1-hydroxyoctan-2-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro studies have been conducted to assess the genotoxicity of 1 -hydroxyoctan-2 -one: two OECD 471 Ames tests (conducted at two different test laboratories) and an OECD 490 L5178Y tk+/- Mouse Lymphoma Assay.

Ames Test 1 (Envigo, 2019)

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD 471 Test Guideline and was 1.5 to 5000 μg/plate. As Experiment 1 was considered to be weakly positive the plate incorporation method was again employed for Experiment 2 in the presence and absence of metabolic activation (S9-mix) to confirm initial findings. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations with an amended dose range employed of 150, 250, 500, 1000, 1500, 2000, 3000 and 5000 μg/plate. Eight test item concentrations per bacterial strain were selected in Experiment 2 in an effort to achieve both reproducibility and a better dose-related response.

A third (confirmatory) experiment was performed in TA100 (without metabolic activation) using plate incorporation methodology following the results from Experiments 1 and 2. The dose range was 750, 1000, 1500, 2000 and 3000 μg/plate. As for Experiment 2, intermediate dose levels were selected in the confirmatory experiment in an effort to confirm and potentially enhance the mutagenic responses observed in Experiments 1 and 2.

Based on the results of this test it was concluded that the test item did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium and one tryptophan-requiring strain (WP2uvrA) Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory test guidelines and a toxic concentration) in the absence and in the presence of a rat liver metabolic activation system (S9).

Ames Test 2 (Covance, 2019)

1 -hydroxyoctan-2 -one was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) ofSalmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9).

Following 1 -hydroxyoctan-2 -one treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any mutagenic activity of the test item in this assay system.

It was concluded that 1 -hydroxyoctan-2 -one did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) ofSalmonella typhimuriumwhen tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory test guidelines and a toxic concentration) in the absence and in the presence of a rat liver metabolic activation system (S-9).

MLA (Envigo, 2019)

The test item 1 -hydroxyoctan-2-one was subjected to testing in accordance with OECD 490 to assay the ability to induce mutation at thetklocus (5-trifluorothymidine [TFT] resistance) in mouse lymphoma cells using a fluctuation protocol.

Based on the results of this study it is concluded that 1 -hydroxyoctan-2 -one did not induce mutation at thetklocus of L5178Y mouse lymphoma cells when tested up to the limit of toxicity in the absence and presence (4 hour exposure) of a rat liver metabolic activation system (S9) following a single mutation experiment.

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:

Feb-April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This is a Klimisch 1 rated OECD 471 guideline study conducted on the registered substance 1-hydroxyoctan-2-one in accordance GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

batch number 1203/16/100
Purity: 96.7%
Referred to in test report as EXPINN PC17032

Target gene:

Histidine (in S. Tryphimurium)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with Aroclor 1254. The S-9 was supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-20°C, and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P 450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).

Test concentrations with justification for top dose:

Calculations of all test article concentrations included a correction factor of 1.34, applied based on impurity content. Retrospectively, it was noted that the correct correction factor should be 1.034. Therefore, for accuracy all concentrations are expressed in terms of actual test article administered into the test system. The highest concentration tested was 6700 μg/plate, which is above the maximum concentration recommended for this assay of 5000 μg/plate. This does not affect the scientific integrity or interpretation of the study.

Experiment 1 treatments of all the tester strains were performed using final concentrations of test item at 6.7, 21.44, 67, 214.4, 670, 2144 and 6700 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity was observed at 6700 µg/plate in all strains in the absence and presence of S-9 except strain TA102 in the absence of S-9 where toxicity was observed at 2144 µg/plate and above.

Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 6700 µg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 214.4-6700 µg/plate, in order to examine more closely those concentrations of test item approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, evidence of toxicity was observed at 3350and/or 6700 µg/plate in all strains except TA100 in the absence of S 9, and at 1675 µg/plate and above in all strains in the presence of S-9.

Vehicle / solvent:

All test item treatments in this study were performed using formulations prepared in anhydrous analytical grade dimethyl sulphoxide (DMSO).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

Five strains of Salmonella typhimurium bacteria (TA98, TA100, TA1535, TA1537 and TA102) were used in this study. Strains TA98, TA1535 and TA1537 were originally obtained from the UK NCTC. Strains TA100 and TA102 were derived from cultures originally obtained from Covance Laboratories Inc., USA. For all assays, bacteria were cultured at 37±1°C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate, to provide bacterial cultures in the range of approximately 108 to 109 cells/mL, based on cell density assessments for each culture. Incubation was carried out with shaking in an anhydric incubator, set to turn on using a timer switch. All treatments were completed within 1.5 hours of the end of the incubation period.

The inocula were taken from master plates or vials of frozen cultures, which had been checked for strain characteristics (histidine dependence, rfa character, uvrB character, if applicable and resistance to ampicillin or ampicillin plus tetracycline).

Evaluation criteria:

Acceptance Criteria

The assay was considered valid if all the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges as defined in ATTACHMENTS
2. The positive control chemicals induced increases in revertant numbers of ≥1.5 fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control confirming discrimination between different strains, and an active S-9 preparation.

Evaluation Criteria

For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
2. Any observed response was reproducible.

The test article was considered positive in this assay if both of the above criteria were met.

The test article was considered negative in this assay if neither of the above criteria were met.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at top two dose concentrations with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at top two dose concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at top two dose concentrations with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at top two dose concentrations with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at top two dose concentrations with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of EXPINN PC17032 at 6.7, 21.44, 67, 214.4, 670, 2144 and 6700 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity in the form of a slight thinning of the background bacterial lawn was observed at 6700 µg/plate in all strains in the absence and presence of S-9 except strain TA102 in the absence of S-9, where a marked reduction in revertant numbers was observed at 2144 µg/plate and a complete killing of the test bacteria was observed at 6700 µg/plate.

Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 6700 µg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 214.4-6700 µg/plate, in order to examine more closely those concentrations of EXPINN PC17032 approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, evidence of toxicity ranging from a slight thinning of the background bacterial lawn to a marked reduction in revertant numbers was observed at 3350 and/or 6700 µg/plate in all strains except strain TA100 in the absence of S-9. Toxicity ranging from a slight thinning of the background bacterial lawn to a complete killing of the test bacteria was observed at 1675 µg/plate and above in all strains in the presence of S-9.

