dimethyl 1,2-benzenedicarboxylate;reaction mass of: 1,2-dimethylpropylidene dihydroperoxide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 13 March 2017. Experimental completion date: date of final thyroid hormone report

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 29 July 2016).

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

442-480-8

EC Name:

-

Cas Number:

182893-11-4

Molecular formula:

Mixture of C5H12O4 and C10H22O6

IUPAC Name:

reaction mass of 1,2-dimethylpropylidene dihydroperoxide and dimethyl 1,2-benzenedicarboxylate

Details on test material:

Identification: Trigonox R-938
Chemical name: Methyl Isopropyl Ketone Peroxide
CASRN: Mixture of 13921-99-8, 33372-83-7, 131-11-3 and 563-80-4
Description: Clear colourless liquid
Batch: 1510-14
Test substance storage: In refrigerator in the dark
Stability under storage conditions: Stable
Expiry date: 01 January 2003

Specific details on test material used for the study:

Test item: Methyl isopropyl ketone peroxide
Test item identity (including alternative names): Butanox P-50. Trigonox R-938 (the R&D name of the
test item)
ELINCS substance: 442-480-8: reaction mass of 1,2-dimethylpropylidene dihydroperoxide and
dimethyl 1,2-benzenedicarboxylate
CAS number: 33372-83-7/13921-99-8
Intended use: Peroxide (which required special handling conditions). Curing agent.
Appearance: Clear colorless liquid
Storage conditions: In a refrigerator (2 to 8 C) protected from light. May be used/formulated in light.
Supplier: Sponsor
Batch number: 1608427802
Expiry date: 31 October 2018
Purity: Main components of the test item are: 1-hydroperoxy-1, 2-dimethylpropyl hydroperoxide
(22.0%) Dimethylphthalate (69.9%)

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Animals
Strain/Species: RccHan™;WIST rat.
Supplier: Envigo RMS (UK) Limited.
Number of animals: 44 males and 48 females. Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization: Males: six days prior to the commencement of treatment. Females: 20 days prior to the commencement of treatment.
Age of the animals at the start of the study: Males 84 to 90 days old. Females 98 to 104 days old.
Weight range of the animals at the start of the study: Males 295 to 336 g. Females 194 to 237 g.

Allocation and Identification
Allocation:
On arrival and non-selective allocation to cages.
Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study.
On Day 1 of study all animals were weighed and body weights were reviewed before dosing commenced by Study Management to ensure variations in body weight of the animals did not exceed 20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.
Identification of animals:
Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.
Identification of cages:
Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Animal Replacement
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before treatment: Irregular estrous cycle, Two females. Body weight range extremes, Two males.

Environmental Control
Rodent facility: Limited access - to minimize entry of external biological and chemical agents.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24ºC and 40-70%.
There were no deviations from these ranges.
Lighting: Artificial lighting, 12 hours light : 12 hours dark
(The females for this study arrived on 5 April 2017. It was noted at the time-clock check on the 7th April that the time clock governing the light cycle was on constant and not the intended ‘12 hours light : 12 hours dark’ regime as required by the Study Plan. See Section 4 for further details).
Electricity supply: Public supply with automatic stand-by generators.

Animal Accommodation
Cages:
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods.
Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Cage distribution:
The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
Bedding:
Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage:
Pre-pairing, up to five animals of one sex
Pairing, one male and one female
Males after mating, up to five animals
Gestation, one female
Lactation, one female + litter

Environmental Enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.

Diet Supply
Diet: SDS VRF1 Certified pelleted diet. A sample (100 g) of each batch of diet used was retained within Pharmacy (frozen -10 to -30C) until finalization of the report. Samples were discarded after finalization of the report. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability: Non-restricted.

Water Supply
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability: Non-restricted.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Method of preparation
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.
Starting with the lowest concentration, approximately 50%, of the final volume of vehicle was added to the test item. The mixture was then magnetically stirred until all test item was uniformly mixed and the
n made up to the required volume with vehicle. The formulation was returned to the container and mixed using a magnetic stirrer until homogenous.

Frequency of preparation
Daily for the first four days and then twice weekly thereafter. (The frequency of preparation was changed as soon as additional stability data became available. However, due to an oversight, the ‘frequency of formulation preparation’ information in the study plan was not updated at this time but this was addressed a few days later, hence for a few days the actual mixing regime differed from that
stated in the Study Plan and therefore a deviation occurred.)

