2,4-dimethylpentan-3-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date (1st administration of the test substance): 17 May 2017; Experimental completion date (evaluation last experiment): 23 Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

No L 142

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Aug 1998

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,4-dimethylpentan-3-one

EC Number:

209-294-7

EC Name:

2,4-dimethylpentan-3-one

Cas Number:

565-80-0

Molecular formula:

C7H14O

IUPAC Name:

2,4-dimethylpentan-3-one

Test material form:

liquid

Details on test material:

- Name of test material (as cited in report): 2,4-Dimethylpentanon-3 / Diisopropyl keton
- Physical state: liquid

Specific details on test material used for the study:

- Name of test substance (as cited in report): Diisopropyl Ketone; 2,4-Dimethyl-3-Pentanone
- Appearance: slightly yellowish liquid
- Batch Number: 151005
- Purity: 99.2%
- Water content: 0.1%
- Impurities: 0.3% 2,4-dimethylpent-1-en-3-one and 0.1% 2-methylpentan-3-one

Method

Target gene:

Histidine revertants (for Salmonella typhimurium strains)
tryptophan revertants (for Escherichia coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S-9 (rat liver), phenobarbital and ß-naphthoflavone induced

Test concentrations with justification for top dose:

1st experiment: 0; 33; 100; 333; 1000; 2500 and 5000 µg/plate
2nd experiment: The doses, strains and test conditions were selected depending on the results of the first investigation. In case of negative results in the standard plate test the preincubation test will be carried out as 2nd Experiment.

Vehicle / solvent:

Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

With S-9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

Without S9

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine (NOPD)

Remarks:

Without S9

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

Remarks:

Without S9

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9

Details on test system and experimental conditions:

STANDARD PLATE TEST:
The experimental procedure of the standard plate test (plate incorporation method) is based on the method of Ames et al.
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCI) and 10 mL amino acid solution wiere kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation) or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (his+ / trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive lnstruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Number of plates: 3 test plates per dose or per control

PREINCUBATION TEST: In case of negative findings in the standard plate test, a repeat experiment following the preincubation method was performed to confirm the data.
The experimental procedure is based on the method described by Yahagi et al. and Matsushima et al.
0.1 mL test solution or vehicle (negative control), 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) was incubated at 37 °C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

DETERMINATION OF CYTOTOXICITY:
Toxicity detected by a;
- decrease in the number of revertants (factor ≤ 0.6)
- clearing or diminution of the background lawn (= reduced his- or trp- background growth) was recorded for all test groups both with and without S9 mix in all experiments and was indicated in the tables. Single values with a factor ≤. 0.6 were not been detected as toxicity in low dose groups.

SOLUBILITY:
If precipitation of the test substance occured, it was recorded and indicated in the tables. As long as precipitation does not interfere with the colony scoring, 5 mg/plate, regarding the purity of the test substance, were generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/ substantiation.

Evaluation criteria:

Mutagenicity
lndividual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative controls in all experiments. 6 doses of the test substance were tested with a maximum of 5 mg/plate regarding the purity of the test substance. Triplicate plating was used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st Experiment.

Acceptance criteria
The experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls has to be within the range of the historical negative control data for each tester strain.
• The sterility controls has to reveal no indication of bacterial contamination.
• The positive control substances both with and without S9 mix have to induce a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture has to be approx. 10E+9 cells per mL.

