estr-4-ene-3,11,17-trione cyclic 3-(1,2-ethanediylmercaptole)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed at a registered GLP site and reported a high standard andis in compliance with recognised OECD standards with no deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

11-KN

IUPAC Name:

11-KN

Constituent 2

Reference substance name:

estr-4-ene-3,11,17-trione cyclic 3-(1,2-ethanediylmercaptole)

Molecular formula:

C20H26O2S2

IUPAC Name:

estr-4-ene-3,11,17-trione cyclic 3-(1,2-ethanediylmercaptole)

Constituent 3

Reference substance name:

920-684-2

EC Number:

920-684-2

IUPAC Name:

920-684-2

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): 11-KN
- Physical state: White powder
- Lot/batch No.: CGG238K1
- Expiration date of the lot/batch: 12 February 2014
- Storage condition of test material: room temperature in the dark
- Purity: >98%

Method

Target gene:

The purpose of the study was to evaluate 11-KN 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.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: TA 1537 Genotype : his C 3076; rfa¯; uvrB¯: TA98 Genotype : his D 3052; rfa¯; uvrB¯; R-factor TAI535 Genotype : his G 46; rfa¯; uvrB¯: TA100 Genotype : his G 46; rfa¯; uvrB¯; R-factor

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: Genotype : trp¯; uvrA¯:

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

1.5, 5, 15, 50, 150, SOD, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water at 50 mg/mL and acetone at 100 mg/mL but was fully soluble in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

Test Procedure
Test for Mutagenicity (Experiment 1) - Plate Incorporation Method
Dose selection
Test items that are not deemed particularly toxic or do not belong to a class of compounds known for their toxicity were tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, SOD, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, vehicle or appropriate positive control was added to 2 mL of trace amino-acid supplemented media (at approximately 45°C) containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Each concentration of the test item, appropriate positive control, and each bacterial strain, was assayed using triplicate plates.
With Metabolic Activation
The procedure was the same as described previously (see 3.5.1.2) except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the trace amino-acid supplemented media instead of phosphate buffer.
All of the plates were incubated at 37 °C ± 3 °C for approximately 48 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). Manual counts were performed at and above 1500 µg/plate because of excessive test item precipitation.

Test for Mutagenicity (Experiment 2) - Pre-Incubation Method
As Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.
Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 50 to 5000 µg/plate.
Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 °C± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. All testing for this experiment was performed in triplicate.
With metabolic activation
The procedure was the same as described previously (see 3.5.2.2) except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 °C± 3° C for 20 minutes (with shaking) and addition of amino-acid supplemented media. All testing for this experiment was performed in triplicate.
All of the plates were incubated at 37 °C± 3 °C for approximately 48 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). Manual counts were performed at and above 1500 ~g/plate because of excessive test item precipitation.

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. Statistical analysis of data as determined by UKEMS (Mahon et aI., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response Cariello and Piegorsch, 1996).

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.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (white and powdery) was observed at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Any other information on results incl. tables

Table 1

Experiment 1
Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
100	(105)	26	(29)	29	(29)	16	(15)	13	(20)
119		19		27		12		24
95		23		31		17		23
Experiment 2
Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
87	(85)	25	(22)	29	(28)	16	(16)	7	(8)
98		20		35		15		8
69		20		21		17		6

