Reaction products of cyclohexanone with buta-1,3-diene and hydrogen peroxide and iron sulphate and methanol, hydrolysed, ammonium salts

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 start and completion dates: 5 January 2021 to 22 February 2021.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

To generate hazard information for the test substance, UB20-LDB(50%), in accordance with the REACH Regulation (Annex VII).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", 21 July 1997 (as corrected in 2020)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Method B13/14 of Commission Regulation (EC) number 440/2008 of May 2008.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

USA, EPA, OCSPP harmonized guideline 870.5100 – Bacterial Reverse Mutation Test, August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) guideline adopted June 2012 (ICH S2(r1) Federal Register. Adopted 2012;77:33748-33749

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethane-1,2-diol

EC Number:

203-473-3

EC Name:

Ethane-1,2-diol

Cas Number:

107-21-1

Molecular formula:

C2H6O2

IUPAC Name:

ethane-1,2-diol

Constituent 2

Reference substance name:

Reaction products of cyclohexanone with buta-1,3-diene and hydrogen peroxide and iron sulphate and methanol, hydrolysed, ammonium salts

EC Number:

854-559-8

Cas Number:

2382963-35-9

Molecular formula:

Not applicable for a UVCB substance

IUPAC Name:

Reaction products of cyclohexanone with buta-1,3-diene and hydrogen peroxide and iron sulphate and methanol, hydrolysed, ammonium salts

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): Sponsor: Suzuki Techno Commercial Corporation (CoA)
- Lot/batch number of test material: 021015
- Purity: 50% (Aliphatic Carboxylic acids(UB-20)ammonium salts: 50wt% and ethylene glycol: 50wt%). Formulated concentrations were adjusted to account for the stated impurity content (50%) of the test item.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately +4 °C, in the dark
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Assumed stable for the duration of the study
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Fully miscible in sterile distilled water at 50 mg/mL. Assumed stable for the duration of the study.
- Reactivity of the test material with the incubation material used (e.g. plastic ware): None

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: None

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Phenobarbitone / β-Naphthoflavone induced S9 Microsomal fractions (Sprague-Dawley). Rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

- source of S9: Moltox; Lot No.’s 4272 (Experiment 1) and 4217 (Experiment 2)

- method of preparation of S9 mix: S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test
S9 fraction: 5.0 mL
1.65 M KCl/0.4 M MgCl2: 1.0 mL
0.1 M Glucose-6-phosphate: 2.5 mL
0.1 M NADP: 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 mL
Sterile distilled water: 14.5 mL

- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix /plate (Final concentration: 10% liver S9 in standard co-factors)

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Sterility controls were conducted for top agar and histidine/biotin or tryptophan in the presence of S9-mix (in triplicate) for both experiments.

Test concentrations with justification for top dose:

Maximum concentration 5000 μg/plate, the OECD TG 471 maximum recommended dose level.

Vehicle / solvent:

- Solvent used: Sterile distilled water
- Justification for choice of solvent/vehicle: Test item fully miscible in sterile distilled water at 50 mg/mL following in-house solubility checks. Sterile distilled water therefore selected as the solvent of choice.

- Justification for percentage of solvent in the final culture medium: 0.1 mL aliquot of test item formulation added. Volumes of solvent when plating out up to 400 µL (0.4 mL) are acceptable in this assay.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

+S9 (TA100, TA1535, WP2uvrA, TA1537)

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

+S9 (TA 98)

Controls 3

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

-S9 (TA100, TA1535, WP2uvrA)

Controls 4

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

-S9 (TA98)

Controls 5

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

-S9 (TA1537)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : Two (Experiment 1: Plate incorporation method, Experiment 2: Pre-incubation method).

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.9 to 9 x 10-9 bacteria per mL.
- Test substance added in sterile distilled water (solvent) to agar (plate incorporation and preincubation method).

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 37 ± 3°C for 20 minutes (with shaking)
- Exposure duration/duration of treatment: 37 ± 3°C for between 48 and 72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
- Any supplementary information relevant to cytotoxicity: None, a lack of cytotoxicity was observed at 5000 μg/plate.

METHODS FOR MEASUREMENTS OF GENOTOXICIY: Presence of revertant colonies using an automated colony counting system.

Rationale for test conditions:

The test item was fully miscible in sterile distilled water at 50 mg/mL. In experiment 1, eight test item concentrations (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed for each tester strain, using the direct plate incorporation method. As no cytotoxicity was observed across the concentration range, a second experiment using a pre-incubation method was performed at six concentrations (15, 50, 150, 500, 1500 and 5000 μg/plate) to achieve at least four non-toxic level and test up to the limit test concentration. All test concentrations, the solvent control and positive controls were prepared in triplicate.