Assessment criteria
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA98, TA100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA1535 and TA1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains is within the historical negative control data range under all experimental conditions in at least 2 experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: test substance is insoluble in water; therefore, DMSO was used as vehicle
- Precipitation: No test substance precipitation was found with and without S9 mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Without S9 mix
TA 1535 (MNNG): 3967 (1541 - 6171, SD 1253.9)
TA 100 (MNNG): 3298 (1126 - 5557, SD 1109.6)
TA 1537 (AAC): 1044 (253 - 2190, SD 404.3)
TA 98 (NOPD): 863 (324 - 1746, SD 206.8)
E.coli (4-NQO): 860 (164 - 1721, SD 431.9)
With S9 mix
TA 1535 (2-AA): 197 (105 - 520, SD 56.3)
TA 100 (2-AA): 1748 (272 - 3021, SD 587.2)
TA 1537 (2-AA): 141 (50 - 399, SD 50.3)
TA 98 (2-AA): 1524 (493 - 3096, SD 529.1)
E.coli (2-AA): 133 (61 - 537, SD 61.8)
- Negative (solvent/vehicle) historical control data:
Without S9 mix
TA 1535: 10 (7 - 16, SD 2.0)
TA 100: 100 (71 - 132, SD 11.4)
TA 1537: 8 (5 - 13, SD 1.7)
TA 98: 21 (14 - 34, SD 3.3)
E.coli: 24 (15 - 34, SD 3.9)
With S9 mix
TA 1535: 10 (6 - 18, SD 2.0)
TA 100: 107 (70 - 147, SD 13.7)
TA 1537: 9 (5 - 16, SD 2.1)
TA 98: 28 (12 - 38, SD 4.5)
E.coli: 25 (17 - 36, SD 4.4)

Any other information on results incl. tables

Table 1: Standard plate test without metabolic activation

Strain	Test group	Dose [µg/plate]	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	DMSO Test item           MNNG	- 33 100 333 1000 2500 5000 5.0	9.3 11.0 8.7 10.7 7.0 7.0 10.0 5654.7	3.1 2.0 2.3 2.1 1.0 1.0 5.2 494.0	- 1.2 0.9 1.1 0.8 0.8 1.1 605.9	6, 12, 10 13, 9, 11 10, 6, 10 9, 13, 10 6, 7, 8 8, 7, 6 16, 7, 7 5472, 6214, 5278
TA 100	DMSO Test item           MNNG	- 33 100 333 1000 2500 5000 5.0	86.0 93.7 86.7 91.7 95.7 97.0 101.0 4315.7	1.0 10.6 22.9 12.7 2.3 2.0 8.7 619.0	- 1.1 1.0 1.1 1.1 1.1 1.2 50.2	87, 86, 85 92, 105, 84 76, 71, 113 100, 77, 98 93, 97, 97 97, 95, 99 91, 107, 105 5022, 4057, 3868
TA 1537	DMSO Test item           AAC	- 33 100 333 1000 2500 5000 100	8.0 11.0 8.7 9.0 8.7 10.0 7.7 403.0	1.0 2.6 4.2 2.6 4.7 2.6 1.5 61.5	- 1.4 1.1 1.1 1.1 1.3 1.0 50.4	8, 7, 9 12, 8, 13 10, 4, 12 6, 10, 11 14, 7, 5 9, 8, 13 9, 8, 6 368, 474, 367
TA 98	DMSO Test item           NOPD	- 33 100 333 1000 2500 5000 10	20.3 18.3 17.0 25.3 19.7 19.7 18.7 606.0	2.9 3.5 5.3 3.2 2.1 8.1 6.4 33.5	- 0.9 0.8 1.2 1.0 1.0 0.9 29.8	17, 22, 22 15, 22, 18 19, 11, 21 23, 24, 29 22, 19, 18 21, 11, 27 16, 26, 14 640, 573, 605
E.coli	DMSO Test item           4-NQO	- 33 100 333 1000 2500 5000 5	24.7 25.3 25.3 28.7 23.3 24.3 25.3 1096.3	5.9 4.0 4.0 5.0 5.5 4.0 5.5 62.7	- 1.0 1.0 1.2 0.9 1.0 1.0 44.4	27, 29, 18 29, 21, 26 29, 26, 21 24, 34, 28 27, 17, 26 28, 25, 20 25, 31, 20 1107, 1029, 1153

