2,4-dimethylpentan-3-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A bacterial reverse mutation assay was conducted GLP-compliant according to OECD Testguideline 471. The test was performed with and without metabolic activation in S. typhimurium TA98, TA100, TA1535, TA1537 and E.coli WP2 uvrA strains. There was no mutagenic effect of test material in the presence or absence of metabolic activation in the Standard Plate Test, nor in the Preincubation Test with or without metabolic activation.

An in vitro mammalian cell micronucleus test was conducted GLP-compliant according to OECD Testguideline 487. The test was performed with and without metabolic activation in human lymphoblastoid cells (TK6). The test material induced micronuclei in the in vitro assay. As the test material contains 0.3 % of a substance with a potent structural alert, it is possible that the result applies to the impurity rather than the main component.

The potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus was investigated GLP-compliant in Chinese hamster ovary (CHO) cells with and without metabolic activation in vitro according to OECD Testguideline 476 (In Vitro Mammalian Cell Gene Mutation Test). No cytotoxicity was observed up to the highest concentrations evaluated for gene mutations. The test substance did not cause any biologically relevant or dose-dependent increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

No L 142

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

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

Target gene:

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

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:

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

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

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

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

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

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.

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

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

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

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

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)

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 21 April 2017 ; Experimental completion date: 02 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

yes

Remarks:

A second 3+27 hour treatment in the presence of S-9 was performed

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

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

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Metabolic activation system:

S-9 Fraction, Aroclor induced

Test concentrations with justification for top dose:

Preliminary solubility assessments were made, a vehicle chosen and dilutions arranged that allow maximum exposure up to the solubility limit, 10 mM or 2000 μg/mL, whichever is lower. For freely soluble test articles, where the molecular weight is unknown, the highest concentration tested is 2000 μg/mL (OECD, 2016).

If there is no information to satisfactorily define the concentrations to be tested in the Micronucleus Experiment, a preliminary toxicity Range-Finder Experiment was performed, using a series of concentrations separated by approximately two-fold intervals, ranging down from the upper limit. All concentration ranges are detailed in the raw data. For freely soluble non-toxic test articles in aqueous solution, where the molecular weight is unknown, the following concentration ranges may be tested.

Range finder experiment:
Concentration of treatment solution (mg/mL) 0.7256, 0.1209, 0.2016, 0.3359, 0.5599, 0.9331, 1.555, 2.592, 4.32, 7.2, 12, 20
Final Concentration (μg/mL): 7.256, 12.09, 20.16, 33.59, 55.99, 93.31, 155.5, 259.2, 432, 720, 1200, 2000

Micronucleus Experiment(s):
To be determined, based on range finding experiment. Concentrations are 10-fold more than those stated to achieve the indicated final concentrations if the vehicle is organic. An appropriate number of test article concentrations was selected for the Micronucleus Experiments, in order that a suitable range of cytotoxicity was achieved.

Vehicle / solvent:

Vehicle controls comprised treatments with the chosen vehicle diluted to the same extent as the test article solutions. The preferred vehicles are water or dimethyl sulphoxide (DMSO). The choice of vehicle is confirmed in the raw data.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Noscapine

Details on test system and experimental conditions:

Cell treatment and harvest times
To provide an accurate method of quantifying the effect on cell proliferation and the cytotoxic or cytostatic activity of a treatment and to ensure that only cells that divided during or after treatment are microscopically scored cytochalasin B is added to the cultures.

The proposed treatment condidtions are as follows:
+ S9 short treament: Cells are exposed for 3 hours. Upon ending of the exposure duration S9 and the test substance are removed and Cytochalasin B added. 27 hours later cells are harvested.
- S9 short treament: Cells are exposed for 3 hours. Upon ending of the exposure duration the test substance is removed and Cytochalasin B added. 27 hours later cells are harvested.
- S9 extended treament: Cells are exposed for 30 hours in the presence of Cytochalasin B. After the exposure period cells are harvested
+ S9 extended treament: Cells are exposed for 30 hours in the presence of S9 and Cytochalasin B. After the exposure period cells are harvested.

Osmolality and pH:
Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit can give rise to chromosome aberrations (Scott et al., 1991; Brusick, 1986). Therefore, osmolality and the effect of the test article on the pH of the culture medium, are assessed during the study. Osmolality and pH are initially measured on post-treatment media in the cytotoxicity Range-Finder Experiment. If marked changes are observed, further measurements may be made in the Micronucleus Experiment(s).