Storage of formulation
Refrigerated (2 to 8°C).

Stability and homogeneity
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 0.5 and 50 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. Formulations were found to be homogenous and stable for 4 days when stored refrigerated (2 to 8°C) and for one day when stored at room temperature (15 to 25°C).

Achieved concentration
Samples of each formulation prepared for administration in Week 1 and Week 4 of treatment (males and females) and on Day 12 of lactation (females only) were analyzed for achieved concentration of
the test item.

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Duration of treatment / exposure:

Males
Two weeks before pairing and up to necropsy after a minimum of five weeks of treatment (animals were killed in Week 6).

Females
Two weeks before pairing, then throughout pairing and gestation until Day 12 of lactation (approximately seven weeks).

Frequency of treatment:

Once daily at approximately the same time each day.

Duration of test:

Males
Two weeks before pairing and up to necropsy after a minimum of five weeks of treatment (animals were killed in Week 6).

Females
Two weeks before pairing, then throughout pairing and gestation until Day 12 of lactation (approximately seven weeks).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

3 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

75 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 10 femals per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection
The doses used in this study (0, 3, 15 and 75 mg/kg/day) were selected in conjunction with the Sponsor.
A subacute 28-day oral toxicity with TRIGONOX R-938 (the R&D name of the test item) by daily gavage in the rat, followed by a 14 day recovery period had been conducted (Ref: NOTOX Project 338726). Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0, 3, 15 and 75 mg/kg/day.
The test item was administered daily for 28 days by oral gavage to SPF-bred Wistar rats (Crl:(WI) BR). One control group and three treated groups were tested, each consisting of five males and five females. An extra five animals per sex in the control and high dose group were allowed 14 days of recovery. The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; ophthalmoscopy at Week 4; clinical pathology and macropathology at termination; organ weights and histopathology on a selection of tissues.
Treatment resulted in minor effects noted at microscopic examination, i.e. an increase of cytoplasmatic vacuolation in the liver of all high dose females. This finding appeared reversible during a 14-day treatment-free period. There were no changes at determination of clinical appearance, performance of functional observations, body weight and food consumption measurements, or alterations during clinical laboratory investigations, macroscopic examination and organ weight determination that were considered to be an effect of treatment. The No Observed Adverse Effect Level (NOAEL) for TRIGONOX R-938 was established at 15 mg/kg/day.
It was therefore considered that the same dose levels could be used for this screening study; 0, 3, 15 and 75 mg/kg/day.

Administration
Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed directly.
Route: Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg/day.
Volume dose: 5 mL/kg body weight.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Control (Group 1): Vehicle at the same volume dose as treated groups.
Frequency: Once daily at approximately the same time each day.
Formulation: To reduce the risk of any irritation of the test formulation on the esophagus during dosing the utmost caution was applied when introducing and removing the dosing catheter. The following procedure was implemented prior to each oral gavage administration on this study:
The dose was drawn up into the catheter and the outside of the catheter was wiped on paper tissue.
The outside of the catheter was then rinsed in a monopot containing tap water and then wiped on a separate paper tissue prior to each gavage administration.
A separate monopot/clean water and clean tissues were used for each group.
A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Examinations

Maternal examinations:

Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
F0 males
Week 1 - daily. Week 2 onwards - once each week.
F0 females
Week 1 - daily. Week 2 - once. Gestation phase - Days 0, 7, 14 and 20. Lactation phase - Days 1, 6 and 12.
Detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation
One to two hours after completion of dosing
As late as possible in the working day.

Clinical Signs
A detailed physical examination was performed on each animal to monitor general health according to the following schedule
F0 males
Weekly.
F0 females
Weekly until pairing. Gestation phase - Days 0, 7, 14 and 20. Lactation phase - Days 1, 6 and 12.

