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Administrative data

Description of key information

Methyl-ethylketone peroxide in TXIB/diacetone alcohol was tested in a 90-day repeated dose toxicity study by oral application in rats according to EU method B.26 and OECD guideline 408. The test item was administered at 20, 50 and 150 mg/kg bw/day. A NOAEL of 150 mg/kg bw/day in males and female rats was determined.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
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
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
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
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)
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
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
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
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
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
Key result
Critical effects observed:
no
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.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2010-01-11 to 2010-04-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Fischer, F344/DuCrl, SPF
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Germany GmbH, 97633 Sulzfeld, Germany
- Age at study initiation: About 8 weeks.
- Weight at study initiation: 198-201 g (male); 141 -145 g (female)
- Housing: Makrolon cages Type IV (33 cm x 55 cm area, 20 cm height).
- Diet: Ssniff R/M-H maintenance diet for rats and mice (item V1534-300) ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: Average of 20.80 °C
- Humidity: Average of 51.70 %
- Air changes (per hr): 12 per hour.
- Photoperiod (hrs dark / hrs light): Artificial light from 6 a.m. to 6 p.m.
Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Mazola
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was administered after being dissolved in the vehicle.
Preparations of the test substance were made freshly every day shortly before the administration to the animals. Appropriate preparations were made to allow a uniform dose volume for all groups.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance was not soluble in water. An attempt to prepare a suspension in an aqueous vehicle did not result in a sufficiently homogeneous preparation. Thus a solution in corn oil was chosen for the preparation of the test substance.
- Amount of vehicle: 5 mL test substance preparation or vehicle per kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
An HPLC-method was developed to determine the test substance in aqueous solutions.
Duration of treatment / exposure:
28 day (Animals of the satellite groups were kept after cessation of dosing without a further administration for additional 15 days.)
Frequency of treatment:
once a day
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
65 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- The doses chosen are derived from and based on the results of the dose range finding study. Investigations performed in the dose range finding study were animal observation, body weight and body weight gain, feed consumption and gross pathological examination at terminal necropsy.
- Based on the dose range finding study, the doses of 20, 65 and 200 mg per kg body weight were selected for the Main Study.
- Day 1 was the day of the first administration of the test substance. The animals of all groups were treated with the test substance solutions or with the vehicle once a day on 28 consecutive days. Animals of the satellite groups KS and CS were kept after cessation of dosing without a further administration for additional 15 days.
Positive control:
no positive control
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 to 28: once a week, all animals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Feed consumption was determined per cage in weekly intervals in all animals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Blood samples were taken from the retrobulbar vein plexus of the left eyes in slight ether anaesthesia in the morning after overnight fasting. Feed was offered again immediately after the blood sampling. Li-heparin was used as anticoagulant. Blood was taken on Day 29 from all animals of groups without recovery and of all animals of groups with recovery on Day 43.
- Parameters determined (abbreviations, when commonly used):
• Red blood cell count (RBC)
• Haemoglobin concentration (Hb)
• Haematocrit (Hct)
• Mean corpuscular haemoglobin (MCH)
• Mean corpuscular haemoglobin concentration (MCHC)
• White blood cell count (WBC)
• Mean cell volume (MCV)
• Platelet count (PLT)
• Differential white blood cell count (% of the different cell species)
• Prothrombin time (Quick) as an indicator of blood clotting capacity.

CLINICAL CHEMISTRY: Yes
Blood samples were taken from the same animals and at the same time as for haematology. Plasma (with Na-EDTA as anticoagulant for the determination of bile acids and Li-heparin for all others) was obtained by centrifugation of the blood.
Parameters determined (abbreviations, when commonly used):
• Alanine aminotransferase (ALT, GPT)
• Albumin
• Alkaline phosphatase (AP)
• Aspartate aminotransferase (AST, GOT)
• Bile acids
• Cholesterol
• Creatinine
• Gamma glutamyl transferase (γGT, GGT)
• Glucose
• Potassium (K+)
• Sodium (Na+)
• Total protein
• Urea

URINALYSIS: Yes
(for details see CLINICAL CHEMISTRY)

