2-Butanone, peroxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-05-06 to 2016-03-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-[(2-hydroperoxybutan-2-yl)peroxy]butane-2-peroxol; butane-2,2-diperoxol

EC Number:

700-954-4

Cas Number:

1338-23-4

Molecular formula:

Mixture of C4H10O4 and C8H18O6

IUPAC Name:

2-[(2-hydroperoxybutan-2-yl)peroxy]butane-2-peroxol; butane-2,2-diperoxol

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: 51 - 54 days males and females
- Weight at study initiation: 180 - 239 g males, 117 - 145 g females
- Housing: 2 - 3 animals per cage
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany, ad libitum
- Water. ad libitum
- Acclimation period: 22 days for females, 85 days for males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: sunflower oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 75 mg/mL, 25 mg/mL and 10 mg/mL. Formulations were prepared in the formulation laboratory of Toxi-Coop Zrt. beforehand not longer than for three days and stored at 5 +/- 3°C until use.

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Amount of vehicle: A constant treatment volume of 2 mL dose preparation/kg body weight was administered.
- Lot/batch no.: 1410-5159, 1503-4337, 1503-4336, 1506-4604

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

MEKP content was determined in sunflower oil formulations using the previously validated reverse phase HPLC method with UV detection on a Kinetex 2.6u C18 100A column (100x4.6 mm). Detector: 205 nm.
MEKP concentrations in the samples varied in the range from 93% to 98 % in comparison to the nominal values.
Recovery of MEKP from sunflower oil formulations at two concentration levels (~10 and ~200 mg/mL) was 96 and 102 %.

Duration of treatment / exposure:

Duration of treatment: 90 or 91 days (depending on the day of necropsy)
Animals assigned to the recovery groups were treated identically up to and including Day 89 then they were observed for further four weeks.

Frequency of treatment:

Once a day on a 7 days/week basis, every day at a similar time (+/- 2 hours)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10 animals per sex per dose in the main study. Additionally, 5 animals per sex in the control and high dose group (recovery group).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose selection rationale: The dose setting with 150, 50 and 20 mg/kg bw/day was based on findings obtained in a previous repeated dose toxicity studies with MEKP in the Rat (28-day oral toxicity study in rats, Report no. SL-LT- 223/10; OECD 407; GLP) and the dose-range-finding study conducted for the dose selection of the 28-day oral toxicity study.
A dose of 150 mg/kg bw/day was selected as highest concentration due to the following reasons:
- A test item concentration of 200 mg/kg bw/day caused unspecific and minor alterations in general appearance, in body weights, feed consumption, heamatology, and clinical biochemistry and organ weights in the 28-day oral toxicity study. Although these effects were considered to be non-adverse, the highest dose used in the 90-day oral toxicity study was set to 150 mg/kg bw/day as the rats were treated over a prolonged exposure period of 90 days.
- As shown in the dose-range-finding study of the 28-day oral toxicity study a steep dose-response curve was observed. While no toxic effects were noted at 200 mg/kg bw/day, 5/5 high dosed (1000 mg/kg bw/day) males and 3/5 high dosed females were found dead during the administration period.

A closer proximity to the lethal dose range over a prolonged period of 90 days was considered to bear too much risk and therefore the highest dose tested was chosen to be 150 mg/kg bw/day in the 90-day oral toxicity study.
The selected dose of 150 mg/kg bw/day is considered to be adequate and met the requirements of the OECD 408 Guideline as slight and reversible elevation in the percentage of reticulocytes along with slight change in the spleen weight were detected. However, these effects were considered to be non-adverse and thus the NOAEL was set to 150.0 mg/kg bw/day.

- Post-exposure recovery period in satellite groups: Animals assigned to the recovery groups were treated identically up to and including Day 89 then they were observed for further four weeks.

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical cage side observations were made once a day, after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage in a standard arena once, prior to the first exposure and once weekly thereafter.
- Parameters checked in table [No.1] were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed in the treatment period with an accuracy of 1 g on Day 0, then weekly. Individual body weight changes were calculated. Fasted body weight was measured on day of necropsy (Days 90 and 91 for the main groups and Day 118 for the recovery groups).