Table 2: Standard plate test with metabolic activation

Strain	Test group	Dose [µg/plate]	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	DMSO Test item           2-AA	- 33 100 333 1000 2500 5000 2.5	9.7 11.3 10.0 11.0 7.0 10.3 12.0 240.3	3.8 1.5 2.0 2.6 2.0 3.1 3.0 45.1	- 1.2 1.0 1.1 0.7 1.1 1.2 24.9	7, 8, 14 13, 10, 11 10, 12, 8 12, 8, 13 9, 7, 5 11, 13, 7 9, 12, 15 284, 243, 194
TA 100	DMSO Test item           2-AA	- 33 100 333 1000 2500 5000 2.5	108.0 97.0 110.7 105.7 106.7 108.0 112.7 2813.0	18.1 7.5 19.1 5.5 3.2 12.1 5.0 37.4	- 0.9 1.0 1.0 1.0 1.0 1.0 26.0	110, 89, 125 90, 105, 96 89, 118, 125 103, 112, 102 109, 103, 108 119, 110, 95 118, 108, 112 2807, 2853, 2779
TA 1537	DMSO Test item           2-AA	- 33 100 333 1000 2500 5000 2.5	6.7 8.7 9.0 5.0 6.7 7.7 7.0 169.0	1.5 1.5 2.6 1.0 0.6 6.0 1.0 23.8	- 1.3 1.4 0.8 1.0 1.2 1.1 25.4	8, 7, 5 9, 10, 7 11, 10, 6 4, 6, 5 7, 7, 6 14, 2, 7 6, 7, 8 178, 187, 142
TA 98	DMSO Test item           2-AA	- 33 100 333 1000 2500 5000 2.5	31.0 26.0 33.7 29.0 23.3 27.7 33.3 1726.7	2.0 7.5 6.7 6.6 2.5 1.5 7.2 208.0	- 0.8 1.1 0.9 0.8 0.9 1.1 55.7	29, 33, 31 19, 25, 34 26, 37, 38 30, 35, 22 21, 23, 26 26, 29, 28 25, 37, 38 1964, 1576, 1640
E.coli	DMSO Test item           2-AA	- 33 100 333 1000 2500 5000 60	21.7 19.3 21.0 27.0 27.0 25.3 28.0 109.3	3.5 3.2 2.0 5.2 7.0 7.5 7.0 14.7	- 0.9 1.0 1.2 1.2 1.2 1.3 5.0	22, 18, 25 17, 23, 18 21, 23, 19 24, 24, 33 20, 27, 34 33, 18, 25 31, 20, 33 98, 126, 104

Table 3: Preincubation test without metabolic activation

Strain	Test group	Dose [µg/plate]	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	DMSO Test item           MNNG	- 33 100 333 1000 2500 5000 5.0	13.3 12.7 11.3 16.3 13.7 15.3 14.0 2754.0	2.3 4.0 2.5 2.3 2.3 4.0 3.5 71.5	- 1.0 0.9 1.2 1.0 1.2 1.1 206.6	12, 12, 16 8, 15, 15 14, 11, 9 15, 19, 15 15, 11, 15 20, 13, 13 10, 16, 16 2683, 2826, 2753
TA 100	DMSO Test item           MNNG	- 33 100 333 1000 2500 5000 5.0	106.0 102.0 97.3 91.3 125.0 102.0 88.0 2623.0	12.0 8.5 5.0 6.0 12.2 2.6 10.1 243.4	- 1.0 0.9 0.9 1.2 1.0 0.8 24.7	106, 94, 118 111, 94, 101 92, 98, 102 85, 92, 97 117, 139, 119 100, 105, 101 86, 99, 79 2342, 2759, 2768
TA 1537	DMSO Test item           AAC	- 33 100 333 1000 2500 5000 100	9.0 11.3 11.7 9.3 10.0 9.7 8.7 737.3	1.7 2.5 2.1 3.1 1.0 3.2 3.8 103.2	- 1.3 1.3 1.0 1.1 1.1 1.0 81.9	10, 7, 10 11, 14, 9 14, 10, 11 12, 10, 6 11, 10, 9 11, 6, 12 6, 7, 13 784, 619, 809
TA 98	DMSO Test item           NOPD	- 33 100 333 1000 2500 5000 10	17.0 19.7 20.0 20.0 19.3 17.7 17.3 490.3	3.5 6.0 3.0 4.4 2.1 1.5 4.9 10.0	- 1.2 1.2 1.2 1.1 1.0 1.0 28.8	15, 21, 15 14, 19, 26 23, 20, 17 23, 22, 15 20, 21, 17 18, 16, 19 15, 14, 23 491, 480, 500
E.coli	DMSO Test item           4-NQO	- 33 100 333 1000 2500 5000 5	23.3 22.7 19.0 19.3 21.7 18.3 23.0 196.3	2.5 5.9 5.2 4.9 8.0 2.5 2.6 9.1	- 1.0 0.8 0.8 0.9 0.8 1.0 8.4	23, 21, 26 16, 25, 27 25, 16, 16 25, 17, 16 30, 21, 14 16, 21, 18 26, 22, 21 188, 195, 206