Harvesting:
At the defined sampling time, cultures were:
• Centrifuged at approx. 200 g, 5 minutes
• Resuspended in 4 mL (hypotonic) 0.075 M KCl at 37°C for 4 minutes
• Fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (7:1 v/v), centrifuged (approx. 200 g, 5 minutes) and resuspended, followed by further centrifugation and fixation (approx. 1250 g, 2-3 minutes) until the cell pellets are clean
• Stored in fixative at 2-8°C prior to slide preparation for a minimum of 3 hours.

Slide Preparation:
• Cells centrifuged (approx. 1250 g, 2-3 minutes) and resuspended in a minimal amount of fresh fixative (if required)
• Several drops of suspension gently spread onto multiple clean, dry microscope slides
• Slides air-dried then stored protected from light at room temperature prior to staining
• Slides stained by immersion in 12.5 μg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 minutes, following by a wash with PBS (with agitation) for a few seconds
• The quality of the stain will be checked. If slides appear over-stained (leaching of orange colouration), slides will be returned to the PBS bath for a further wash and the check repeated
• Stained slides will be air-dried and stored protected from light at room temperature prior to analysis
• Immediately prior to analysis 1-2 drops of PBS will be added to the slides before mounting with glass coverslips.

Evaluation criteria:

For valid data, the test article is considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations is observed
2. An incidence of cells with micronuclei at such a concentration that exceeds the normal range in both replicates is observed
3. A concentration-related increase in the proportion of cells with micronuclei is observed (positive trend test).
The test article is considered positive in this assay if all of the above criteria are met.
The test article is considered negative in this assay if none of the above criteria are met.
Results which only partially satisfy the above criteria are dealt with on a case-by-case basis.

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No marked changes in osmolality or pH were observed at the highest concentration tested in the Range-Finder (1150 μg/mL), compared to the concurrent vehicle controls

Remarks on result:

other: after 30h exposure

 

Data for 3+27 Hour Treatments -S-9, Micronucleus Experiment, Trial 2

 

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	48	397	55	500	1.01
	B	65	381	54	500	0.98
	C	61	376	63	500	1.00
	D	69	398	33	500	0.93
	Total	243	1552	205	2000	0.98	-
200.0	A	64	374	62	500	1.00
	B	61	388	51	500	0.98
	Total	125	762	113	1000	0.99	0
400.0	A	98	366	36	500	0.88
	B	111	349	40	500	0.86
	Total	209	715	76	1000	0.87	12 #
600.0	A	165	321	14	500	0.70
	B	156	324	20	500	0.73
	Total	321	645	34	1000	0.71	27 #
800.0	A	265	233	2	500	0.47
	B	210	280	10	500	0.60
	Total	475	513	12	1000	0.54	45
900.0	A	236	258	7	501	0.54
	B	193	299	8	500	0.63
	Total	429	557	15	1001	0.59	40 #
950.0	A	284	214	2	500	0.44
	B	282	213	5	500	0.45
	Total	566	427	7	1000	0.44	55
1000	A	234	265	1	500	0.53
	B	218	276	6	500	0.58
	Total	452	541	7	1000	0.56	43
1025	A	232	265	3	500	0.54
	B	204	283	13	500	0.62
	Total	436	548	16	1000	0.58	41
1050	A	206	289	5	500	0.60
	B	152	333	15	500	0.73
	Total	358	622	20	1000	0.66	33
1075	A	240	252	8	500	0.54
	B	297	201	2	500	0.41
	Total	537	453	10	1000	0.47	52 #
1100	A	448	51	1	500	0.11
	B	427	73	0	500	0.15
	Total	875	124	1	1000	0.13	87
1150	A	238	259	3	500	0.53
	B	297	201	2	500	0.41
	Total	535	460	5	1000	0.47	52 #
MMC, 0.10	A	206	289	5	500	0.60
	B	291	209	0	500	0.42
	Total	497	498	5	1000	0.51	48 #
MMC, 0.20	A	262	237	1	500	0.48
	B	298	198	4	500	0.41
	Total	560	435	5	1000	0.45	55 #

Mono = Mononucleate Bi = Binucleate Multi = Multinucleate RI = Replication index

# Highlighted concentrations selected for analysis

 

Data for 3+27 Hour Treatments +S-9, Micronucleus Experiment, Trial 3

 