Table 2

Test Period		From: 05 April 2013					To: 08 April 2013
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)	91	(89) 4.0#	28	(23) 4.7	21	(18) 3.1	11	(18) 6.6	15	(16) 2.3
		91		19		19		24		15
		84		21		15		19		19
	1.5 µg	91	(97) 11.8	24	(23) 1.7	21	(20) 12.0	8	(20) 12.0	11	(10) 2.1
		90		21		23		19		12
		111		24		17		32		8
	5 µg	88	(93) 5.6	28	(21) 7.5	19	(19) 0.6	9	(11) 2.1	24	(16) 6.7
		92		21		19		13		13
		99		13		20		12		12
	15 µg	91	(92) 14.5	13	(18) 4.2	11	(18) 8.9	11	(13) 2.5	11	(13) 6.8
		78		19		28		13		21
		107		21		15		16		8
	50 µg	108	(109) 3.1	19	(20) 1.2	17	(18) 1.2	13	(14) 1.7	15	(10) 4.2
		106		21		19		16		7
		112		21		17		13		9
	150 µg	76	(88) 25.8	27	(24) 3.1	16	(19) 2.9	21	(17) 3.5	8	(9) 1.5
		118		25		21		15		9
		71		21		21		15		11
	500 µg	90 P	(91) 0.6	20 P	(24) 4.0	21 P	(18) 4.6	25 P	(20) 4.6	12 P	(14) 4.0
		91 P		24 P		21 P		17 P		12 P
		91 P		28 P		13 P		17 P		19 P
	1500 µg	98 P	(101) 19.2	19 P	(22) 2.5	18 P	(20) 1.7	17 P	(17) 0.6	12 P	(11) 1.0
		122 P		24 P		21 P		18 P		10 P
		84 P		22 P		21 P		17 P		11 P
	5000 µg	81 P	(88) 11.8	18 P	(22) 3.8	24 P	(23) 0.6	12 P	(16) 5.1	14 P	(13) 1.2
		82 P		25 P		23 P		22 P		12 P
		102 P		24 P		23 P		15 P		14 P
Positive controls S9-Mix (-)	Name Dose level No.of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80µg
		750	(795) 45.5	599	(706) 93.0	1002	(953) 65.2	171	(207) 58.1	523	(700) 262.6
		841		770		879		274		576
		795		748		978		176		1002

ENNG = N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-l-oxide

9AA = 9-Aminoacridine

P = Test item precipitate

# = Standard deviation

Table 3

Test Period		From: 05 April 2013					To: 08 April 2013
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)	103	(97) 4.9#	19	(16) 3.1	41	(35) 7.2	28	(20) 6.7	21	(14) 7.0
		94		13		27		16		7
		95		15		37		17		15
	1.5 µg	91	(89) 6.2	15	(14) 2.3	32	(28) 7.5	12	(21) 8.5	12	(12) 0.6
		94		15		32		21		12
		82		11		19		29		13
	5 µg	92	(100) 7.6	12	(15) 4.4	29	(31) 1.5	19	(18) 2.6	11	(15) 3.5
		107		13		32		20		17
		102		20		31		15		17
	15 µg	96	(104) 10.6	13	(12) 1.2	23	(33) 8.7	24	(21) 2.5	17	(14) 3.1
		116		11		37		21		11
		100		11		39		19		13
	50 µg	104	(96) 6.8	16	(16) 1.0	19	(27) 6.9	23	(22) 2.1	13	(12) 3.6
		94		17		31		20		15
		91		15		31		24		8
	150 µg	96	(104) 7.2	19	(16) 3.5	44	(32) 10.8	19	(21) 2.1	13	(13) 0.6
		106		16		29		20		12
		110		12		23		23		13
	500 µg	108	(98) 15.6	9 P	(12) 3.1	27 P	(30) 2.3	19 P	(19) 0.0	11 P	(11) 0.6
		80		11 P		31 P		19 P		11 P
		106		15 P		31 P		19 P		10 P
	1500 µg	74 P	(83) 8.5	11 P	(10) 1.2	25 P	(28) 4.2	20 P	(19) 2.3	14 P	(13) 1.2
		91 P		9 P		27 P		16 P		12 P
		84 P		11 P		33 P		20 P		12 P
	5000 µg	99 P	(97) 4.0	11 P	(12) 1.0	18 P	(22) 8.7	15 P	(22) 6.2	9 P	(9) 1.0
		92 P		12 P		16 P		24 P		10 P
		99 P		13 P		32 P		27 P		8 P
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
		1445	(1450) 24.4	237	(254) 23.4	262	(283) 20.6	378	(369) 12.1	354	(301) 47.2
		1429		245		303		373		287
		1477		281		285		355		263