Body Weight
The body weight of animals was recorded as follows:
F0 males
Weekly during acclimatization. Before dosing on the day that treatment commenced (Day 1) and weekly thereafter. On the day of necropsy.
F0 females
Weekly during acclimatization. Before dosing on the day that treatment commenced (Day 1) and weekly before pairing. Days 0, 7, 14 and 20 after mating. Day 1, 4, 7 and 13 of lactation. On the day of necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 males
Weekly before pairing. (Food consumption was not reliably recorded for the first part of Week 1).
F0 females
Weekly before pairing. (Food consumption was not reliably recorded for the first part of Week 1).
After mating the food consumption was performed to match the body weight recording:
Days 0-6, 7-13 and 14-19 after mating. Days 1-3, 4-6 and 7-12 of lactation.
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

Thyroid Hormone Analysis
Blood samples were collected as follows:
At termination:All adults (Except those females which were not pregnant).
Day 4 of age: F1 offspring, two females per litter (where possible).
No pups were allocated to these procedures if the resultant live litter size fell below eight pups/litter or if the resultant number of female pups fell below three offspring.
- one for T4 (serum)#
- one for TSH (plasma)
# priority given to serum sample
Day 13 of age: F1 offspring, two males and two females per litter (where possible)
- two for T4 (serum): where possible one male and one female#
- two for TSH (plasma): where possible one male and one female)
# priority given to serum sample
Sequence of blood sampling on each occasion: In order to minimize any potential confounding effect of the time of day of blood sampling, the order of blood sampling was controlled to allow satisfactory inter-group comparisons.
Conditions: F1 offspring: No overnight deprivation of food. F0 animals: No overnight deprivation of food.
Anesthetic: F1 offspring: None. F0 animals: Isoflurane.
Blood sample site: F1 offspring: Decapitation. F0 animals: Sublingual vein.
Anticoagulant: Plasma samples: K2 EDTA. Microtainers used for collection of samples did not contain separator gel. Serum samples: Greiner Minicollect tubes containing clot activator.
Blood volume: F1 offspring: maximum possible. F0 animals: 2 x 0.5 mL.
Processing: Plasma samples: Samples were kept on wet ice prior to centrifugation and commenced within 30 minutes of sampling. Serum samples: Samples were kept at ambient temperature (15 to 25C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions: At 2000g for ten minutes at 4°C. All available plasma/serum transferred to appropriately labelled polypropylene tubes using micropipettes.
Final storage conditions: Deep frozen (approximately -60 to -90ºC).
Fate of samples: Dispatched to the Department of Biomarkers, Bioanalysis and Clinical Sciences, Envigo.
Thyroid hormone analysis: Performed by the Department of Bioanalysis, Envigo.
Initially samples from offspring on Day 13 of age and main phase adult males were assessed for levels of Thyroxine (T4). As no effects of treatment were seen no further analyses were undertaken. All samples were discarded after finalization of the report.

Method of Kill
All adult animals: Carbon dioxide asphyxiation with subsequent exsanguination.
Offspring - selected for thyroid hormone sampling: Decapitation.
Offspring - all other: Intraperitoneal injection of sodium pentobarbitone.
Sequence: To allow satisfactory inter-group comparison.

Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Time of Necropsy
F0 males: During Week 6 of treatment (see Section 4: this was a deviation from Study Plan which stated that the time of necropsy would be during Week 5).
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females: Day 13 of lactation.
F1 offspring: Selected offspring for thyroid hormone analysis - Day 4 of age. Terminal kill - Day 13 of age.

Females
The following were recorded:
Each uterine horn: Number of implantation sites was counted and confirmed if none were visible at visual inspection.

Organ Weights
For bilateral organs, left and right organs were weighed together.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of the testes which were preserved in modified Davidson’s fluid.

Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List (including abnormalities): All adult animals in Groups 1 and 4 at scheduled termination.
Abnormalities only: All adult animals in Groups 2 and 3.
Routine staining: Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Light Microscopy
Tissues preserved for examination were examined as follows:
All adult males and females in Groups 1 and 4: All specified below table.
All adult males and females in Groups 2 and 3: Abnormalities only.
Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
For the assessment of the testes, a detailed qualitative examination was made, 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, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.

Ovaries and uterine content:

Parturition Observations and Gestation Length
Duration of gestation
Time elapsing between the detection of mating and commencement of parturition.
Parturition observations
From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Estrous Cycles
Dry and wet smears were taken as follows:
Dry smears
For 15 days before pairing using cotton swabs.
Wet smears
Using pipette lavage during the following phases:
For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycl
es were not allocated to study.
After pairing until mating.
For four days before scheduled termination (nominally Days 10 to 13 of lactation).

Fetal examinations:

Records Made During Littering Phase
Clinical observations
Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size
Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter
Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights
Days 1, 4, 7 and 13 of age.
Ano-genital distance
Day 1 - all F1 offspring.
Nipple/areolae count
Day 13 of age - male offspring.