As less than 5 analysable concentrations were available in Experiment 2 due to complete toxicity observed at 3350 µg/plate and above in all strains in the presence of S 9, an additional experiment was conducted using the pre-incubation method at a concentration range of 67-6700 µg/plate. Following these treatments, evidence of toxicity ranging from a slight thinning of the background bacterial lawn, with or without a concurrent marked reduction in revertant numbers, to a complete killing of the test bacteria was observed at 1675 µg/plate and above in all strains.

Precipitation was observed at 6700 µg/plate in all strains in the absence and presence of S-9 in Experiment 1.

Data Acceptibility and Validity
The individual mutagenicity plate counts were averaged to give mean values. Vehicle control counts fell within the laboratory’s historical ranges with the exception of two vehicle control counts in strain TA100 in the presence of S-9 in Experiment 2. These counts were slightly above, but sufficiently close, to the historical control range and were comparable to the vehicle control replicate counts and the laboratory historical control range, and therefore accepted as characteristic and valid. The positive control chemicals all induced increases in revertant numbers of ≥1.5-fold (in strain TA102), ≥2 fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle controls confirming discrimination between different strains, and an active S-9 preparation. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.

Retrospectively, it was noted that the correct correction factor should be 1.034. Therefore, for accuracy all concentrations are expressed in terms of actual test article administered into the test system. The highest concentration tested was 6700 μg/plate, which is above the maximum concentration recommended for this assay of 5000 μg/plate. This does not affect the scientific integrity or interpretation of the study.

Mutation
Following test item treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any mutagenic activity of the test item in this assay system.

Raw Plate Counts and Calculated Mutagenicity Data, Experiment 1, ‑S‑9

Strain	Compound	Conc. Level (µg/plate)		Mean	Standard Deviation	Fold Increase			Revertant Numbers Per Plate
TA98	DMSO	-	24.0		5.3		-	26, 28, 18
	EXPINN PC17032	6.7	20.3		1.5		0.8	22, 20, 19
		21.44	22.7		0.6		0.9	22, 23, 23
		67	22.0		6.1		0.9	18, 29, 19
		214.4	17.0		2.0		0.7	15, 19, 17
		670	19.0		2.0		0.8	17, 19, 21
		2144	15.7		0.6		0.7	15, 16, 16
		6700	5.7		4.6		0.2	11 S P, 3 S P, 3 S P
	2NF	5	897.3		67.4		37.4	841, 879, 972
TA100	DMSO	-	119.7		12.0		-	132 M B, 108, 119
	EXPINN PC17032	6.7	113.0		13.7		0.9	98, 116, 125
		21.44	114.0		11.8		1.0	117, 124, 101
		67	114.3		15.6		1.0	129, 116, 98
		214.4	118.3		6.7		1.0	126, 114, 115
		670	126.3		2.1		1.1	124, 128, 127
		2144	159.3		14.8		1.3	163, 143, 172
		6700	181.7		26.6		1.5	207 S P, 184 S P, 154 S P
	NaN3	2	1318.3		19.2		11.0	1301, 1339, 1315
TA1535	DMSO	-	15.0		1.7		-	16, 16, 13
	EXPINN PC17032	6.7	19.0		1.7		1.3	20, 17, 20
		21.44	13.3		0.6		0.9	14, 13, 13
		67	13.3		7.0		0.9	20, 6, 14
		214.4	11.3		4.5		0.8	11, 7, 16
		670	15.3		1.2		1.0	14, 16, 16
		2144	18.7		4.7		1.2	15, 24, 17
		6700	9.7		4.0		0.6	14 S P, 9 S P, 6 M B S P
	NaN3	2	1020.7		60.0		68.0	962, 1018, 1082
TA1537	DMSO	-	12.7		3.1		-	12 M B, 16, 10
	EXPINN PC17032	6.7	12.0		4.4		0.9	9, 17, 10
		21.44	16.0		3.0		1.3	19, 13, 16 M B
		67	15.3		6.4		1.2	19, 8, 19
		214.4	21.0		5.3		1.7	25, 15, 23
		670	12.3		2.1		1.0	10, 13, 14
		2144	11.0		3.0		0.9	11, 8, 14
		6700	2.7		1.5		0.2	4 S P, 3 S P, 1 M B S P
	AAC	50	1076.0		113.0		84.9	1078, 1188, 962
TA102	DMSO	-	289.3		10.0		-	278, 297, 293
	EXPINN PC17032	6.7	276.0		14.0		1.0	270, 292, 266
		21.44	299.3		4.2		1.0	296, 298, 304
		67	284.7		8.1		1.0	280, 280, 294
		214.4	283.3		15.2		1.0	286, 297, 267
		670	279.7		12.5		1.0	280, 267, 292
		2144	154.7		27.3		0.5	130, 184, 150
		6700	-		-		-	- T P, - T P, - T P
	MMC	0.2	989.0		18.0		3.4	1009, 984, 974

 