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicit y Based on RI (%)
Vehicle	A	73	295	132	500	1.12
	B	48	332	120	500	1.14
	C	67	324	109	500	1.08
	D	60	339	101	500	1.08
	Total	248	1290	462	2000	1.11	-
200.0	A	57	349	94	500	1.07
	B	55	344	101	500	1.09
	Total	112	693	195	1000	1.08	2
400.0	A	48	366	86	500	1.08
	B	83	392	25	500	0.88
	Total	131	758	111	1000	0.98	11 #
600.0	A	75	382	43	500	0.94
	B	91	376	33	500	0.88
	Total	166	758	76	1000	0.91	18
700.0	A	95	350	55	500	0.92
	B	135	325	40	500	0.81
	Total	230	675	95	1000	0.87	22
750.0	A	96	388	16	500	0.84
	B	89	355	56	500	0.93
	Total	185	743	72	1000	0.89	20
800.0	A	91	376	33	500	0.88
	B	77	386	37	500	0.92
	Total	168	762	70	1000	0.90	19
850.0	A	85	371	44	500	0.92
	B	127	327	46	500	0.84
	Total	212	698	90	1000	0.88	21
900.0	A	130	346	24	500	0.79
	B	119	362	19	500	0.80
	Total	249	708	43	1000	0.79	28 #
950.0	A	111	356	33	500	0.84
	B	132	340	28	500	0.79
	Total	243	696	61	1000	0.82	26
1000	A	161	326	13	500	0.70
	B	125	331	44	500	0.84
	Total	286	657	57	1000	0.77	30
1050	A	152	312	36	500	0.77
	B	169	308	23	500	0.71
	Total	321	620	59	1000	0.74	33
1150	A	200	294	6	500	0.61
	B	205	285	10	500	0.61
	Total	405	579	16	1000	0.61	45 #
CPA, 3.00	A	157	329	14	500	0.71
	B	176	311	13	500	0.67
	Total	333	640	27	1000	0.69	37
CPA, 5.00	A	205	284	11	500	0.61
	B	179	316	5	500	0.65
	Total	384	600	16	1000	0.63	43 #

Mono = Mononucleate Bi = Binucleate Multi = Multinucleate RI = Replication index

# Highlighted concentrations selected for analysis

Data for 30+0 Hour Treatments -S-9, Micronucleus Experiment, Trial 1

 

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	57	302	141	500	1.17
	B	50	341	109	500	1.12
	C	48	337	115	500	1.13
	D	57	312	131	500	1.15
	Total	212	1292	496	2000	1.14	-
100.0	A	43	327	130	500	1.17
	B	44	336	120	500	1.15
	Total	87	663	250	1000	1.16	0
200.0	A	48	365	87	500	1.08
	B	69	329	102	500	1.07
	Total	117	694	189	1000	1.07	6
300.0	A	65	387	48	500	0.97
	B	60	347	93	500	1.07
	Total	125	734	141	1000	1.02	11
400.0	A	57	378	65	500	1.02
	B	42	355	103	500	1.12
	Total	99	733	168	1000	1.07	6
450.0	A	55	381	64	500	1.02
	B	44	401	55	500	1.02
	Total	99	782	119	1000	1.02	11
500.0	A	73	388	39	500	0.93
	B	80	351	69	500	0.98
	Total	153	739	108	1000	0.96	16
550.0	A	58	399	43	500	0.97
	B	53	385	62	500	1.02
	Total	111	784	105	1000	0.99	13
600.0	A	62	387	51	500	0.98
	B	70	365	65	500	0.99
	Total	132	752	116	1000	0.98	14 #
650.0	A	131	356	13	500	0.76
	B	127	356	17	500	0.78
	Total	258	712	30	1000	0.77	32
700.0	A	87	387	26	500	0.88
	B	96	364	40	500	0.89
	Total	183	751	66	1000	0.88	23
750.0	A	286	211	3	500	0.43
	B	123	359	18	500	0.79
	Total	409	570	21	1000	0.61	46
800.0	A	167	327	6	500	0.68
	B	115	362	23	500	0.82
	Total	282	689	29	1000	0.75	35 #
1000	A	265	230	5	500	0.48
	B	242	255	3	500	0.52
	Total	507	485	8	1000	0.50	56 #

 

