:
- Males: approximately six weeks
Feamles: Up to eight weeks - Frequency of treatment:
- Daily
- Details on study schedule:
- Chronological Sequence of Study
i. Groups of twelve male and twelve 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/behavioral toxicity.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43 or Day 44.
ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically. - Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Control
- Dose / conc.:
- 50 mg/kg bw/day (actual dose received)
- Remarks:
- Low dose level treatment group. Concentration 12.5 mg/mL
- Dose / conc.:
- 150 mg/kg bw/day (actual dose received)
- Remarks:
- Intermediate dose level treatment group. Concentration: 37.5 mg/mL
- Dose / conc.:
- 450 mg/kg bw/day (actual dose received)
- Remarks:
- High dose level treatment group: Concentration: 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 dose levels were chosen in consultation with the sponsor and were based on the results of previous toxicity work (Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study (Envigo Study Number: BX90DW)) where an overall reduction in body weight gain was noted in male animals treated with 1000 mg/kg bw/day (78% lower than control). Reductions in food consumption and food efficiencies were also apparent throughout the treatment period. Male animals treated with 500 or 250 mg/kg bw/day also showed reductions in body weight gain and food consumption. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
- Positive control:
- Not applicable
- Parental animals: Observations and examinations:
- 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.
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.
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++) - Oestrous cyclicity (parental animals):
- A vaginal smear was prepared for each female and the stage of oestrous or the presence of sperm was recorded.
- Sperm parameters (parental animals):
- Teses weight and histopathology
- Litter observations:
- Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)
Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum. - Postmortem examinations (parental animals):
- Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 43 or Day 44. 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
Prostate and Seminal Vesicles
Epididymides
Spleen
Heart
Testes
Kidneys
Thymus
Liver
Thyroid (weighed post-fixation with Parathyroid)
Ovaries
Uterus (weighed with Cervix)
.
Histopathology
Samples of the following tissues were removed from 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)
Prostate
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 (W Henderson). A peer review of the findings observed was conducted by V Mowat at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS, UK. - Postmortem examinations (offspring):
- All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
- Statistics:
- Please refer to "Any other information of materials and methods including tables"
- Reproductive indices:
- Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
i. Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii. Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated / Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females / Number of animals mated) x 100
Gestation and Parturition Data
The following parameters were calculated 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 - 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 their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
i. Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
Pre–implantation loss (%) = ((Number of corpora lutea) - Number of implantation sites) / Number of corpora lutea) x100
Post–implantation loss (%) = ((Number of implantation sites - Total number of offspring born) / Number of implantation sites) x100
ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4 / Number of offpsring alibve on Day 1) x 100
iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:
Number of male offspring / Number of female offspring x 100 - 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):
- effects observed, non-treatment-related
- 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.
There were no treatment-related effects detected in the Blood chemical parameters examined. Statistical analysis of the data did not reveal any significant intergroup differences. - Clinical biochemistry findings:
- no effects observed
- 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
- 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.
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.
There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Reproductive function: oestrous cycle:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment-related effects were detected in fertility.
Two females treated with 150 mg/kg bw/day and one female treated with 450 mg/kg bw/day were not pregnant following positive evidence of mating. One male animal treated with 150 mg/kg bw/day that was paired with a female that failed to achieve pregnancy had small testes and microscopic examination of this male revealed marked tubular atrophy in the testes and subsequent aspermia in the epididymis. This was considered the reason for the lack of pregnancy, however, as no similar effects were noted in any other treated male animals, this effect was considered to be incidental and unrelated to treatment. The remaining animals showed no histological changes which could account for the lack of pregnancy. - Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- No treatment-related effects were detected in mating performance.
- 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: 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.
- Remarks on result:
- other: reproductive and developmental toxicity
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 50 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- histopathology: non-neoplastic
- Remarks on result:
- other: Systemic toxicity
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 150 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- gross pathology
- Remarks on result:
- other: Systemic toxicity
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 450 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Please refer to remarks on results
- Remarks on result:
- other: Please refer to later section: Any other remarks on results
- Clinical signs:
- no effects observed
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- For animals treated with 450 mg/kg bw/day the litter weights were slightly lower than controls from Days 1 to 4. However, this was considered to be mainly attributable to one female which only gave birth to four offspring.
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Necropsy findings apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 50, 150 and 450 mg/kg bw/day.
- Histopathological findings:
- no effects observed
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOEL
- Generation:
- F1
- Effect level:
- 450 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No effects reported on the F1 generation
- Key result
- Reproductive effects observed:
- no
- Lowest effective dose / conc.:
- 450 mg/kg bw/day (actual dose received)
- Treatment related:
- no
- 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.
Reference
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.
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 450 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- GLP study.
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Effects on developmental toxicity
Description of key information
No test substance-related adverse effects were observed in reproductive performance, mating, fertility, gestation lengths. Additionally, no test item related adverse changes were noted in litter responses (offspring litter size, sex ratio and viability) or offspring growth and development. Within the confines of the OECD 422 study in rats, the ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was considered to be 450 mg/kg bw/day.
Link to relevant study records
- 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
- 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
- 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 - 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. - 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 - Dose / conc.:
- 50 mg/kg bw/day (actual dose received)
- Remarks:
- Low dose level treatment group: 12.5 mg/mL
- Dose / conc.:
- 150 mg/kg bw/day (actual dose received)
- Remarks:
- Intermediate dose level treatment group - 37.5 mg/mL
- 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.
- 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 - 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
- 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
- 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
- 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.
- 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
- 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
- Key result
- Developmental effects observed:
- no
- Lowest effective dose / conc.:
- 450 mg/kg bw/day (actual dose received)
- Treatment related:
- no
- 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.
Reference
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.
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 450 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- GLP study
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
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, which was the highest dose tested. No adverse effects were observed and therefore no classification of the substance is needed. Data are complete, but not sufficient for classification.
Additional information
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