Raw Plate Counts and Calculated Mutagenicity Data, Experiment 1, +S‑9

Strain	Compound	Conc. Level (µg/plate)	Mean	Standard Deviation	Fold Increase	Revertant Numbers Per Plate
TA98	DMSO	-	37.0	1.7	-	36 M B, 36, 39
	EXPINN PC17032	6.7	37.3	6.8	1.0	32, 35, 45
		21.44	32.7	4.6	0.9	38, 30, 30
		67	40.7	5.7	1.1	36, 39, 47
		214.4	38.0	6.6	1.0	31, 39, 44
		670	34.0	2.0	0.9	34, 36, 32
		2144	34.3	5.0	0.9	39, 29, 35
		6700	21.0	2.0	0.6	21 S P, 19 S P, 23 S P
	B[a]P	10	393.0	28.1	10.6	366, 422, 391
TA100	DMSO	-	138.7	14.0	-	150, 143, 123
	EXPINN PC17032	6.7	141.0	3.6	1.0	144, 137, 142
		21.44	127.3	11.2	0.9	137, 115, 130
		67	136.3	18.8	1.0	126, 125, 158
		214.4	133.3	9.1	1.0	123, 140, 137
		670	139.0	5.6	1.0	144, 133, 140
		2144	142.7	10.6	1.0	133, 141, 154
		6700	150.3	11.1	1.1	162 S P, 149 S P, 140 S P
	AAN	5	3468.7	112.3	25.0	3460, 3361, 3585
TA1535	DMSO	-	14.3	2.3	-	13 M B, 17, 13
	EXPINN PC17032	6.7	16.0	2.0	1.1	18, 14, 16
		21.44	10.7	3.1	0.7	10, 14, 8
		67	14.0	6.9	1.0	6, 18, 18
		214.4	16.3	2.9	1.1	18, 13, 18
		670	15.7	4.0	1.1	11, 18, 18
		2144	18.3	3.8	1.3	20, 21, 14
		6700	10.0	1.0	0.7	11 S P, 9 S P, 10 M B S P
	AAN	5	386.0	31.5	26.9	395, 412, 351
TA1537	DMSO	-	21.0	2.6	-	18, 22, 23
	EXPINN PC17032	6.7	29.0	9.2	1.4	39, 21, 27
		21.44	25.3	4.0	1.2	21, 26, 29
		67	24.0	6.6	1.1	23, 18, 31
		214.4	25.3	1.5	1.2	27, 25, 24
		670	23.3	5.1	1.1	19, 29, 22
		2144	24.0	7.0	1.1	29, 27, 16
		6700	9.7	1.2	0.5	9 S P, 11 S P, 9 S P
	AAN	5	322.3	9.3	15.3	312, 325, 330
TA102	DMSO	-	306.0	35.8	-	273, 301, 344
	EXPINN PC17032	6.7	355.3	24.8	1.2	384, 341, 341
		21.44	359.3	4.6	1.2	354, 362, 362
		67	383.0	16.8	1.3	370, 377, 402
		214.4	377.7	13.7	1.2	363, 380, 390
		670	381.3	16.3	1.2	374, 400, 370
		2144	288.7	19.1	0.9	283, 310, 273
		6700	18.3	9.5	0.1	18 M P V, 28 M P V, 9 M P V
	AAN	20	3919.7	761.0	12.8	4492, 4211, 3056

Raw Plate Counts and Calculated Mutagenicity Data, Experiment 2, ‑S‑9

Strain	Compound	Conc. Level (µg/plate)		Mean	Standard Deviation	Fold Increase			Revertant Numbers Per Plate
TA98	DMSO	-	15.7		5.5		-	10, 21, 16
	EXPINN PC17032	214.4	19.0		4.4		1.2	16, 17, 24
		402	19.3		3.8		1.2	21, 15, 22
		837.5	17.3		4.0		1.1	21, 18, 13
		1675	16.0		1.7		1.0	17, 14, 17
		3350	16.0		7.0		1.0	23, 16, 9
		6700	5.3		2.1		0.3	7 S, 3 S, 6 S
	2NF	5	1131.3		67.6		72.2	1179, 1161, 1054
TA100	DMSO	-	114.3		12.2		-	117, 101, 125
	EXPINN PC17032	214.4	129.3		18.3		1.1	115, 123, 150
		402	118.3		29.1		1.0	115, 91, 149
		837.5	112.3		19.3		1.0	124, 123, 90
		1675	119.7		10.7		1.0	132, 113, 114
		3350	146.3		14.4		1.3	163, 138, 138
		6700	134.7		5.0		1.2	134, 140, 130
	NaN3	2	1007.7		49.6		8.8	999, 963, 1061
TA1535	DMSO	-	17.3		1.5		-	19, 17, 16
	EXPINN PC17032	214.4	19.0		4.4		1.1	16, 24, 17
		402	25.3		5.5		1.5	25, 31 M C, 20
		837.5	21.0		2.6		1.2	23, 22, 18
		1675	19.3		7.1		1.1	27, 18, 13
		3350	16.3		2.5		0.9	19, 16, 14
		6700	5.7		1.2		0.3	5, 7, 5
	NaN3	2	649.7		13.3		37.5	661, 653, 635
TA1537	DMSO	-	8.7		4.6		-	6, 14, 6
	EXPINN PC17032	214.4	9.0		2.0		1.0	9, 11, 7
		402	14.3		2.9		1.7	11, 16, 16
		837.5	11.7		6.7		1.3	10, 19, 6
		1675	9.0		1.7		1.0	11, 8, 8
		3350	8.0		1.0		0.9	9, 8, 7
		6700	3.3		2.3		0.4	6, 2, 2
	AAC	50	343.7		70.3		39.7	397, 370, 264
TA102	DMSO	-	264.0		3.5		-	260, 266, 266
	EXPINN PC17032	214.4	262.0		24.6		1.0	241, 256, 289
		402	289.3		17.2		1.1	271, 305, 292
		837.5	272.7		32.0		1.0	305, 272, 241
		1675	194.0		28.6		0.7	209, 212, 161
		3350	65.0		5.0		0.2	70 S, 65 S, 60 S
		6700	-		-		-	- T, - T, - T
	MMC	0.2	948.3		121.6		3.6	1088, 891, 866

 