NOS, 10.0	A	89	370	41	500	0.90
	B	86	378	36	500	0.90
	Total	175	748	77	1000	0.90	21
NOS, 20.0	A	111	301	88	500	0.95
	B	118	277	105	500	0.97
	Total	229	578	193	1000	0.96	16 #

Mono = Mononucleate Bi = Binucleate Multi = Multinucleate RI = Replication index

# Highlighted concentrations selected for analysis

 

Conclusions:

The test material induced micronuclei in the in-vitro assay. As the test material contains 0.3% of a substance with a potent structural alert, it is possible that the result applies to the impurity rather than the main component.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2016

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Type of assay:

other: Gene Mutation Assay in Mammalian Cells in vitro (HPRT)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: BASF SE

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: No test substance precipitation in culture medium occured up to the highest applied concentration in the absence and presence of S9 mix at the end of treatment.
- Solubility and stability of the test substance in the solvent/vehicle: Due to the good solubility of the test substance in water, the aqueous culture medium (Ham's F12) was selected as vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The substance was emulsified in culture medium (Ham's F12). The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. To achieve homogeneity of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted from according to the planned doses. All test substance formulations were prepared immediately before administration. To keep the test substance homogeneously in the vehicle, the test substance preparation was carefully pipetted before removal.

Target gene:

hprt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: The CHO cell line is a permanent cell line derived from the Chinese hamster and has a high proliferation rate, high plating efficiency (about 90 %) and karyotype with a modal number of 20 chromosomes.
- Cell cycle length, doubling time or proliferation index: 12 - 16 hours
- Number of passages if applicable: At least 2 passages were performed before cells were taken for the experiment. A further passage was also necessary in order to prepare test cultures.
- Methods for maintenance in cell culture if applicable: For cell cultivation, deep-frozen cell suspensions were thawed at 37 °C in a water bath, and volumes of 0.5 mL were transferred into 25 cm² plastic flasks containing about 5 mL Ham's F12 medium including 10 % (v/v) FCS. Cells were grown with 5 % (v/v) CO2 at 37 °C and ≥ 90 % relative humidity up to approximate confluence and subcultured twice weekly (routine passage in 75 cm² plastic flasks).
- Modal number of chromosomes: 20
- Normal (negative control) cell cycle time: 12 - 16 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10 % (v/v) fetal calf serum (FCS), 1 % (v/v) penicillin / streptomycin (stock solution: 10000 IU / 10000 µg/mL) and 1 % (v/v) amphotericine B (stock solution: 250 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: no

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and ß-naphthoflavone induced rat liver S9 fraction

Test concentrations with justification for top dose:

According to an initial range-finding cytotoxicity test for the determination of the experimental doses the top concentration was determined to be the limit concentration of 10 mM (1150 µg/mL). In the main experiments the following concentrations were tested.
1st experiment
without S9 mix
0; 71.9; 143.8; 287.5; 575.0; 1150.0 µg/mL
with S9 mix
0; 71.9; 143.8; 287.5; 575.0; 1150.0 µg/mL
2nd experiment
without S9 mix
0; 100.0; 200.0; 400.0; 800.0; 1150.0 µg/mL
with S9 mix
0; 100.0; 200.0; 400.0; 800.0; 1150.0 µg/mL
All test groups except for 100.0 µg/mL in the 2nd experiment were evaluated for gene mutations.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqueous culture medium (Ham's F12)
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, the aqueous culture medium (Ham's F12) was selected as vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 20 x 10^6 cells in 40 mL

DURATION
- Preincubation period: about 20 - 24 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 - 9 days
- Selection time (if incubation with a selection agent): 6 - 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 16 days

SELECTION AGENT (mutation assays): 6-thioguanine

STAIN (for cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.

NUMBER OF CELLS EVALUATED: 200 cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative survival after treatment; mutant frequency

Evaluation criteria:

The HPRT assay is considered valid if the following criteria are met:
- The absolute cloning efficiencies of the negative controls should not be less than 50 % (with and without S9 mix).
- The background mutant frequency in the negative controls should be within the historical negative control data range (95 % control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not "under control".
- The positive controls both with and without S9 mix should induce a distinct, statistically significant increase in mutant frequencies in the expected range.
A test substance is considered to be clearly positive if all following criteria are met:
- A statistically significant increase in mutant frequencies is obtained.
- A dose-related increase in mutant frequencies is observed.
- The corrected mutation Frequencies exceeds both the concurrent negative / vehicle control value and the range of the laboratory's historical negative control data (95 % control limit).
Isolated increases of mutant frequencies abouve the historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
A test substance is considered to be clearly negative if the following criteria are met:
- Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
- The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of the laboratory's historical negative control data (95 % control limit).

