2-Butanone, peroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is not considered to be classified for genetic toxicity and no further tests are required for methyl-ethylketone peroxide.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-11-27 to 2009-11-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Bacterial strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used.
His-mutations D6610 (TA97a) and D3052 (TA98) are frameshift mutations, reversion can occur by addition or deletion of base-pairs. His-mutation G46 (TA100) is a base pair mutation, reversion occurs by the substitution of a single base. In G428 (his-mutation of TA102) th codon CAA, coding for gutamine is mutated to the stop-codon TAA. The functionality of the wild type can be restored by base pair mutation as well as by deletion of 3 or 6 bases.
The rfa mutation leads to a reduced lipopolysaccharide barrier in the cell wall and allows larger molecules to pass the cell wall. Bacteria with this mutation are sensitive to crystal violet.
UvrB results in a loss of DNA-excision repair system. This increases the sensitivity to mutagenic influences. Bacteria with this mutation are sensitive to UV light.
The plasmid pkM101 also disturbs the ability of the bacteria to repair genetic damage and therefore increases their sensitivity to mutagens. Bacteria with this plasmid are resistant against ampicillin. TA102 is also resistant against tetracycline.

Species / strain / cell type:

other: S. typhimurium TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

In preliminary toxicity test, the following concentrations of test substance solutions are used: 5000, 1667, 556, 185, 62 and 21 µg/plate.
Main test: 556 µg/plate was chosen as highest concentration which could be in the toxic range, and a total of 6 concentrations was tested.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

2-nitrofluorene

sodium azide

other: 4-Nitro-o-phenylenediamine (TA97, without S9); t-Butyl-hydroperoxide (TA102, without S9); 2-aminoanthracene (TA98, TA100, TA1535, with S9); 1,8-dihydroxy-anthraquinone (TA102, with S9)

Details on test system and experimental conditions:

One day before the Ames test was performed, a small amount from each of the frozen bacterial cultures was transferred to nutrient. The liquid cultures were incubated in a shaker overnight at 37 °C and then used for the exposure. The mean number of viable cells in the overnight-cltures is 2 to 3 x10e9 cells per mL, based on historical data.

Evaluation criteria:

Calculations, criteria for a positive result:
Means and standard deviations were calculated for the number of mutants in every concentration group.
The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
- For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2 1/2 fold of the amount of the spontaneous revertants.
- For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 1 2/3 fold of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of historic data of the Ames test.

Statistics:

Means and standard deviations were calculated for the number of mutants in every concentration group.

Key result

Species / strain:

S. typhimurium TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the main test, the test substance was again toxic to bacteria at 556 µg/plate and some of the 185 µg/plate samples. At 62 µg/plate and beneath the bacterial background was normal. Only strain TA102 was not affected.
There was no such increase in the number of mutants in any of the tested bacterial strains at any of tested concentrations. The addition of an external metabolising system did not change these results.

Conclusions:

According to these results, methyl-ethylketone peroxide in TXIB/diacetone alcohol is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolising system up to 556 µg/plate, which is the limit of toxicity.

Executive summary:

Methyl-ethylketone peroxide in TXIB/diacetone alcohol was tested in the Salmonella typhimurium reverse mutation test (Ames test) according to OECD guideline 471 and EU method B.13/14. Five bacterial strains, Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used to investigate the mutagenic potential of methyl-ethylketone peroxide in two independent experiments. These experiments were carried out with and without metabolic activation (S9 mix). Triplicate repetitions were run for each dose group in each of two seperate experiments that were conducted, for the control groups six-fold repetitions were run. The exposure for the first experiment was performed according to the Plate Incorporation Assay. The exposure for the second experiment was performed according to the Preincubation Assay. All positive and negative (solvent) control groups were in the range of the historical control range and demonstrated the sensitivity and validity of the test. There was no biologically relevant increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change the results.