Raw Plate Counts and Calculated Mutagenicity Data, Experiment 2, +S‑9

Strain	Compound	Conc. Level (µg/plate)		Mean	Standard Deviation	Fold Increase			Revertant Numbers Per Plate
TA98	DMSO	-	27.0		1.7		-	25, 28, 28
	EXPINN PC17032	214.4	36.7		11.9		1.4	50, 33, 27
		402	34.7		1.2		1.3	34, 34, 36
		837.5	29.7		5.5		1.1	35, 24, 30
		1675	27.7		10.5		1.0	17 S, 28 S, 38 M B S
		3350	-		-		-	- T, - T, - T
		6700	-		-		-	- T, - T, - T
	B[a]P	10	404.3		2.1		15.0	405, 402, 406
TA100	DMSO	-	158.0		33.4		-	186, 121, 167
	EXPINN PC17032	214.4	153.0		6.9		1.0	145, 157, 157
		402	138.3		8.5		0.9	135, 132, 148
		837.5	146.7		19.1		0.9	131, 168, 141
		1675	104.7		3.5		0.7	108 S, 101 S, 105 S
		3350	-		-		-	- T, - T, - T
		6700	-		-		-	- T, - T, - T
	AAN	5	2689.0		132.2		17.0	2745, 2538, 2784
TA1535	DMSO	-	27.0		11.4		-	32, 14, 35
	EXPINN PC17032	214.4	17.3		1.5		0.6	16, 19, 17
		402	15.0		4.4		0.6	10, 17, 18
		837.5	17.3		5.1		0.6	23, 16, 13
		1675	17.7		3.5		0.7	14 S, 21 S, 18 S
		3350	-		-		-	- T, - T, - T
		6700	-		-		-	- T, - T, - T
	AAN	5	272.0		40.6		10.1	280, 228, 308
TA1537	DMSO	-	14.0		7.5		-	6, 21, 15
	EXPINN PC17032	214.4	18.3		2.1		1.3	19, 20, 16
		402	19.7		6.0		1.4	26, 19, 14
		837.5	12.0		2.6		0.9	11, 10, 15
		1675	9.0		0.0		0.6	9 S, 9 S, 9 S
		3350	-		-		-	- T, - T, - T
		6700	-		-		-	- T, - T, - T
	AAN	5	295.3		12.2		21.1	298, 282, 306
TA102	DMSO	-	316.7		22.0		-	295, 339, 316
	EXPINN PC17032	214.4	391.0		19.5		1.2	406, 398, 369
		402	361.3		13.4		1.1	367, 346, 371
		837.5	304.0		38.2		1.0	260, 324, 328
		1675	195.7		25.7		0.6	172 S, 223 S, 192 S
		3350	-		-		-	- T, - T, - T
		6700	-		-		-	- T, - T, - T
	AAN	20	1750.0		185.1		5.5	1939, 1742, 1569

 

Raw Plate Counts and Calculated Mutagenicity Data, Experiment 3, +S‑9

Strain	Compound	Conc. Level (µg/plate)		Mean	Standard Deviation	Fold Increase			Revertant Numbers Per Plate
TA98	DMSO	-	45.0		3.5		-	47, 47, 41
	EXPINN PC17032	67	36.3		7.2		0.8	40, 41, 28
		134	41.3		9.1		0.9	45, 48, 31
		214.4	42.0		7.0		0.9	35, 49, 42
		402	42.0		2.6		0.9	45, 41, 40
		837.5	47.0		6.0		1.0	41, 53, 47
		1675	26.0		2.6		0.6	29 S, 24 S, 25 S
		3350	-		-		-	- T, - T, - T
		6700	-		-		-	- T, - T, - T
	B[a]P	10	352.0		8.7		7.8	342, 356, 358
TA100	DMSO	-	128.0		6.1		-	131, 121, 132
	EXPINN PC17032	67	129.0		4.0		1.0	129, 125 M B, 133
		134	131.7		21.2		1.0	108, 138, 149
		214.4	119.0		13.1		0.9	105, 131, 121
		402	134.3		14.2		1.0	143, 118, 142
		837.5	127.0		14.5		1.0	126, 142, 113
		1675	92.3		10.0		0.7	93 S, 102 S, 82 S
		3350	-		-		-	- T, - T, - T
		6700	-		-		-	- T, - T, - T
	AAN	5	2713.0		82.9		21.2	2736, 2621, 2782
TA1535	DMSO	-	14.7		6.7		-	13, 22, 9
	EXPINN PC17032	67	9.0		1.7		0.6	10, 7, 10
		134	13.0		3.5		0.9	11, 17, 11
		214.4	15.3		5.9		1.0	11, 22, 13
		402	14.0		6.2		1.0	16, 7, 19
		837.5	14.3		5.1		1.0	10, 20, 13
		1675	9.0		4.6		0.6	5 S, 8 S, 14 S
		3350	-		-		-	- T, - T, - T
		6700	-		-		-	- T, - T, - T
	AAN	5	204.7		19.4		14.0	192, 195, 227
TA1537	DMSO	-	18.3		3.8		-	14, 20, 21
	EXPINN PC17032	67	16.3		5.5		0.9	20, 19, 10
		134	15.3		3.8		0.8	18, 11, 17
		214.4	16.3		3.2		0.9	15, 20, 14
		402	16.0		1.0		0.9	16, 15, 17
		837.5	16.0		4.6		0.9	11, 17, 20
		1675	10.0		4.6		0.5	14 S, 5 S, 11 S
		3350	-		-		-	- T, - T, - T
		6700	-		-		-	- T, - T, - T
	AAN	5	239.7		12.7		13.1	235, 254, 230
TA102	DMSO	-	324.7		14.0		-	338, 326, 310
	EXPINN PC17032	67	314.7		29.4		1.0	328, 281, 335
		134	314.7		29.7		1.0	281, 326, 337
		214.4	242.0		42.6		0.7	231, 289, 206
		402	324.7		9.9		1.0	318, 320, 336
		837.5	312.7		14.6		1.0	329, 301, 308
		1675	-		-		-	- S, - S, - S
		3350	-		-		-	- T, - T, - T
		6700	-		-		-	- T, - T, - T
	AAN	20	2080.3		203.1		6.4	2258, 2124, 1859

Conclusions:

It was concluded that the test item did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 6700 µg/plate (a concentration that exceeded the maximum recommended concentration of 5000 μg/plate, according to current regulatory test guidelines) in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

Following 1 -hydroxyoctan-2 -one treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any mutagenic activity of the test item in this assay system.

