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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Gene mutation in bacteria (equivalent to EPA OPPTS 970.5265): S. typhimurium TA 1535, TA 1537, TA 98, and TA 100, with and without metabolic activation, all strains negative

- Gene mutation in mammalian cells (equivalent to OECD 476): mouse lymphoma L5178Y, with and without metabolic activation, positive only at cytotoxic concentrations in the absence of S9

- DNA damage and/or repair (OECD 482): hepatocytes of male Fischer F344 rats, negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
GLP compliance:
yes
Type of assay:
other: Damage and Repair, Unscheduled DNA Synthesis in mammalian cells in vitro
Species / strain / cell type:
hepatocytes: male Fischer F344 rats
Test concentrations with justification for top dose:
1500, 500, 150, 50 and 15 pg/ml
Vehicle / solvent:
- Vehicle: water
- Justification for choice of solvent/vehicle:based on a solubility and stability determination of the test article and compatibility with the target cells
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
- Exposure duration: 18-20 hours


NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 150 cells total (50 per replicate)

DETERMINATION OF CYTOTOXICITY
- Method: other: LDH determinations of media

OTHER: nuclear grains were counted in fifty cells in random areas on each of three coverslips for a total of 150 cells per treatment. The net nuclear counts were determined by counting three nucleus-sized areas adjacent to each nucleus and subtracting the average cytoplasmic count from the nuclear count. Replicative synthesis was identified by nuclei completely blackened with grains and such cells were not counted. Nuclei exhibiting toxic effects of treatment, such as dark or uneven staining, disrupted membranes, or irregular shape, were not counted
Evaluation criteria:
If the mean net nuclear count was increased by at least five counts over the vehicle control, the results for a particular dose level were considered significant. A test article was judged positive if it induced a dose-related response and at least one dose produced a significant increase in the average net nuclear grains when compared to that of the vehicle control. In the absence of a dose response, a test article which showed a significant increase in the mean net nuclear grain count in at least two successive doses was considered positive.
Species / strain:
hepatocytes: male F344 rats
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 and 1500 pg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
The results of the UDS assay indicate that under the test conditions, the test article did not cause a significant increase in the unscheduled DNA synthesis as measured by the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the vehicle control), at any dose level. In this study the positive control, DMBA, induced a significant increase in the mean number of net nuclear grain counts over that in the vehicle control. All criteria for a valid test were met. Therefore, the test article is considered to be negative in this study
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
other: L5178Y TK+/- Mouse lymphoma assay
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared from Aroclor 1254-induced male Fisher 344 rats.
Test concentrations with justification for top dose:
0.0, 2.8, 3.6, 4.6, 5.5 and 6.5 µl/ml (without S9 activation)
0.0, 1.7, 2.8, 3.6, 4.6, 5.5 and 6.5 µl/ml (with S9 activation)
Vehicle / solvent:
Dimethyl sulfonate (DMSO)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
other: positive only with cytotoxicity in the absence of S9
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

MEKO showed evidence of mutagenic activity in the absence of S9 activation, but in the presence of cytotoxicity (it was noted that there was a dose response for both mutant frequency and relative total colony growth (see table below).  No evidence of mutagenic activity was observed in the presence of S9 activation.

Dose ul/ml – Relative Total Growth (% of control) – mutant fequency (100,000 survivors)

0.0                     100                                                       0.50

2.8                      50.0                                                     0.75

3.6                      35.5                                                     0.95

4.6                      28.5                                                     1.3

5.5                      12.5                                                     2.0

6.5                       7.5                                                     2.65

 

Conclusions:
Methyl ethyl ketoxime was negative for mutagenic activity in mouse lymphoma L5178Y cells in the presence of S9 metabolic activation, but was positive in the absence of S9 metabolic activation and in the presence of cytotoxicity.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his and trp operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver, induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
0, 313, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
Acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation)
NUMBER OF REPLICATIONS: Two (3 plates/test)




Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg plate and higher
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Toxicity was observed at 5000 µg/plate.
Conclusions:
Methyl ethyl ketoxime did not induce gene mutations in Salmonella or E.Coli strains with or without metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- Cytogenicity (equivalent to EPA OPPTS 870.5385): male and female Sprague-Dawley rats, 300 – 1200 mg/kg bw/day, negative

- Gene mutation (equivalent to EPA OTS 798.5275): Drosophilia melanogaster, 7500 ppm, negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.5385 (In Vivo Mammalian Cytogenetics Tests: Bone Marrow Chromosomal Analysis)
GLP compliance:
yes
Type of assay:
mammalian bone marrow chromosome aberration test
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Frederick, MD
- Age at study initiation: 6-8 weeks
- Weight at study initiation: Males, 175-200 g; Females, 138-161 g
- Assigned to test groups randomly: yes
- Housing: Housed one per cage during study in plastic autoclavable cage
- Diet (e.g. ad libitum): Certified laboratory rodent chow, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2-27.7
- Humidity (%): 50 +/- 20% relative humidity
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
oral: gavage
Vehicle:
Distilled water
- Amount of vehicle (if gavage or dermal): dose volumes were 0.326, 0.652 and 1.304 mL/kg bw.
Duration of treatment / exposure:
Single oral dose by gavage
Frequency of treatment:
Once
Post exposure period:
6, 24 and 48 hours
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
in water
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
in water
Dose / conc.:
1 200 mg/kg bw/day (nominal)
Remarks:
in water
No. of animals per sex per dose:
5/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
A single oral dose of cyclophoshamide at 25 mg/kg body weight; a dose volume of 1.2 mL/kg body weight was used.
Tissues and cell types examined:
Bone marrow cells, arrested in metaphase and collected at 6, 24 and 48 hours after administration were examined microscopically for structural chromosome aberrations.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No statistically significant increases in percentage of aberrant cells were observed in the test article treated groups, regardless of treatment or bone marrow collection time.
Clinical signs of toxicity were noted within 4 hours of dosing at each dose level tested.
Conclusions:
Methyl ethyl ketoxime did not induce chromosomal aberrations in bone marrow cells of male or female rats.
Endpoint:
in vivo mammalian germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OTS 798.5275 (Sex-linked Recessive Lethal Test in Drosophila melanogaster)
GLP compliance:
yes
Type of assay:
Drosophila SLRL assay
Species:
Drosophila melanogaster
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Wisconsin Genetics Laboratory
- Age at study initiation: 8-30 hours old
- Fasting period before study: 4 hours
- Diet (e.g. ad libitum): Standard Drosophila culture medium
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22