FOOD CONSUMPTION: Yes
The food consumption was determined in the treatment phase with an accuracy of 1 g on Day 7, then weekly by reweighing the non-consumed diet.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During the acclimation period, prior to test termination (Day 89)
- Dose groups that were examined: Repeated on all control and high dose test animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: One day after the last treatment (Days 90 and Day 91) and on recovery animals at the end of recovery period (Day 118)
- Anaesthetic used for blood collection: Yes, under isofluran anesthesia
- Animals fasted: Yes
- How many animals: all animals at each dose group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: One day after the last treatment (Days 90 and Day 91) and on recovery animals at the end of recovery period (Day 118)
- Animals fasted: Yes
- How many animals: all animals at each dose group
- Parameters checked in table [No.3] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During the last exposure week (Day 86)
- Dose groups that were examined: all animals
- Battery of functions tested: Different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity

ESTROUS CYCLE: Yes
- Time schedule for examinations: During the last two weeks of the treatment period (from Day 76 up to and including Day 90 or 91)
- Dose groups that were examined: all female animals
- Examinations: A vaginal smear was prepared from each female. After drying, smears were stained with 1 % aqueous methylene blue solution and were examined by a light microscope. The type of cycle (regular or irregular), number of days in pro-estrous, estrous and diestrous, number of cycle during the two weeks, number of animals with prolonged diestrus, number of animals with prolonged estrus were determined.

SPERM EXAMINATION
- Time for examinations: at Necropsy
- Quantitative examinations: The total number of homogenization of one side testis was enumerated. Testes and epididymides were frozen at the necropsy and enumeration was performed later.
- Qualitative examinations: Sperm motility was determined from ductus deferens at the necropsy. For the determination of the sperm motility the mean percentage of motile sperms was determined. The total sperm count and number of immotile sperms were recorded. Two samples were prepared from each animal.
A morphological evaluation of ductus deferens sperms sample was performed from the same animals. Sperm was examined as fixed, wet preparations and classified as either normal or abnormal (isolated heads, misshapen heads and/or tails).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table No. 4), including organ weights
HISTOPATHOLOGY: Yes (see table No. 5)

Other examinations:

not applicable

Statistics:

Statistical analysis was done with SPSS PC+ software package for the following data:
- body weight
- food consumption
- estrous cycle
- hematology
- blood coagulation
- clinical chemistry
- organ weight data
- sperm parameters

The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences.

Where significant heterogeneity was found, we examined the normal distribution of data by Kolmogorov-Smirnov test. In case of not normal distribution, we applied the non-parametric method of Kruskal-Wallis One-Way analysis of variance. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.

For the evaluation of data in the recovery group, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed.

The rate of mortality, frequency of clinical signs, ophthalmological data, pathology and histopathology findings was calculated.
Results were evaluated in comparison with values of control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value in paragraph “Results”. The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p < 0.05 and < 0.01. Male and female rats were evaluated separately

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One male and one female animal died probably due to intra-tracheal applications of the test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male and one female animal died probably due to intra-tracheal applications of the test item.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A slight and reversible elevation of the percentage of reticulocytes was observed in a dose related manner in male and female animals at 150 and 50 mg/kg bw/day.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased spleen weights (absolute and relative to body and brain weight) in male and females at 150 mg/kg bw/day and in females at 50 mg/kg bw/day.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

REFERENCES

G.A. Boorman, R.E.Chapin and K. Mitsumori: Testis and Epididymis Pp: 405-417. In: Pathology of the Fischer Rat. Reference and Atlas. Edited by Boorman, G. A., Montgomery, C. A and Mackenzie, W. F. Academic Press Inc. San Diego, New York, Boston, London, Sidney, Tokyo, Toronto, 1990

Vidal et al.: Reproductive System and Mammary Gland, pp 717-830. In: Toxicologic Pathology, Edited by Sahota et al: CRC Press, Taylor and Francis Group, 2013

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for Methyl-ethylketone peroxide (MEPK) was 150 mg/kg bw /day for male and females rats.