NEUROBEHAVIOURAL EXAMINATION: Yes
Assessment of the behaviour, the motor activities, and the sensory reactivity to different stimuli (acoustic, tactile, visual and proprioceptive) was performed outside the home cage in a standard arena.
On Day 27: all animals of all groups.
On Day 41: all animals of groups KS and CS.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Other examinations:
No other examinations
Statistics:
Statistical method: analysis of variance followed by the Scheffè-test, t-test, H-test of Kruskal and Wallis followed by the test of Nemenyi, Chi2-test and Fisher`s exact test
Clinical signs:
no effects observed
Description (incidence and severity):
All animals survived until their scheduled sacrifice. As in the daily observations, statistically significant signs of reduced well-being were noted in the high dose groups (200 mg/kg b.w. with and without recovery).
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced mean body weights (statistically significant) were found in male animals of the high dosed group, as well as in male animals of the high dosed group with recovery. Reduced and statistically significant differences were also determined in female animals of the high dosed group, as well as in female animals of the high dosed group with recovery. The high dose group animals gained significantly less weight during the administration period.
During the follow-up observation period the high dose satellite animals of both sexes showed significantly increased body weight gains.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A trend for reduced feed consumption was observed in both sexes in the high dosed groups. At least in the high dosed groups the connection between reduced feed consumption and reduced body weight was obvious.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The group differences in mean cellular haemoglobin (males) and monocytes (males) were noted only at the end of the follow-up period and lack corresponding effects at the end of the dosing period. Thus they are not regarded as test substance related and may rather be incidental. The increase in white blood cell count (males) and the trend towards a higher number of monocytes may indicate inflammatory processes. Erythrocyte related alterations (males and females) are too small to be given toxicological relevance. At the end of the follow-up period, values returned to normal or even below normal (monocytes). These effects could just reflect biological variations. Increases in platelet count seen in females of the high dose group are outside the historical control range and may be related to the test substance. In the recovery period the number of platelets goes back on a normal rate.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Changes in alkaline phosphatase (both sexes) at the end of the dosing period may be related to the lower body weights, higher activities at the end of the follow-up period are taken as indication of recovery. Elevated Na+ (females) is not given toxicological importance as to small difference to
the control. The effect for sodium is inside the historical control range and could just reflect biological variation. Elevated GGT (females) may indicate hepatic or biliary alteration as a test substance related effect but does not show an correlated finding at organ weights or at histopathology of the potential target organs (kidneys and/or liver).
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test substance related findings were made nor were there any significant group differences at the functional observations. All results represent a normal pattern of behaviour and normal reactions of rats of the strain and age examined. There was no significant group difference in the grip strengths of the males. In the females, grip strength of the low dosed group was significantly increased at the end of the administration period. This can be classified as incidental because differences were not seen in the mid and high dosed groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The higher relative brain and testicle weights in males may be based on the lower body weight thus not affecting organ weights. In the tendency this statement applies also on the spleen of the females of the high dose, however with the effect that at the end of the dosing period no significance could be calculated. The significance at the end of the recovery period is without toxicological relevance. In summary can be noted: as the absolute organ weights are not affected for brain, testes and spleen the increase in relative organ weight is associated with the reduced body weight gain.
Lower thymus weights in females are most likely caused by a stress related involution, which was too poorly pronounced to be noted histopathologically. There is no correlate found in histopathology indicating a tissue and/or organ damage.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test substance related or uncommon spontaneous findings were made at the gross examination during the necropsy. A single tissue mass in one female animal of the control group was a fatty tissue necrosis, i.e. not test substance related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There was no test substance related alteration noted histopathologically. Some isolated findings in histopathology are regarded as part of the background pathology, inconspicuous in type and incidence.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test item related adverse effects observed.
Key result
Critical effects observed:
no
Conclusions:
The No-observed-adverse-effect-level (NOAEL) of methyl-ethylketone peroxide was 200 mg per kg body weight for male and female animals.
Executive summary:

Methyl-ethylketone peroxide (in TXIB and diacetonalcohol) was tested in a 28 day repeated dose toxicity study by oral application in rats according to Council Regulation (EC) No. 440/2008, Annex B.7 and OECD guideline 407. The test item was administered at 20, 65 and 200 mg per kg body weight in corn oil. The test substance caused a series of specific and minor alterations in general appearance (reduced well-being), on body weights, feed consumption, haematology, clinical biochemistry and organ weights. None of the alterations gives a clear evidence for a target organ; none bears severe or life-threatening effects and were therefore considered to be test item-related but not adverse. Alterations partially returned to normal during the recovery period. No test substance related findings were made in the low and mid dosed groups (20 and/or 65 mg/kg bw). There was no sex difference in the response to the test substance. At necropsy no test substance related or uncommon spontaneous findings were observed. There was no test substance related alteration noted histopathologically. Therefore, the No-observed-adverse-effect-level (NOAEL) of methyl-ethylketone peroxide was 200 mg/kg bw/day for male and female animals.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Two repeated dose toxicity studies with the test item according to GLP and the respective OECD/EU guidelines are available.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral route:

Key study (90 -day)

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 wasnot 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 noabnormalities 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 chemistryexaminations 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.

 

Supporting study (28 -day)

Methyl-ethylketone peroxide (in TXIB and diacetonalcohol) was tested in a 28 day repeated dose toxicity study by oral application in rats according to Council Regulation (EC) No. 440/2008, Annex B.7 and OECD guideline 407. The test item was administered at 20, 65 and 200 mg per kg body weight in corn oil. The test substance caused a series of specific and minor alterations in general appearance (reduced well-being), on body weights, feed consumption, haematology, clinical biochemistry and organ weights. None of the alterations gives a clear evidence for a target organ; none bears severe or life-threatening effects and were therefore considered to be test item related but not adverse. Alterations partially returned to normal during the recovery period. No test substance related findings were made in the low and mid dosed groups (20 and/or 65 mg/kg bw). There was no sex difference in the response to the test substance. At necropsy no test substance related or uncommon spontaneous findings were observed. There was no test substance related alteration noted histopathologically. Therefore, the No-observed-adverse-effect-level (NOAEL) of methyl-ethylketone peroxide was 200 mg/kg bw/day for male and female animals.

 

Inhalation route:

Repeated dose toxicity testing by inhalation route was waived for methyl-ethylketone peroxide as data for the oral route are available.

 

Dermal route:

Methyl-ethylketone peroxide (in DMP) was tested in two dermal 90-day repeated dose toxicity studies to mice and rats (Reliability: 3). None of those studies are suitable for risk assessment of systemic toxicity after dermal contact, as severe local tissue (skin) damage was noted due to methyl-ethylketone peroxide`s corrosivity. Consequently, systemic toxicity after dermal application is extrapolated from oral studies by route to route extrapolation according to the guidance documents. Performing of repeated dermal toxicity studies is not in line with animal welfare ideas. Therefore a new 90 day oral toxicity study is proposed to gain more information on the repeated dose toxicity of methyl-ethylketone peroxide.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on repeated dose toxicity, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the seventeenth time in Regulation (EU) No 2021/849.