Premature death: Where possible a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
F1 offspring on Day 4 of age: Blood sampling was undertaken.
Externally normal offspring were discarded without examination.
Externally abnormal offspring were examined and retained pending possible future examination.
F1 offspring on Day 13 of age: Blood sampling was undertaken
All animals (but not including those selected for thyroid hormone analysis) were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Animals observed with external abnormalities were retained pending possible future examination.
Thyroid glands were preserved from one male and one female in each litter.
Animals selected for thyroid hormone analysis: externally normal offspring were discarded without examination. Externally abnormal offspring were examined.

Statistics:

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

Indices:

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No consistent signs were seen in the animals in association with the dosing procedure. Only two signs were recorded: one female receiving 15 mg/kg/day had noisy breathing after dosing on Day 2 of treatment (prior to pairing) and one female receiving 75 mg/kg/day appeared to be ‘unsteady’ on Day 1 of lactation.

Mortality:

no mortality observed

Description (incidence):

No animals died prematurely and there no signs were seen at the routine clinical examinations that were considered related to treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The overall body weight gains (Day 1 to 36) for males receiving 15 or 75 mg/kg/day were marginally l ow, when compared with the controls (92 and 89% of control, respectively). At 15 mg/kg/day the effect was seen in the first week of treatment (the gain was 62% of control) and at 75 mg/kg/day the effect was seen from Week 3 to termination (79% of control).
There was no clear effect of treatment on the body weights of females at any stage of the study.
However, during the gestation phase the weight gains of females receiving 3 or 15 mg/kg/day were lower than controls (Day 0-20 change was 90 and 86% of control, respectively) but the weight gains of females receiving 75 mg/kg/day were similar to the controls and, in the absence of a dose-related effect across all groups, these body weight changes are considered to have arisen by chance.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The food consumption of the males was not affected by treatment.
There was no effect of treatment on food consumption in the females prior to pairing or during gestation. During the lactation phase of the study females receiving 75 mg/kg/day ate more food than the
controls in the period Day 7 to 12 (+13%).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age. There was therefore no requirement to measure T4 or TSH (thyroid stimulating hormone) in the samples obtained from offspring on Day 4 of age or from the adult females.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The requirement was to weigh the male reproductive organs. Epididymides, prostate, seminal vesicles and testes were weighed for all animals. No effects of treatment were apparent.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examination of adult males and females did not reveal any findings related to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment related findings.

Other effects:

no effects observed

Description (incidence and severity):

All females allocated to the study showed normal 4/5 day estrous cycles during the acclimatisation period.
Estrous cyclicity, pre-coital interval, fertility, mating performance, gestation length and index were unaffected by treatment.
All females were not cycling before termination (Days 10 – 13 of lactation) and were in diestrous at termination, with the exception of one female treated at 15 mg/kg/day that was found to be in estrous at termination.

Maternal developmental toxicity

Number of abortions:

not examined

Description (incidence and severity):

Not applicable, as rats do not abort.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the number of implantations

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There was no effect of treatment on survival indices

Early or late resorptions:

no effects observed

Description (incidence and severity):

There was no effect of treatment on survival indices

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

gestation length and index were unaffected by treatment.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Estrous cyclicity, pre-coital interval, fertility, mating performance, gestation length and index were unaffected by treatment.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical biochemistry

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

maternal abnormalities

mortality

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

On Day 1 of age the body weights of male and female offspring in all the Methyl isopropyl ketone peroxide treated groups were essentially similar to the controls and considered unaffected by parental treatment.
Thereafter, the body weights of males and females in the 3 mg/kg/day group remained similar to the controls but in the 15 and 75 mg/kg/day groups the male and female offspring body weights were marginally low from Day 4 of age. This resulted in the mean overall body weight gains (Day 1 to 13) of male and female offspring in these groups being slightly low when compared with the controls (95 and 93% of control for males in the 15 and 75 mg/kg/day groups, respectively, and 93 and 91% of control for females in the 15 and 75 mg/kg/day groups, respectively).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no effect of treatment on survival indices of the offspring.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the sex ratio of the offspring.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the number of implantations or litter size.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