Evaluation criteria:

Any, one, or all of the following can be used to determine the overall result of the study:

A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
A reproducible increase at one or more concentrations.
A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments give clear positive or negative results, in some instances the data generated prohibit making a definite judgment about test item activity. Results of this type are reported as equivocal.

Statistics:

Statistical significance was not included as part of the result evaluation.

Results and discussion

Test results

Test results 1

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

Positive controls validity:

valid

Remarks:

9AA (-S9), 2AA (+S9)

Test results 2

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

Positive controls validity:

valid

Remarks:

4NQO (-S9), BP (+S9)

Test results 3

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

Positive controls validity:

valid

Remarks:

EENG (-S9), 2AA (+S9)

Test results 4

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

Positive controls validity:

valid

Remarks:

EENG (-S9), 2AA (+S9)

Test results 5

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

Positive controls validity:

valid

Remarks:

ENNG (-S9), 2AA (+S9)

Any other information on results incl. tables

Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From:25 January 2021 29 January 2021†					To: 28 January 2021 01 February 2021†
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA†			TA98		TA1537
	Solvent Control (Water)	143 123 140	(135) 10.8#	10 12 14	(12) 2.0	30 24 26		(27) 3.1	25 14 16	(18) 5.9	6 7 11	(8) 2.6
	1.5 µg	158 127 126	(137) 18.2	6 12 16	(11) 5.0	26 17 24		(22) 4.7	22 24 21	(22) 1.5	12 14 15	(14) 1.5
	5 µg	129 128 144	(134) 9.0	14 14 13	(14) 0.6	29 27 26		(27) 1.5	28 16 17	(20) 6.7	12 10 13	(12) 1.5
	15 µg	136 156 125	(139) 15.7	20 10 28	(19) 9.0	18 18 19		(18) 0.6	20 22 21	(21) 1.0	12 12 10	(11) 1.2
	50 µg	152 149 143	(148) 4.6	12 11 21	(15) 5.5	21 16 14		(17) 3.6	16 18 16	(17) 1.2	13 9 15	(12) 3.1
	150 µg	120 149 105	(125) 22.4	19 15 13	(16) 3.1	24 28 21		(24) 3.5	24 18 19	(20) 3.2	11 15 7	(11) 4.0
	500 µg	143 128 132	(134) 7.8	7 11 13	(10) 3.1	22 20 22		(21) 1.2	29 18 20	(22) 5.9	19 5 13	(12) 7.0
	1500 µg	129 136 132	(132) 3.5	6 13 13	(11) 4.0	25 21 20		(22) 2.6	17 16 19	(17) 1.5	14 11 4	(10) 5.1
	5000 µg	112 126 123	(120) 7.4	14 13 11	(13) 1.5	24 10 33		(22) 11.6	20 26 11	(19) 7.5	9 10 6	(8) 2.1
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	500 478 503	(494) 13.7	512 556 652	(573) 71.6	738 743 682		(721) 33.9	154 142 169	(155) 13.5	527 121 358	(335) 203.9

†: Experimental procedure repeated at a later date due to no bacterial growth in the original test
ENNG:  N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO:  4-Nitroquinoline-1-oxide:
9AA: 9-Aminoacridine
#:  Standard deviation

Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From:25 January 2021 29 January 2021†					To: 28 January 2021 01 February 2021†
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA†			TA98		TA1537
	Solvent Control (Water)	147 183 169	(166) 18.1#	13 11 16	(13) 2.5	26 22 35		(28) 6.7	25 25 26	(25) 0.6	16 10 13	(13) 3.0
	1.5 µg	166 169 149	(161) 10.8	17 2 12	(10) 7.6	15 28 17		(20) 7.0	21 41 27	(30) 10.3	11 14 8	(11) 3.0
	5 µg	159 135 175	(156) 20.1	8 13 10	(10) 2.5	23 19 27		(23) 4.0	21 22 30	(24) 4.9	14 13 19	(15) 3.2
	15 µg	166 171 163	(167) 4.0	11 13 12	(12) 1.0	16 25 24		(22) 4.9	35 20 32	(29) 7.9	9 18 14	(14) 4.5
	50 µg	152 173 170	(165) 11.4	17 16 17	(17) 0.6	30 18 18		(22) 6.9	21 29 25	(25) 4.0	8 14 7	(10) 3.8
	150 µg	170 145 166	(160) 13.4	10 8 8	(9) 1.2	30 17 16		(21) 7.8	25 19 26	(23) 3.8	12 11 14	(12) 1.5
	500 µg	152 169 147	(156) 11.5	8 13 9	(10) 2.6	24 21 36		(27) 7.9	22 19 25	(22) 3.0	9 7 10	(9) 1.5
	1500 µg	147 137 119	(134) 14.2	6 12 8	(9) 3.1	25 30 20		(25) 5.0	22 22 19	(21) 1.7	8 8 3	(6) 2.9
	5000 µg	123 119 102	(115) 11.2	15 7 11	(11) 4.0	24 16 36		(25) 10.1	19 26 19	(21) 4.0	8 10 11	(10) 1.5
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	1916 1944 2023	(1961) 55.5	285 247 266	(266) 19.0	205 192 221		(206) 14.5	158 170 194	(174) 18.3	219 203 230	(217) 13.6