It was concluded that 1 -hydroxyoctan-2 -one did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) ofSalmonella typhimuriumwhen tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory test guidelines and a toxic concentration) in the absence and in the presence of a rat liver metabolic activation system (S-9).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

August-Sept 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This is a Klimisch 1 rated OECD 471 guideline study conducted on the registered substance 1-hydroxyoctan-2-one in accordance GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch: 1203/16/100
Purity: 96.7%

Target gene:

Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor

Escherichia coli
Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution

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:

S9 Microsomal fractions (CD Sprague-Dawley) were pre-prepared using standardized in-house procedures

Test concentrations with justification for top dose:

Experiment 1
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Experiment 2
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 150, 250, 500, 1000, 1500, 2000, 3000 and 5000 ug/plate.

Experiment 3
The dose range was determined by the results of Experiments 1 and 2 and was 750, 1000, 1500, 2000 and 3000 µg/plate.

Vehicle / solvent:

The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide (DMSO) at the same concentration in solubility checks performed in house. DMSO was therefore selected as the vehicle in this study

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

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

Without Metabolic Activation
A 0.1 mL aliquot of the appropriate concentration of test item, solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overplayed onto a Vogel Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously (see 3.3.2.2) except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two fold the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 in the number of revertants when compared to the concurrent vehicle control.
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989) may be used in conjunction with the above criteria to further support the result.

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnett’s Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test item induced visible reduction in growth of bacterial background lawns of all Salmonella strains dosed without S9, from 3000 µg/plate (TA100 and TA1537) and at 5000 µg/plate (TA1535 and TA98) and to TA100 and TA1535 at 5000 µg/plate dosed with S9

Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
122		25		25		25		15
115	(123)	14	(19)	18	(21)	40	(35)	13	(14)
132		18		19		39		14

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
119		17		28		31		12
137	(125)	11	(15)	34	(27)	13	(22)	12	(11)
120		17		19		21		9

 

Experiment 3 – Confirmatory Test

 

Number of revertants (mean number of colonies per plate)
Base-pair substitution type
TA100
143
133	(135)
130

Experiment 1 (plate incorporation)

The maximum dose level of the test item in the first experiment was selected as the OECD 471 Test Guideline recommended dose level of 5000 µg/plate. 

In the first mutation test (plate incorporation method), the test item induced a visible reduction in the growth of the bacterial background lawns of all of theSalmonellastrains dosed in the absence of metabolic activation from 1500 µg/plate (TA1537) at 5000 µg/plate (remainingSalmonellastrains) and to TA100 and TA1535 at 5000 µg/plate dosed in the presence of metabolic activation.

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).

In Experiment 1, the test item induced a 2.5-fold increase over the concurrent solvent control in TA100 at 1500 µg/plate in the absence of metabolic activation only.

Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From:03 September 2018					To: 06 September 2018
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	123 121 127	(124) 3.1#	11 16 10	(12) 3.2	33 22 34		(30) 6.7	23 14 23	(20) 5.2	9 11 10	(10) 1.0
	1.5 µg	108 114 116	(113) 4.2	10 7 11	(9) 2.1	21 21 20		(21) 0.6	10 18 20	(16) 5.3	17 18 6	(14) 6.7
	5 µg	110 85 95	(97) 12.6	20 17 15	(17) 2.5	24 13 14		(17) 6.1	19 26 21	(22) 3.6	9 10 8	(9) 1.0
	15 µg	106 96 101	(101) 5.0	14 14 12	(13) 1.2	21 18 22		(20) 2.1	29 27 21	(26) 4.2	20 9 11	(13) 5.9
	50 µg	97 120 93	(103) 14.6	17 14 22	(18) 4.0	24 18 19		(20) 3.2	27 20 13	(20) 7.0	11 13 3	(9) 5.3
	150 µg	121 147 126	(131) 13.8	14 17 22	(18) 4.0	22 11 19		(17) 5.7	21 43 27	(30) 11.4	12 17 13	(14) 2.6
	500 µg	211 200 243	*** (218) 22.3	22 12 16	(17) 5.0	22 14 20		(19) 4.2	30 51 23	(35) 14.6	17 18 24	(20) 3.8
	1500 µg	366 240 313	*** (306) 63.3	21 13 17	(17) 4.0	37 14 20		(24) 11.9	27 17 12	(19) 7.6	17 S 9 S 9 S	(12) 4.6
	5000 µg	156 S 205 S 218 S	** (193) 32.7	6 S 9 S 8 S	(8) 1.5	30 26 35		(30) 4.5	20 S 29 S 35 S	(28) 7.5	0 V 0 V 0 V	(0) 0.0
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		607 733 662	(667) 63.2	978 658 746	(794) 165.3	805 768 784		(786) 18.6	189 226 234	(216) 24.0	218 149 409	(259) 134.7

Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From:03 September 2018					To: 06 September 2018
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	150 128 110	(129) 20.0#	13 16 14	(14) 1.5	35 20 44		(33) 12.1	15 25 11	(17) 7.2	6 11 7	(8) 2.6
	1.5 µg	102 103 103	(103) 0.6	14 11 8	(11) 3.0	20 26 24		(23) 3.1	17 24 19	(20) 3.6	7 12 8	(9) 2.6
	5 µg	124 120 110	(118) 7.2	7 10 16	(11) 4.6	25 25 28		(26) 1.7	20 36 30	* (29) 8.1	5 5 11	(7) 3.5
	15 µg	129 120 113	(121) 8.0	10 15 11	(12) 2.6	38 32 36		(35) 3.1	25 24 24	(24) 0.6	25 10 11	(15) 8.4
	50 µg	122 126 134	(127) 6.1	13 20 10	(14) 5.1	45 32 25		(34) 10.1	24 23 26	(24) 1.5	9 9 10	(9) 0.6
	150 µg	111 105 121	(112) 8.1	9 11 16	(12) 3.6	36 33 23		(31) 6.8	25 27 20	(24) 3.6	11 15 9	(12) 3.1
	500 µg	144 169 180	(164) 18.4	9 10 19	(13) 5.5	28 21 41		(30) 10.1	33 33 32	** (33) 0.6	8 12 11	(10) 2.1
	1500 µg	187 170 148	* (168) 19.6	9 8 8	(8) 0.6	21 20 19		(20) 1.0	16 19 24	(20) 4.0	14 11 11	(12) 1.7
	5000 µg	122 S 182 S 114 S	(139) 37.2	12 S 9 S 5 S	(9) 3.5	21 20 13		(18) 4.4	10 20 24	(18) 7.2	6 12 6	(8) 3.5
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		1905 1717 1532	(1718) 186.5	319 301 246	(289) 38.0	188 196 255		(213) 36.6	132 164 150	(149) 16.0	357 315 233	(302) 63.1

Experiment 2 (plate incorporation) – Table 4 and Table 5

The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 µg/plate). Intermediate dose levels were selected in order to confirm and potentially enhance the mutagenic responses observed in Experiments 1.