BP = Benzo(a)pyrene

2AA = 2-Aminoanthracene

P = Test item precipitate

# = Standard deviation

Table 4

Test Period		From: 05 April 2013					To: 08 April 2013
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)	75	(75) 7.5#	25	(20) 6.4	27	(24) 6.4	19	(14) 5.0	3	(7) 3.2
		68		13		29		13		8
		83		23		17		9		9
	50 µg	84	(81) 5.8	29	(23) 9.5	33	(30) 2.3	19	(18) 1.2	8	(9) 2.3
		74		12		29		17		8
		84		28		29		17		12
	150 µg	87	(77) 9.3	29	(23) 5.2	25	(28) 3.1	11	(14) 3.1	8	(7) 1.7
		74		20		27		17		5
		69		20		31		15		8
	500 µg	88 P	(84) 3.2	17 P	(19) 1.5	21 P	(23) 2.1	15 P	(15) 2.0	8 P	(9) 1.7
		82 P		20 P		24 P		13 P		8 P
		83 P		19 P		25 P		17 P		11 P
	1500 µg	72	(80) 9.8	21 P	(23) 4.9	31 P	(25) 6.0	20 P	(17) 3.1	7 P	(11) 3.2
		77 P		20 P		19 P		16 P		13 P
		91 P		29 P		25 P		14 P		12 P
	5000 µg	68 P	(72) 5.1	18 P	(21) 6.1	26 P	(22) 3.6	15 P	(16) 1.2	11 P	(9) 3.8
		71 P		17 P		19 P		15 P		5 P
		78 P		28 P		21 P		17 P		12 P
Positive controls S9-Mix (-)	Name Dose level No.of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80µg
		470	(463) 17.0	345	(536) 177.3	422	(371) 45.2	239	(207) 32.0	1658	(1213) 386.5
		476		695		335		206		961
		444		569		357		175		1020

ENNG = N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-l-oxide

9AA = 9-Aminoacridine

P = Test item precipitate

# = Standard deviation

Table 5

Test Period		From: 05 April 2013					To: 08 April 2013
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)	96	(89) 18.1#	24	(21) 5.2	35	(32) 3.6	23	(19) 3.5	17	(8) 7.8
		68		15		28		17		4
		102		24		33		17		3
	50 µg	88	(83) 4.6	21	(17) 4.0	33	(28) 6.1	15	(15) 2.0	9	(11) 4.4
		79		13		29		13		8
		82		17		21		17		16
	150 µg	74	(84) 13.1	17	(20) 4.6	35	(32) 3.5	17	(20) 3.5	13	(11) 5.7
		99		25		28		20		5
		80		17		32		24		16
	500 µg	90 P	(88) 1.5	16 P	(16) 0.6	24 P	(28) 3.6	11 P	(17) 6.0	9 P	(12) 3.6
		88 P		17 P		29 P		23 P		16 P
		87 P		16 P		31 P		17 P		11 P
	1500 µg	73 P	(70) 5.2	15 P	(20) 5.7	28 P	(31) 3.8	26 P	(25) 1.0	7 P	(7) 1.5
		64 P		26 P		29 P		25 P		9 P
		73 P		18 P		35 P		24 P		6 P
	5000 µg	84 P	(78) 6.0	13 P	(15) 3.2	33 P	(30) 6.4	17 P	(22) 6.4	11 P	(8) 2.5
		79 P		19 P		23 P		29 P		8 P
		72 P		14 P		35 P		19 P		6 P
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
		1148	(1119) 29.0	374	(375) 22.5	238	(249) 9.3	203	(205) 19.6	190	(209) 24.8
		1120		353		255		186		200
		1090		398		253		255		237

BP = Benzo(a)pyrene

2AA = 2-Aminoanthracene

P = Test item precipitate

# = Standard deviation

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

11-KN was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLWand MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA

(TSCA) OCSPP harmonized guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods 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 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 50 to 5000 µg/plate.

Results

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of

5000 µg/plate. A test item precipitate (white and powdery) was observed at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

Conclusion

11-KN was considered to be non-mutagenic under the conditions of this test.