Methyl-ethylketone peroxide is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolising system up to 556 ug/plate, which is the limit of toxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies:

Three new (2010) and valid in vitro studies are available conducted with the test material as specified in IUCLID5, section 1.2. These studies were selected as key studies:

Methyl-ethylketone peroxide in TXIB/diacetone alcohol was tested in the Salmonella typhimurium reverse mutation test (Ames test) according to OECD guideline 471 and EU method B.13/14. Five bacterial strains, Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used to investigate the mutagenic potential of methyl-ethylketone peroxide in two independent experiments. These experiments were carried out with and without metabolic activation (S9 mix). Triplicate repetitions were run for each dose group in each of two seperate experiments that were conducted, for the control groups six-fold repetitions were run. The exposure for the first experiment was performed according to the Plate Incorporation Assay. The exposure for the second experiment was performed according to the Preincubation Assay. All positive and negative (solvent) control groups were in the range of the historical control range and demonstrated the sensitivity and validity of the test. There was no biologically relevant increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change the results. Methyl-ethylketone peroxide is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolising system up to 556 µg/plate, which is the limit of toxicity.

 

Methyl-ethylketone peroxide in TXIB/diacetone alcohol was tested in a mammalian cell gene mutation assay at the hprt locus. In this study, V79 cells were exposed to methyl-ethylketone peroxide in DMSO for 3 hours at 19.53 to 78.12 µg/mL without S9 mix and at 19.53 to 136.71 µg/mL with S9 mix (experiment 1). In the second experiment test conditions were 20 hours at 19.53 to 78.12 µg/mL without S9 mix and 3 hours at 19.53 to 136.71 µg/mL with S9 mix. Methyl-ethylketone peroxide was tested up to cytotoxic concentrations. The positive controls induced the appropriate response in this assay. There was no evidence of induced mutant colonies over background in experiments 1 and 2.

Methyl-ethylketone peroxide (MEKP) tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test. Therefore, MEKP is considered not mutagenic in this in vitro mammalian cell gene mutation test performed with V79 (Chinese hamster lung) cells.

 

In a mammalian cell cytogenetic assay for chromosome aberration, V79 cell cultures were exposed to methyl-ethylketone peroxide (in TXIB/diacetone alcohol) in DMSO at (i) 9.76, 19.53, 29.29, 39.06 and 58.59 µg/mL (without and without S9 mix) with 3 hours treatment and 20 hours sampling time, at (ii) 4.88, 9.76, 19.53, 39.06 and 58.59 µg/mL (without S9 mix) with 20 hours treatment and 20 hours sampling time or with 20 hours treatment and 28 hours sampling time and at (iii) 4.88, 9.76, 19.53, 39.06, 58.59 and 78.12 µg/mL with 3 hours treatment and 28 hours sampling time. The results of a pretest on cytotoxicity were used as basis for dose level selection. Methyl-ethylketone peroxide was tested up to cytotoxic concentrations with and without metabolic activation. Positive controls induced the appropriate responses. There was no evidence of chromosome aberration induced over background.

 

Besides that, older in vitro studies with methyl-ethylketone peroxide in dimethyl phthalate (DMP) are also available. Methyl-ethylketone peroxide was not specified in detail in the study reports as given in IUCLID5 section 1.2:

 

Methyl-ethylketone peroxide was examined for mutagenic activity in the Ames test using the histidine requiring S. typhimurium mutants (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and a liver microsome fraction of Aroclor-induced rats for metabolic activation. No indications of toxicity were obtained within the range of concentrations (0, 1.9, 5.6, 16.7, 50 and 150 µg/plate) tested. Methyl-ethylketone peroxide did not reveal any indication for mutagenicity under these chosen test conditions.

 

In a second study, methyl-ethylketone peroxide in DMP was tested for mutagenic activity in the Ames test using four S. typhimurium strains at the concentration range of 1 to 333 µg/plate. Methyl-ethylketone peroxide was not mutagenic in this study.