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0.
In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R.
If the results of these tests were statistically significant compared with the respective vehicle control, labels are printed in the tables.
However, both, biological and statistical significance are considered together.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: In this study, in the absence and presence of S9 mix, no precipitation in culture medium was observed macroscopically up to the highest applied test substance concentration.

RANGE-FINDING/SCREENING STUDIES: In the pretest for toxicity based on the molecular weight of the test substance 1150 µg/mL (approx. 10 mM) was used as top concentration both with and without S9 mix 4-hour exposure time. In the pretest the pH value was not influenced by the addition of the test substance preparation to the culture medium at the concentrations tested. The highest applied concentration of 1150.0 µg/mL was a homogeneous emulsion in the vehicle (culture medium). No test substance precipitation in culture medium occured up to the highest applied concentration in the absence and presence of S9 mix at the end of treatment. After 4 hours treatment in the absence and presence of S9 mix, no cytotoxicity was observed as indicated by a reduced RS of about or below 20 % of control up to the highest applied concentration.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: without S9 mix: MF(corr.): 42.47 - 419.90 per 10^6 cells; with S9 mix: MF(corr.): 21.52 - 270.48 per 10^6 cells)
- Negative (solvent/vehicle) historical control data: MF(corr.): 0.00 - 6.84 per 10^6 cells)

Table 1: Summary of results - experimental parts without and with S9 mix

Experiment	Exposure period [h]	Test groups [µg/mL]	S9 mix	Prec.*	Genotoxicity** MFcorr.[per 10^6cells]	Cytotoxicity***
						RS [%]	CE2[%]
1	4	Negative control^1 71.9 143.8 287.5 575.0 1150.0 Positive control^2	- - - - - - -	n.d. - - - - - n.d.	5.97 1.07 3.55 2.51 2.72 1.80 178.95^s	100.0 95.3 107.2 120.8 101.7 93.4 70.1	100.0 146.9 106.3 112.9 115.4 105.0 83.6
2	4	Negative control^1 100.0 200.0 400.0 800.0 1150.0 Positive control^2	- - - - - - -	n.d. - - - - - n.d.	3.46 n.c.^1 0.29 1.34 1.20 2.66 125.48^s	100.0 99.6 93.6 92.7 96.6 82.4 75.2	100.0 n.c.^1 106.9 117.0 105.0 106.3 82.7
1	4	Negative control^1 71.9 143.8 287.5 575.0 1150.0 Positive control^3	+ + + + + + +	n.d. - - - - - n.d.	2.28 1.71 0.74 0.00 1.21 3.97 162.80^s	100.0 85.6 109.3 116.2 122.8 100.6 88.2	100.0 95.1 87.6 89.9 80.5 82.1 67.4
2	4	Negative control^1 100.0 200.0 400.0 800.0 1150.0 Positive control^3	+ + + + + + +	n.d. - - - - - n.d.	4.57 n.c.^1 6.95 3.91 0.30 3.17 102.70^s	100.0 108.0 100.4 98.0 92.2 92.5 94.7	100.0 n.c.^1 100.9 93.6 101.5 96.0 67.7

*: Macroscopically visible precipitation in culture medium at the end of exposure period

**: Mutant frequency MF_corr.: mutant colonies per 10⁶cells corrected with the CE₂value

***: Cloning efficiency related to the respective vehicle control

^s: Mutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)

n.c.¹: Culture was not continued since a minimum of only four analyzable concentrations is required

n.d.: Not determined

¹: Medium

²: EMS 400 µg/mL

³: DMBA 1.25 µg/mL

Conclusions:

In the absence and the presence of metabolic activation, the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and ß-naphthoflavone induced rats (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses the top concentration was determined to be the limit concentration of 10 mM (1150 µg/mL). In the main experiments the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:

1st Experiment

without S9 mix

0; 71.9; 143.8; 287.5; 575.0; 1150.0 µg/mL

with S9 mix

0; 71.9; 143.8; 287.5; 575.0; 1150.0 µg/mL

2nd Experiment

without S9 mix

0; 100.0; 200.0; 400.0; 800.0; 1150.0 µg/mL

with S9 mix

0; 100.0; 200.0; 400.0; 800.0; 1150.0 µg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6 -thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12 -dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

In this study, in both experiments in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations evaluated for gene mutations.