The test item induced a visible reduction in the growth of the bacterial background lawns of all of theSalmonellastrains dosed in the absence of metabolic activation, initially from 3000 µg/plate (TA100 and TA1537) and at 5000 µg/plate (TA1535 and TA98) and to TA100 and TA1535 at 5000 µg/plate dosed in the presence of metabolic activation.

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).

In Experiment 2, no increases greater than two-fold the concurrent solvent control were observed in any of the strains tested, in either the presence or absence of S9-mix. However, similar to Experiment 1, small but statistically significant increases in the frequency of TA100 revertant colonies were recorded in the absence of metabolic activation from 1000 to 2000 µg/plate. Whilst it is recognised that statistical significance should not be the sole factor to determine a positive response, the reproducibility of the result obtained was investigated further.

Test Results: Experiment 2 – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 11 September 2018					To: 14 September 2018
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	134 121 99	(118) 17.7#	17 19 19	(18) 1.2	20 30 40		(30) 10.0	16 40 19	(25) 13.1	27 26 19	(24) 4.4
	150 µg	132 114 120	(122) 9.2	26 15 12	(18) 7.4	28 21 19		(23) 4.7	29 24 17	(23) 6.0	21 26 21	(23) 2.9
	250 µg	136 117 115	(123) 11.6	15 25 20	(20) 5.0	21 14 21		(19) 4.0	13 21 20	(18) 4.4	12 11 11	(11) 0.6
	500 µg	141 130 124	(132) 8.6	24 27 27	(26) 1.7	41 34 20		(32) 10.7	23 13 16	(17) 5.1	17 16 17	(17) 0.6
	1000 µg	177 154 154	** (162) 13.3	26 27 30	(28) 2.1	19 31 41		(30) 11.0	16 23 19	(19) 3.5	29 8 18	(18) 10.5
	1500 µg	148 177 183	*** (169) 18.7	15 15 30	(20) 8.7	30 31 17		(26) 7.8	19 23 25	(22) 3.1	18 26 12	(19) 7.0
	2000 µg	181 178 173	*** (177) 4.0	19 22 14	(18) 4.0	28 35 13		(25) 11.2	33 27 21	(27) 6.0	15 13 18	(15) 2.5
	3000 µg	172 S 218 S 177 S	*** (189) 25.2	30 27 18	(25) 6.2	19 26 22		(22) 3.5	44 19 19	(27) 14.4	8 S 9 S 9 S	(9) 0.6
	5000 µg	0 V 0 V 0 V	(0) 0.0	16 S 21 S 19 S	(19) 2.5	31 21 29		(27) 5.3	32 S 23 S 25 S	(27) 4.7	0 T 0 T 0 T	(0) 0.0
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		551 506 572	(543) 33.7	329 314 298	(314) 15.5	515 611 570		(565) 48.2	165 201 178	(181) 18.2	134 206 245	(195) 56.3

Test Results: Experiment 2 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 11 September 2018					To: 14 September 2018
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	121 120 113	(118) 4.4#	14 15 14	(14) 0.6	41 49 21		(37) 14.4	20 23 23	(22) 1.7	13 15 15	(14) 1.2
	150 µg	99 109 135	(114) 18.6	20 17 14	(17) 3.0	38 32 34		(35) 3.1	31 22 27	(27) 4.5	15 16 19	(17) 2.1
	250 µg	135 137 149	(140) 7.6	22 15 16	(18) 3.8	30 23 28		(27) 3.6	27 37 31	(32) 5.0	18 17 17	(17) 0.6
	500 µg	142 136 156	(145) 10.3	13 16 16	(15) 1.7	32 15 34		(27) 10.4	30 22 32	(28) 5.3	12 16 12	(13) 2.3
	1000 µg	155 138 132	(142) 11.9	10 12 10	(11) 1.2	36 35 26		(32) 5.5	34 34 30	(33) 2.3	19 10 12	(14) 4.7
	1500 µg	123 127 152	(134) 15.7	8 8 21	(12) 7.5	27 39 32		(33) 6.0	40 32 24	(32) 8.0	12 13 16	(14) 2.1
	2000 µg	148 122 153	(141) 16.6	11 10 19	(13) 4.9	28 31 28		(29) 1.7	11 26 18	(18) 7.5	16 18 16	(17) 1.2
	3000 µg	159 153 126	(146) 17.6	13 10 9	(11) 2.1	37 40 24		(34) 8.5	11 24 30	(22) 9.7	10 13 19	(14) 4.6
	5000 µg	90 S 134 S 126 S	(117) 23.4	13 S 8 S 11 S	(11) 2.5	21 24 32		(26) 5.7	26 22 19	(22) 3.5	11 10 5	(9) 3.2
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		2173 1980 2222	(2125) 127.9	332 326 314	(324) 9.2	274 200 243		(239) 37.2	203 199 221	(208) 11.7	270 234 268	(257) 20.2

Experiment 3 Confirmatory (plate incorporation)

The maximum dose level of the test item in the confirmatory experiment was 3000 µg/plate. Intermediate dose levels were again selected in order to confirm the reproducibility of the responses observed in Experiments 1 and 2. The test item induced a visible reduction in the growth of the bacterial background lawns of TA100 at 3000 µg/plate dosed in the absence of metabolic activation.

No test item precipitate was observed on the plates at any of the doses tested in the absence of metabolic activation (S9-mix).