 

In a third study, methyl-ethylketone peroxide in DMP was examined for mutagenic activity in an Ames test using the histidine requiring S. typhimurium mutants (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and a liver microsome fraction of Aroclor-induced rats for metabolic activation. The concentration range of 0, 3.1, 9.3, 27.8, 83.3 and 250 µg/plate was used. The test substance did not increase the number of his+ revertants with TA 1537, TA 1538, TA 98 and TA 100, either in the absence or in presence of the S9 mix. A slight increase in the number of his+ revertants was observed with TA 1535 in the presence of S9 mix, only. Within the range of concentrations tested, no indications of toxicity were obtained. The background lawn of bacterial growth in control and test plates was comparable.

It is concluded that methyl-ethylketone peroxide does not show mutagenic activity in Salmonella typhimurium TA 1537, TA 1538, TA 98 and TA 100. A slight mutagenic effect (the revertant colony number in the highest and second highest dose was three times the number of the solvent control) has been observed in TA 1535 in the presence of the S9 mix, only whereas all other tests were negative. A second, confirmatory test had also been conducted (using the strains TA 1535 and 1537) but the results are not given in the study report and therefore it is not clear if the positive result was reproduced. Nevertheless these effects were not seen in two other experiments with similar or even higher concentrations. Thus, this effect is considered as an individual finding and the overall conclusion (of the weight of evidence-approach for the Ames tests) is no mutagenic effect.

 

Methyl-ethylketone peroxide in DMP was tested for mutagenic activity in the mouse lymphoma L5178Y test. The concentration ranged from 2 to 10 nL/mL in ethanol. Methyl-ethylketone peroxide induced trifluorothymidine-resistent cells in the mouse lymphoma L5178Y test without S9 activation. But, as no details on the test material (specification) and on cytotoxicity were given the study was report was considered as disregarded.

 

Methyl-ethylketone peroxide in DMP was also tested in cultured Chinese hamster ovary (CHO) for induction of chromosomal aberrations in presence and in the absence of Aroclor 1254 -induced male Spargue-Dawley rat liver S9 and cofactor mix. Methyl-ethylketone peroxide was tested in the range of 1.6 - 50 µg/mL without and 1.6 - 75 µg/mL with S9 mix.

A positive response was only noted at the highest test concentration (with S9) at which also a pronounced cytotoxicity was observed. This study report was also disregarded as no substance specification is given.

 

Methyl-ethylketone peroxide in DMP was tested in cultured Chinese hamster ovary (CHO) cells for induction of sister chromatid exchanges in presence and in the absence of Aroclor 1254 -induced male Sprague-Dawley rat liver S9 and cofactor mix. In this cytogenetic test methyl-ethylketone peroxide in dimethyl sulfoxide induced sister chromatid exchanges in concentration-related manner in the absence of S9 mix and at the highest, severely cytotoxic concentration (50 µg/mL) in the presence of metabolic activation system. Again, due to the missing substance specification and due to the fact that it is not clear if the results are caused by cytotoxicity only the study was disregarded.

 

 

In vivo studies:

The potential of methyl-ethylketone peroxide in DMP to induce micronuclei in the peripheral blood of mice was investigated after a 13 week dermal exposure of 0.357 to 3.57 mg methyl-ethylketone peroxide/animal. At this concentration severe skin damage was observed. Thus it can be assumed that the test item became systemically available. No increase was observed in frequency of micronucleated erythrocytes in peripheral blood samples obtained from male and female mice at the end of the 13-week dermal study.

 

The ability of methyl ethyl ketone peroxide (MEKP) in DMP to produce CAA->CTA transversions in codon 61 of the mouse Ha-ras gene was evaluated when administered to female Sencar mice topically twice weekly for four weeks. Epidermal and dermal hyperplasia and total dermal cellularity were measured at day 2 and day 4 after last dosing. Ha-ras gene mutation was determined in DNA isolated from 25 paraffin sections (8μm) using MSP-32P assay. When applied repetitively, to the dorsal skin of Sencar mice in doses of 10, 100 and 200 µmol/mouse, MEKP caused an statistically significant increase in epidermal thickness over the threshold in the two higher doses but no Ha-ras gene mutation was detected in DNA isolated from the epidermis 4 days after repetitive applications of 200 µmol/mouse MEKP.