Table 4: Preincubation test with metabolic activation

Strain	Test group	Dose [µg/plate]	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	DMSO Test item           2-AA	- 33 100 333 1000 2500 5000 2.5	8.7 11.3 9.3 7.3 11.3 9.7 12.0 254.0	2.1 1.2 2.3 2.1 2.5 4.6 6.1 17.4	- 1.3 1.1 0.8 1.3 1.1 1.4 29.3	8, 7, 11 12, 12, 10 12, 8, 8 8, 5, 9 9, 11, 14 15, 7, 7 15, 5, 16 274, 246, 242
TA 100	DMSO Test item           2-AA	- 33 100 333 1000 2500 5000 2.5	99.0 104.0 90.7 92.0 99.0 94.3 100.0 2640.3	7.9 11.3 9.0 2.6 8.9 5.5 11.3 32.7	- 1.1 0.9 0.9 1.0 1.0 1.0 26.7	102, 105, 90 117, 98, 97 101, 85, 86 95, 90, 91 96, 109, 92 100, 89, 94 93, 94, 113 2644, 2606, 2671
TA 1537	DMSO Test item           2-AA	- 33 100 333 1000 2500 5000 2.5	11.0 12.0 11.3 11.7 8.3 6.0 7.0 63.7	4.6 3.0 2.1 2.5 2.9 1.0 1.0 9.9	- 1.1 1.0 1.1 0.8 0.5 0.6 5.8	15, 12, 6 9, 12, 15 12, 9, 13 14, 12, 9 10, 5, 10 6, 7, 5 8, 7, 6 57, 59, 75
TA 98	DMSO Test item           2-AA	- 33 100 333 1000 2500 5000 2.5	28.7 28.3 25.3 30.3 23.0 27.3 16.0 1587.0	3.5 8.1 1.2 4.2 5.3 9.0 5.2 226.0	- 1.0 0.9 1.1 0.8 1.0 0.6 55.4	29, 32, 25 34, 19, 32 26, 26, 24 27, 29, 35 19, 29, 21 17, 33, 32 22, 13, 13 1512, 1408, 1841
E.coli	DMSO Test item           2-AA	- 33 100 333 1000 2500 5000 60	21.3 19.7 22.3 23.7 25.7 23.0 23.0 84.0	5.5 5.7 4.0 2.1 7.1 5.0 5.0 20.5	- 0.9 1.0 1.1 1.2 1.1 1.1 3.9	27, 21, 16 15, 26, 18 20, 20, 27 26, 23, 22 32, 27, 18 23, 18, 28 28, 18, 23 64, 105, 83

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen here, it is concluded that the test substance is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

Strains: TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA

Dose range: 33 - 5000 µg/plate (SPT and PIT)

Test conditions: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

Solubility: No precipitation of the test substance was found with and without S9 mix.

Toxicity: No bacteriotoxic effect was observed.

Mutagenicity: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.