In Experiment 3, no increases greater than two-fold the concurrent solvent control were observed in TA100, in the absence of S9‑mix. Whilst small but statistically significant increases in the frequency of revertant colonies were recorded in the absence of metabolic activation from 750 to 2000 µg/plate, these increases were deemed not biologically relevant as no increases in revertant colony frequency were observed that were more than two-fold the concurrent control.

Following test item treatments of all tester strains in the absence and presence of S9-mix, overall, it can be concluded that no increases in revertant colony frequency were observed that were more than 2-fold (strains TA98, TA100 and WP2uvrA) or 3-fold (strains TA1535 and TA1537) the concurrent solvent control. This study was , therefore, considered to have provided no evidence of any test item mutagenic activity in this assay system.

Test Results: Experiment 3 (confirmatory) – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 18 September 2018	To: 21 September 2018
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type
		TA100
-	Solvent Control (DMSO)	134 131 118 137 133	(131) 7.4#
	750	176 158 179	*** (171) 11.4
	1000	192 185 217	*** (198) 16.8
	1500	191 186 208	*** (195) 11.5
	2000	243 204 223	*** (223) 19.5
	3000	229 S 233 S 217 S	*** (226) 8.3
Positive controls S9-Mix -	Name	ENNG
	Concentration	3 µg/plate
	No. colonies per plate	551 579 612	(581) 30.5

Conclusions:

It was concluded that the test item did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium and one tryptophan-requiring strain (WP2uvrA) Escherichia coli when tested under the conditions of this study, with or without metabolic activation.

Executive summary:

The purpose of the study was to evaluate the test item for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria. This study is based upon the general provisions indicated by OECD Guidelines for Testing of Chemicals No. 471 “Bacterial Reverse Mutation Test”, 21 July 1997.

It was concluded thatEXPINN PC17032did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) ofSalmonella typhimuriumand one tryptophan-requiring strain (WP2uvrA)Escherichia coliwhen tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory test guidelines and a toxic concentration) in the absence and in the presence of a rat liver metabolic activation system (S9).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 2018 - Jan 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This is a Klimisch 1 rated OECD 490 guideline study conducted on the registered substance 1-hydroxyoctan-2-one in accordance GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Batch: 1203-16-100
Purity: 96.7%
Referred to in test report as EXPINN PC17032

Target gene:

tk (thymidine kinase) locus in mouse lymphoma L5178Y cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y tk+/- 3.7.2C mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Metabolic activation:

with and without

Metabolic activation system:

phenobartione/β-naphthoflavone-induced rat liver post-mitochondrial fraction (S9). Lot No. PB/BNF 31/08/18 was used in this study, and was pre-prepared in house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test.

Test concentrations with justification for top dose:

Following solubility checks performed in-house, the test item was accurately weighed and formulated in DMSO prior to serial dilutions being prepared. The molecular weight of the test item was 144.21 g/mol, therefore the maximum dose level was 1442 µg/mL which was equivalent to 10 mM (an acceptable maximum concentration for in vitro genetic toxicity studies according to current regulatory guidelines). The purity of the test item was 96.7% and was therefore accounted for when formulating the dosing solutions. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al. 1991). The pH and osmolality readings are in the following table:

µg/mL 0 5.64 11.27 22.54 45.07 90.13 180.25 360.5 721 1442
pH 7.21 7.21 7.21 7.23 7.23 7.23 7.24 7.24 7.27 7.22
mOsm 466 464 467 461 - 460 - 458 450 450

Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values markedly less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.

Vehicle / solvent:

The test article was formulated in dimethyl sulphoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Preliminary Toxicity Test

A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9). The dose range used in the preliminary toxicity test was 5.63 to 1442 µg/mL for both of the exposure groups. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 105 cells/mL. Where the mean cell count was less than 3 x 105 cells/mL, cells were maintained. Single cultures only were used and positive controls were not included, and no cultures were plated out for viability or mutation assessment

The cultures were incubated at 37°C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity test. Maximum dose levels were selected using the following criteria:

i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 µL/mL whichever is the lowest.
ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to 10 to 20 % of survival. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

Mutagenicity Test

Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% (v/v) S9 final concentration) at eight dose levels of the test item (11.25 to 480 µg/mL in the absence of metabolic activation, and 22.5 to 720 µg/mL in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 0.2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL.

The exposure vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.


Measurement of Survival, Viability and Mutant Frequency

At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 105 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained.
On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.

The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data, a Relative Total Growth (RTG) value.

Plate Scoring

Microtitre plates were scored using a magnifying mirror box after twelve days incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al., 1990). Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.

Evaluation criteria:

Data Evaluation

An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 mutants per 106 viable cells for microwell assays, which is based on the analysis of the distribution of the vehiclecontrol MF data from participating laboratories.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

Statistics:

The experimental data was analyzed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989). The statistical package used indicates the presence of statistically significant increases and linear trend events.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed in top two doses in experiments with and without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: test item does not induce mutation at the tk locus of L5178Y up to the limit of cytotoxicity in absence and presence of rat liver metabolic activation system (S9)

Preliminary Cytotoxicity Test

In the Preliminary Cytotoxicity Test (4 hour treatment) nine concentrations were tested in the absence and presence of S9 ranging from 5.63 to 1442 µg/mL equivalent to 10 mM at the highest concentration tested (an acceptable maximum concentration forin vitrogenetic toxicity studies according to current regulatory guidelines). Precipitate of the test item was observed at 1442 µg/mL in both of the exposure groups at the end of the exposure period. The highest concentration to provide >10% RSG was 360.5 µg/mL in the absence of S9, which gave 16% RSG. Severe toxicity (≤5% RSG) was observed at concentrations ≥721 μg/mL in the presence of S9, with a steep toxicity curve observed. A concentration of 360.5 μg/mL gave 56% RSG (see following table).

Dose (mg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure
0	100	100
5.63	96	107
11.27	94	97
22.53	93	101
45.06	92	103
90.13	88	100
180.25	70	82
360.5	16	56
721	0	1
1442	0	0

 

No significant changes in osmolality or pH were observed in the 3 hour treatments compared to the concurrent vehicle controls. Therefore, following the recommendations of the OECD 490 guideline, the maximum dose levels in the subsequent Mutagenicity Test were limited by test item-induced toxicity.