Based on the results of the present study, the test substance did not cause any biologically relevant or dose-dependent increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The positive result in the in-vitro study would normally require a testing proposal for an in-vivo study. In this case, the substance is handled at an industrial site under strictly controlled conditions. Therefore, no further testing is proposed.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames Test

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.

Micronucleus Test

An in vitro mammalian cell micronucleus test was performed with and without metabolic activation (Aroclor induced S9 fraction) using human lymphoblastoid cells (TK6).

To provide an accurate method of quantifying the effect on cell proliferation and the cytotoxic or cytostatic activity of a treatment and to ensure that only cells that divided during or after treatment are microscopically scored Cytochalasin B is added to the cultures.

The proposed treatment conditions are as follows:

+ S9 short treatment: Cells are exposed for 3 hours. Upon ending of the exposure duration S9 and the test substance are removed and Cytochalasin B added. 27 hours later cells are harvested.

- S9 short treatment: Cells are exposed for 3 hours. Upon ending of the exposure duration the test substance is removed and Cytochalasin B added. 27 hours later cells are harvested.

- S9 extended treatment: Cells are exposed for 30 hours in the presence of Cytochalasin B. After the exposure period cells are harvested.

+ S9 extended treatment: Cells are exposed for 30 hours in the presence of S9 and Cytochalasin B. After the exposure period cells are harvested.

Changes in osmolality and of more than 50 mOsm/kg and fluctuations in pH of more than one unit can give rise to chromosome aberrations (Scott et al., 1991; Brusick, 1986). Therefore, osmolality and the effect of the test article on the pH of the culture medium are assessed during the study. Osmolality and pH are initially measured on post-treatment media in the cytotoxicity Range-Finder Experiment. If marked changes are observed, further measurements may be made in the Micronucleus Experiment(s).

Harvesting: At the defined sampling time, cultures were

- centrifuged at approx. 200 g, 5 minutes

- resuspended in 4 mL (hypotonic) 0.075 M KCl at 37 °C for 4 minutes

- fixed by dropping the KCl suspension into fresh, cold methanol / glacial acetic acid (7:1 v/v), centrifuged (approx. 200 g, 5 minutes) and resuspended, followed by further centrifugation and fixation (approx. 1250 g, 2 - 3 minutes) until the cell pellets are clean

- stored in fixative at 2 - 8 °C prior to slide preparation for a minimum of 3 hours.

Slide preparation:

- cells centrifuged (approx. 1250 g, 2 - 3 minutes) and resuspended in a minimal amount of fresh fixative (if required)

- several drops of suspension gently spread onto multiple clean, dry microscope slides

- slides air-dried then stored protected from light at room temperature prior to staining

- slides stained by immersion in 12.5 µg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 minutes, following by a wash with PBS (with agitation) for a few seconds

- the quality of the stain will be checked. If slides appear over-stained (leaching of orange colouration), slides will be returned to the PBS bath for a further wash and the check repeated

- stained slides will be air-dried and stored protected from light at room temperature prior to analysis

- immediately prior to analysis 1 - 2 drops of PBS will be added to the slides before mounting with glass coverslips.

The test material induced micronuclei in the in vitro assay. As the test material contains 0.3 % of a substance with a potent structural alert, it is possible that the result applies to the impurity rather than the main component.

HPRT

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and ß-naphthoflavone induced rats (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses the top concentration was determined to be the limit concentration of 10 mM (1150 µg/mL). In the main experiments the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:

1st Experiment

without S9 mix

0; 71.9; 143.8; 287.5; 575.0; 1150.0µg/mL

with S9 mix

0; 71.9; 143.8; 287.5; 575.0; 1150.0µg/mL

2nd Experiment

without S9 mix

0;100.0;200.0; 400.0; 800.0; 1150.0µg/mL

with S9 mix

0;100.0;200.0; 400.0; 800.0; 1150.0µg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6 -thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12 -dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

In this study, in both experiments in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations evaluated for gene mutations.

Based on the results of the present study, the test substance did not cause any biologically relevant or dose-dependent increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

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 1272/2008. No indication of genotoxicity was observed in the Ames Test (OECD 471, GLP) and in the HPRT Test (OECD 476, GLP). As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the seventh time in Regulation (EC) No. 2015/1221.