 

Additionally the ability of the test substance to produce DNA damage, 8-Hydroxy-2'-deoxyguanosine (8-OH-dG), formation was evaluated when administered to female Sencar mice topically twice weekly for four weeks. Epidermal and dermal hyperplasia and total dermal cellularity were measured at day 2 and day 4 after last dosing. 8-OH-dG/dG ratio was determined by HPLC/ECD using DNA isolated from frozen skins. When applied repetitively, to the dorsal skin of Sencar mice in doses of 10, 100 and 200 µmol/mouse, MEKP caused an increase in epidermal thickness over the threshold. However, no statistically significant increase of 8-OH-dG was detected in DNA isolated from the epidermis after repetitive applications of 200 µmol/mouse test substance. The positive control, DMBA (100 nmoles), effectively induced DNA damage (8-OH-dG), epidermal hyperplasia and dermal hyperplasia. It was concluded that the test substance did not produce the modified DNA base 8-OH-dG.

 

The above mentioned study reports (MEKP in DMP) are older and no EU or OECD Guideline was followed. No detailed information about the substance composition and no impurities are given in the reports (except for the study on the induction of sustained skin hyperplasia and DNA damage). Therefore the potential effects at toxic doses in the CA, SCE and MLA, but not confirmed in the in vivo assays, were not considered to be relevant for MEKP as produced and sold today. In addition, the fact that no effects were noted in a state of the art test battery in vitro with well specified material showing no hints towards genotoxic or mutagenic effects allows the overall conclusion that MEKP is not mutagenic or clastogenic.

 

 

Conclusion

Methyl-ethylketone peroxide in TXIB/diacetone alcohol was not mutagenic in the Ames test, in an HPRT test and in a chromosome aberration test allowing the conclusion that the test item to be registered (of the quality as specified in IUCLID section 1.2) is not a genotoxic or clastogenic substance. Data on genetic toxicity are also available for less characterized methyl-ethylketone peroxide in DMP. Two Ames tests revealed a negative result and one Ames test was ambiguous. Positive results were obtained in several in vitro studies (MLA, CA, SCE). As already mentioned above, the older studies do not contain a detailed description of the impurities present in the test substance. Positive results were not confirmed by in vivo assays for methyl-ethylketone peroxide in DMP or for DMP as such, thus the relevance of these in vitro findings even to the less characterized material is highly questionable. The three available in vivo results address all relevant endpoints which were found positive in the older in vitro assays. The in vivo micronucleus assay clearly shows the lack of clastogenic effects. In addition, in the Ha-ras assay and when investigating the oxidative DNA-damage 8-OHdG it was demonstrated that the substance has no genotoxic potential in vivo with regard to DNA base damage and mutation.

Studies with MEKP in TXIB/diacetone alcohol according to the state of the art OECD guidelines clearly showed that this substance lacks of any mutagenic potential in vitro.

 

MEKP (CAS 1338-23-4) is (voluntarily) recommended to be classified as mutagen by the Japanese MHLW (Ministry of Health, Labour and Welfare). This recommendation is exclusively based on the older studies with less characterized material and not based on the state-of-the-art studies conducted in 2010 which are all negative. The in vivo data (Ha-ras assay, 8-OHdG induction) investigating the mutagenic and DNA damaging potential were also not taken into account by the Japanese authorities. In addition, it should be mentioned that even the older material did not cause mutagenic/clastogenic effects in vivo. In conclusion, this recommendation based on old data with MEKP of unknown purity is of no relevance for this REACH dossier.

 

Consequently, the substance is not considered to be classified for genetic toxicity and no further tests are required for 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 genotoxicity, 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.