Route of administration:
oral: feed
Vehicle:
5% aqueous sucrose
Details on exposure:
Drosophila were treated in groups of 15 in shell vials (23 mm x 90 mm) plugged with rayon fiber balls. The base of each vial was covered with three discs of glass fiber filter material (Whatman, Grade GF/D) onto which approximately 1.0 mL of feeding solution was pipetted. The males were transferred to new treatment vials with freshly prepared feeding solution daily for 3 days. At the end of each 24-hour treatment period, the number of dead flies and ingestion was noted. Negative control flies were fed on 5% aqueous sucrose alone by the same procedure.
Duration of treatment / exposure:
3 days
Dose / conc.:
7 500 ppm (nominal)
Remarks:
in 5% sucrose in aqueous solution
No. of animals per sex per dose:
15 male flies/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Dimethylnitrosomine at 25 ppm in 5% aqueous sucrose. Given for 24 hours only.
Tissues and cell types examined:
Two runs were completed. A standard gemetic scheme (Basc females crossed to Canton-S wild type males) was employed and germ cells which were post-meiotic at the time of exposure were tested for lethal mutations.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Induced mortality was 33% in the first run and 65% in the second run. No increase in sterility was noted. The sex-linked recessive lethal results showed no statistical difference between the treated samples and the negative controls. All frequencies, except that of the positive control, are well within the range of historical negative control values of approximately 0.01 - 0.16%.
Conclusions:
Methyl ethyl ketoxime did not induce mutations in the post-meiotic germ cells of male Drosophila melanogaster when administered in their feed for three consecutive days.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro studies

Six reverse bacterial mutation assays (three of which were conducted by NTP (1999) and reported as a single study) conducted by several methods in standard bacterial strains did not find a mutagenic response in the presence or absence of rat liver activating enzymes (Allied Corporation, 1983; Ministry of Health & Welfare (MHW) of Japan, 1996; Rogers-Back et al., 1988b; NTP, 1999b). The tests were conducted up to the limit dose recommended by modern guidelines and cytotoxicity was usually noted at the highest dose level. A single reverse mutation bacterial assay conducted by the preincubation method reported a mutagenic response in only tester strain TA1535 and only in the presence of high (not standard) levels of hamster liver activating enzymes (NTP, 1999).  A mouse lymphoma study found evidence of mutagenic activity in the absence of rat liver activating enzymes but in the presence of cytotoxicity. Thus, the positive result in the absence of a metabolic activation system was considered to be due to an increase in unspecific cytotoxicity rather than solely genotoxicity. Following rat liver enzyme activation, a negative response was observed (Rogers-Back et al., 1988a). In addition, an unscheduled DNA Synthesis (UDS) test in rat primary hepatocytes (maximal tested concentration 1500 pg/mL) was negative (Microbiological Associates, Inc., 1995).

 

In vivo studies

A cytogenic assay in male and female rats given single gavage doses of 300, 600 or 1200 mg/kg bw/day gave no significant increase in chromosomal aberrations in the bone marrow. The high dose level was set as the maximum tolerated dose. Bone marrow cells, arrested in metaphase and collected at 6, 24, and 48 hours after administration, were examined microscopically for structural chromosome aberrations (Microbiological Associates Inc., 1990).

A mouse micronucleus assay performed on blood samples obtained from male and female mice at the end of the 13-week drinking water study conducted by the NTP was negative. Exposure was by drinking water for 7d/week for 13 weeks at 0, 625, 1250, 2500, 5000 or 10000 ppm (average daily doses of approximately 110, 200, 515, 755, 1330 mg/ kg bw/day to males and 145, 340, 630, 1010, 3170 mg/kg bw/day for females) (NTP, 1999c).

A Drosophila melanogaster sex-linked recessive lethal (SRL) assay with the test substance in adult males exposed by feeding of 7500 ppm orally for 3 days was negative (University of Wisconsin, 1991). Induced mortality was 33% in the first run and 65% in the second run. No increase in sterility was noted. The sex-linked recessive lethal results showed no statistical difference between the treated samples and the negative controls. All frequencies, except that of the positive control, are well within the range of historical negative control values of approximately 0.01 - 0.16%.

Concentrations of 8-oxodeoxyguanosine and 8-aminodeoxyguanosine were not increased in DNA isolated from the test substance inhalation exposed rats 1350 to 3600 mg/m3 (375 - 1000 ppm for 6 hours). These modifications and N2-aminodeoxyguanosine were, however, present in liver DNA from rats treated with 2-nitropropane as positive control (Volkel et al, 1999).

 

The data on toxicokinetics and the results of systemic toxicity in repeated dose toxicity studies all support that the test substance and/or metabolites were available to the target tissues investigated in the in vivo genotoxicity tests.

Based on all available results, the test substance is considered to be non-genotoxic.

Justification for classification or non-classification

There is no concern for genotoxic properties of the test substance based on the available data. Classification according to Regulation (EC) No 1272/2008 is not warranted.