Mutation Experiment

In the Mutation Experiment (4 hour treatment) eight concentrations, ranging from 11.25 to 480 µg/mL, were tested in the absence of S9 and eight concentrations ranging from 22.5 to 720 µg/mL were tested in the presence of S9.Precipitate of the test item was not observed at any of the dose levels, in either of the exposure groups, at the end of the exposure period. Two days after treatment the highest concentrations selected to determine viability and TFT resistance were 240 µg/mL in the absence of S9 and 480 µg/mL in the presence of S9.A summary of the results from the test is presented in Table 1.

The results of the microtitre plate counts and their analysis are presented in Tables 2 to 7.

There was evidence of marked dose related toxicity following exposure to the test item in both of the exposure groups, as indicated by the %RSG and RTG values (Tables 3 and 6). There was also evidence of a reduction in viability (%V) in the absence of metabolic activation, indicating that residual toxicity had occurred (Table 3). Based on the %RSG values and / or the RTG values, optimum levels of toxicity were considered to have been achieved in both the absence and presence of metabolic activation. Acceptable levels of toxicity were seen with both of the positive control substances (Tables 3 and 6).

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at thetk^+/-locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional (Tables 3 and 6).Marked increases in the number of both small and large colony mutants were observed following treatment with the positive control chemicals EMS and CP (refer toTables 4 and 7).

The MF of the concentrations plated were all less than the sum of the mean control MF plus the global evaluation factor (GEF, 126 mutants/10⁶ viable cells).

Table 1            Summary of Results

 

Main Experiment

Concentration (µg/mL)			4-Hours-S9					Concentration (µg/mL)			4-Hours+S9
			%RSG	RTG[DB2]	MF§						%RSG	RTG	MF§
0			100	1.00	132.94			0			100	1.00	110.54
11.25			87	0.98	98.14			22.5			89	1.01	90.97
22.5			92	0.89	135.55			45			86	0.83	124.46
45			96	1.14	101.70			90			89	0.96	110.77
90			94	0.99	109.73			180			81	0.84	103.29
180			70	0.69	135.55			360			52	0.53	126.31
240			49	0.19	145.42			480			10	0.09	120.63
360		Ø	1					600		Ø	0
480		Ø	0					720		Ø	0
MF threshold for a positive response = 258.94								MF threshold for a positive response = 236.54
Positive control								Positive control
EMS								CP
400			71	0.63	1095.65			1.5			69	0.57	854.74

 

Table 2          Cell and 96-Well Plate Counts: Mutagenicity Test (-S9) 4-Hour Exposure

 

Concentration (µg/mL)		Cell counts $			Viability § after day 2 2 cells/well				Resistant mutants § after day 2 2000 cells/well
		0h	24h	48h
0	A B	12.29 11.54	7.59 8.17	8.44 7.02	74 73	72 76	77 76	78 77	18 18	17 14	20 23	16 16
11.25	A B	12.41 11.33	7.12 6.25	7.58 8.28	79 77	79 81			15 14	18 13
22.5	A B	11.68 11.76	7.58 7.00	7.77 7.88	74 81	70 72			17 17	18 18
45	A B	12.08 11.66	7.29 6.78	8.03 8.65	82 84	79 77			14 17	18 16
90	A B	10.85 11.28	8.23 6.29	7.40 9.57	73 79	77 80			15 18	13 17
180	A B	8.62 9.68	6.98 6.16	8.55 8.47	74 71	76 76			20 14	20 16
240	A B	8.00 8.77	5.76 4.19	7.17 9.96	47 42	46 42			7 8	9 9
360	A B	4.74 3.30	1.37 1.17	2.47(1.37) 1.79(1.16)	NP NP	NP NP			NP NP	NP NP
480	A B	1.54 1.58	1.36(1.54) 1.18(1.58)	0.35(1.36) 0.36(1.18)	NP NP	NP NP			NP NP	NP NP
Positive control EMS (µg/mL)
400	A B	11.39 10.58	7.23 6.77	6.59 6.72	68 70	72 76			76 64	77 81

 

Table 3            Summary Analysis: Mutagenicity Test (-S9) 4-Hour Exposure

 

Concentration (µg/mL)		SG	%RSG	%V	RTG	MF§
0		15.23	100	76.89	1.00	132.94
11.25		13.25	87	86.56	0.98	98.14
22.5		14.26	92	74.24	0.89	135.55
45		14.67	96	91.18	1.14	101.70
90		15.40	94	81.66	0.99	109.73
180		13.98	70	74.24	0.69	135.55
240		10.65	49	30.90	0.19	145.42
360	Ø	0.68	1
480	Ø	0.11	0
Positive control EMS
Concentration (µg/mL)		SG	%RSG	%V	RTG	MF§
400		11.65	71	68.28	0.63	1095.65

 

GEF =126, therefore MF threshold for a positive response = 258.94

 

Table 4            Large and Small ColoniesAnalysis: Mutagenicity Test (-S9) 4-Hour Exposure

 

Concentration (µg/mL)		Viability # after day 2				Small colonies # after day 2				Large colonies # after day 2
0	A B	74 73	72 76	77 76	78 77	8 8	9 6	10 11	11 9	10 10	8 8	10 12	5 7
11.25	A B	79 77	79 81			6 6	7 8			9 8	11 5
22.5	A B	74 81	70 72			10 10	6 9			7 7	12 9
45	A B	82 84	79 77			6 8	5 8			8 9	13 8
90	A B	73 79	77 80			6 9	7 6			9 9	6 11
180	A B	74 71	76 76			10 8	9 5			10 6	11 11
240	A B	47 42	46 42			5 3	5 6			2 5	4 3
400 EMS	A B	68 70	72 76			39 29	39 44			37 35	38 37

Mutation frequencies

Concentration (µg/mL)				Small colonies			Large colonies			Proportion small colony mutants
		Viable		Mutants			Mutants
		Yv	Nv	Ym	Nm	MF§	Ym	Nm	MF§
0		165	768	696	768	64.0	698	768	62.1	0.51
11.25