†:  Experimental procedure repeated at a later date due to no bacterial growth in the original test
BP:  Benzo(a)pyrene
2AA:  2-Aminoanthracene
#:  Standard deviation

Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From:18 February 2021					To: 21 February 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	126 128 129	(128) 1.5#	18 12 18	(16) 3.5	19 17 23		(20) 3.1	21 17 13	(17) 4.0	21 12 20	(18) 4.9
	15 µg	135 129 127	(130) 4.2	14 20 13	(16) 3.8	17 28 31		(25) 7.4	19 14 25	(19) 5.5	12 17 19	(16) 3.6
	50 µg	121 118 114	(118) 3.5	16 14 16	(15) 1.2	17 20 19		(19) 1.5	21 17 25	(21) 4.0	14 20 16	(17) 3.1
	150 µg	132 130 118	(127) 7.6	18 8 9	(12) 5.5	37 21 17		(25) 10.6	23 21 22	(22) 1.0	14 19 13	(15) 3.2
	500 µg	118 144 145	(136) 15.3	18 17 17	(17) 0.6	15 18 15		(16) 1.7	13 15 20	(16) 3.6	14 16 18	(16) 2.0
	1500 µg	141 118 113	(124) 14.9	9 11 15	(12) 3.1	24 19 22		(22) 2.5	15 16 21	(17) 3.2	16 11 7	(11) 4.5
	5000 µg	107 103 97	(102) 5.0	23 13 21	(19) 5.3	19 14 14		(16) 2.9	14 19 20	(18) 3.2	14 7 11	(11) 3.5
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	787 807 814	(803) 14.0	2000 1737 1829	(1855) 133.5	1099 894 896		(963) 117.8	325 303 288	(305) 18.6	866 711 654	(744) 109.7

ENNG:  N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO:  4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
#: Standard deviation

Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period		From:18 February 2021					To: 21 February 2021
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	131 130 126	(129) 2.6#	11 7 12	(10) 2.6	17 17 17		(17) 0.0	33 32 27	(31) 3.2	12 18 21	(17) 4.6
	15 µg	131 130 128	(130) 1.5	13 8 6	(9) 3.6	31 26 19		(25) 6.0	19 21 24	(21) 2.5	17 13 14	(15) 2.1
	50 µg	128 120 132	(127) 6.1	12 10 15	(12) 2.5	23 11 14		(16) 6.2	27 26 24	(26) 1.5	12 15 15	(14) 1.7
	150 µg	145 111 128	(128) 17.0	12 14 8	(11) 3.1	21 19 28		(23) 4.7	29 21 27	(26) 4.2	9 19 19	(16) 5.8
	500 µg	144 137 136	(139) 4.4	10 18 12	(13) 4.2	19 22 14		(18) 4.0	27 24 24	(25) 1.7	11 11 8	(10) 1.7
	1500 µg	126 103 102	(110) 13.6	9 15 12	(12) 3.0	15 32 15		(21) 9.8	23 20 29	(24) 4.6	10 7 8	(8) 1.5
	5000 µg	88 84 98	(90) 7.2	19 5 9	(11) 7.2	23 16 24		(21) 4.4	21 26 17	(21) 4.5	13 13 11	(12) 1.2
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	2425 2344 2318	(2362) 55.8	330 337 353	(340) 11.8	326 330 324		(327) 3.1	112 105 115	(111) 5.1	546 515 534	(532) 15.6

BP:  Benzo(a)pyrene
2AA: 2-Aminoanthracene
#:  Standard deviation

 

Applicant's summary and conclusion

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item UB20-LDB(50%) did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test UB20-LDB(50%) was considered to be non-mutagenic.

In accordance with EU CLP Regulation (EC) No 1272/2008 UN GHS) further testing in mammalian cells is required to confirm no classification for mutagenicity.

Executive summary:

In a OECD 471 "Ames Test" performed to GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with UB20-LDB(50%) in two independent tests (a plate incorporation method and the pre-incubation method). Plates were prepared in triplicate with and without S9 fraction. Negative and positive control were also used. The concentration range in experiment 1 was 1.5 to 5000 μg/plate (8 doses). As the result was negative with no observed cytotoxicity, experiment 2 was performed using a modified dose range of 15 to 5000 μg/plate (6 doses). In both experiments there was no notable reduction in bacterial background lawn at any dose level, either in the presence or in the absence of S9-mix. The negative control was within or close to the historical range and the positive control showed amarked increases in revertant colonies.and positive controls. Under the conditions of this study, the test item, UB20-LDB(50%) was considered to be non-mutagenic.