Executive summary:

The objective of this study was to obtain information on the possible health hazards likely to arise from repeated exposure with Methyl-ethylketone peroxide (MEKP) at three dose levels over a prolonged period of time (90 days) followed by a 28-day recovery period in order to assess reversibility, persistence or delayed occurrence of potential toxicological effects. The test item was administered orally (by gavage) to Hsd.Han: Wistar rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 150, 50 and 20 mg/kg bw/day doses corresponding to concentrations of 0, 75, 25 and 10 mg/mL, applied in a dose volume of 2 mL/kg bw for 90 or 91 days. 5 animals/ sex in the control and high dose groups assigned to the recovery groups were treated identically up to Day 89 then they were observed without administration for four weeks (recovery observations).

The suitability of the chosen vehicle for the test item and sufficient stability of Methyl-ethylketone peroxide (MEKP) in the vehicle was analytically verified up front. Methyl-ethylketone peroxide (MEKP) was stable in the applied concentrations in sunflower oil at room temperature for 4 hours and in a refrigerator (5 +/-3°C) for 3 days. Concentrations of the test item in the dosing formulations varied from 93 % to 98 % of nominal concentrations at each analytical occasion, thereby confirming proper dosing. Animals were observed for mortality twice a day in the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of treatment. The body weight and food consumption were measured and evaluated weekly. Clinical pathology examinations (including hematology and clinical chemistry) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. Further sperm and estrous cycle examination were performed. The absolute and relative weights of selected organs were measured. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups including recovery groups. In addition, epididymidis, testes and skin were also processed histologically in a single male animals of the 20 mg/kg bw/day dose group due to macroscopic observations at the necropsy. The results of study were interpreted comparing test item treated groups with respect to controls, which were administered concurrently with vehicle only.

Results:

One male and one female animal administered with 150 mg/kg bw/day died on Day 76 and 36 of the study, respectively. Decreased activity, dyspnea or cyanotic skin were noted for these animals 1-2 days before the death. In accordance with necropsy findings (dark colored or dark reddish mottled lungs, dark red liver), histological examinations revealed in both cases acute catarrhal-purulent tracheitis accompanied with abundant fibrinous exudation into the lumen and diffuse congestion and edema in the lungs as cause of the death. These lesions were probably due to intra-tracheal applications of the test item in both animals.Toxic signs related to the test item were not detected at any dose level (150, 50 or 20 mg/kg bw/day) at the daily and detailed weekly clinical observations and in the course of the functional observation battery.The body weight development of male and female animals was not affected by the test item. No test item related body weight, or body weight gain changes were observed with respect to controls at any dose level during the course of the study (150, 50 or 20 mg/kg bw/day). The daily mean food consumption was similar in animals of the control and test item treated groups (150, 50 and 20 mg/kg bw/day).There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment (150 mg/kg bw/day). A slight and reversible elevation of the percentage of reticulocytes was observed in a dose related manner in male and female animals at 150 and 50 mg/kg bw/day at the termination of the treatment period. Although all values were lying well within the historical background range, the slight elevation correlated with the slight changes in the splenic weight, therefore a test item influence cannot be excluded. Clinical chemistry examinations did not reveal test item related toxic changes in the evaluated parameters (150, 50 and 20 mg/kg bw/day). A test item influence on the estrous cycle was not detected (150, 50 and 20 mg/kg bw/day).Sperm analysis did not reveal test item influence on the sperm cells (count, motility and morphology) at 150 mg/kg bw/day dose.Specific macroscopic alterations related to treatment with the test item were not observed at the terminal necropsy or at the end of the recovery period. A test item influence cannot be excluded in changes of the weights of spleen (absolute and relative to body and brain weight) in male and female animals at 150 mg/kg bw/day and in female animals at 50 mg/kg bw/day. Although all values remained within the historical control ranges, along with the elevated percentage of reticulocytes a test item effect might be supposed. There were no histological lesions related to the test item effect.

Conclusion:

Based on these observations the no observed adverse effect level (NOAEL) for Methyl-ethylketone peroxide (MEPK) was 150 mg/kg bw /day for male and females rats.