There was no effect of treatment on survival indices of the offspring.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Three of the Control litters had male offspring with nipples (one pup in Litter 42, three pups in Litter 44 and one pup in Litter 48). A Group 4 Litter (Litter 76) also had two male offspring with nipples on Day 13 of age. No nipples were observed in all other male offspring. The presence of nipples in these offspring is considered fortuitous and is not related to treatment.
There were no signs in the decedent offspring, or offspring at termination on Day 13 of age that were considered related to parental treatment.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, daily oral administration of Methyl isopropyl ketone peroxide to Han Wistar rats at dose levels of 3, 15 or 75 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 13 of lactation in females was well-tolerated in the adult animals but did elicit a slight reduction in body weight gain in adult male animals receiving 15 or 75 mg/kg/day.
Reproductive performance, fertility and offspring survival were unaffected by parental treatment. There was no effect of treatment on the number of implantations or litter size. From Day 4 of age growth was slightly impaired for male and female offspring in the groups receiving 15 or 75 mg/kg/day but this was not of sufficient magnitude to be considered adverse.
In the context of this study, Methyl isopropyl ketone peroxide showed no evidence of being an endocrine disruptor.
The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive/developmental toxicity was considered to be 75 mg/kg/day.

Executive summary:

This study was a screening test for reproductive/developmental effects and included the assessment of endpoints potentially relevant for detecting endocrine active compounds. The test item, Methyl isopropyl ketone peroxide, a curing agent, was administered daily by oral gavage administration for at least four weeks.

Three groups of 10 male and 10 female rats received Methyl isopropyl ketone peroxide at doses of 3, 15 or 75 mg/kg/day by oral gavage administration. Males were treated daily for 15 days before pairing and up to necropsy after a minimum of four consecutive weeks. Females were treated daily for 15 days before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, propylene glycol, at the same volume dose as the treated groups.

During the study, clinical condition, body weight, food consumption, thyroid hormone analysis (T4) for adult males and Day 13 offspring, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

Results

Parental (F0) responses

No animals died prematurely and there no signs were seen at the routine clinical examinations that were considered related to treatment. No consistent signs were seen in the animals in association with the dosing procedure.

The overall body weight gains (Day 1 to 36) for males receiving 15 or 75 mg/kg/day were marginally low, when compared with the controls (92 and 89% of control, respectively). At 15 mg/kg/day the effect was seen in the first week of treatment and at 75 mg/kg/day the effect was seen from Week 3 to termination. There was no clear effect of treatment on the body weights of females at any stage of the study.

The food consumption of the males was not affected by treatment. There was no effect of treatment on food consumption in the females prior to pairing or during gestation but during the lactation phase of the study females receiving 75 mg/kg/day ate more food than the controls in the period Day 7 to 12.

Estrous cyclicity, pre-coital interval, fertility, mating performance, gestation length and index were unaffected by treatment. There was no effect of treatment on the number of implantations or litter size.

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in the Day 13 offspring.

There was no effect of treatment on the male organ weights.

The macroscopic and microscopic examination of adult males and females did not reveal any findings related to treatment.

F1 Litter Responses

The clinical condition of the offspring, offspring survival and sex ratio were unaffected by parental treatment.

There was no effect of parental treatment on the body weights of the offspring on Day 1 of age. The body weights of male and female offspring in the 15 and 75 mg/kg/day groups were, however, marginally low from Day 4 to 7 of age, resulting in slightly low overall gains when compared with the controls.

Ano-genital distance of both male and female offspring on Day 1 of age showed no adverse effects of parental treatment.

Macroscopic examination of offspring that died prior to the scheduled termination or were killed on Day 13 of age did not reveal any findings that were considered related to parental treatment at any dose level.

Conclusion

In conclusion, daily oral administration of Methyl isopropyl ketone peroxide to Han Wistar rats at dose levels of 3, 15 or 75 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 12 of lactation in females was well-tolerated in the adult animals but did elicit a slight reduction in body weight gain in adult male animals receiving 15 or 75 mg/kg/day.

Reproductive performance, fertility and offspring survival were unaffected by parental treatment. There was no effect of treatment on the number of implantations or litter size. From Day 4 of age growth was slightly impaired for male and female offspring in the groups receiving 15 or 75 mg/kg/day but this was not of sufficient magnitude to be considered adverse.

In the context of this study, Methyl isopropyl ketone peroxide showed no evidence of being an endocrine disruptor.

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive/developmental toxicity was considered to be 75 mg/kg/day.