13-ethyl-11-methylene-gon-4-ene-3,17-dione

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:

8 June 2020 - 10 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

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:

(3aS,3bS,9aR,9bS,11aS)‐11a‐ethyl‐10‐methylidene‐1H,2H,3H,3aH,3bH,4H,5H,7H,8H,9H,9aH,9bH,10H,11H,11aH‐cyclopenta[a]phenanthrene‐1,7‐dione

Cas Number:

54024-17-8

Molecular formula:

C20 H26 O2

IUPAC Name:

(3aS,3bS,9aR,9bS,11aS)‐11a‐ethyl‐10‐methylidene‐1H,2H,3H,3aH,3bH,4H,5H,7H,8H,9H,9aH,9bH,10H,11H,11aH‐cyclopenta[a]phenanthrene‐1,7‐dione

Test material form:

solid: particulate/powder

Details on test material:

Storage Conditions: In refrigerator (2-8°C)

Specific details on test material used for the study:

Physical Description: Off-white powder
Storage conditions: In refrigerator (2-8°C)
Test item handling: No specific handling conditions required

Method

Target gene:

Salmonella typhimurium strain: histidine locus
Escherichia coli: tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Rat liver microsomal enzymes (S9 homogenate) prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution. The above solution was filter (0.22 μm)-sterilized.
- concentration S9 in the S9-mix: 5% (v/v) S9-fraction

Test concentrations with justification for top dose:

Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
First experiment: 52, 164, 512, 1600 and 5000 μg/plate
Second experiment:
- Without S9-mix: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (TA1535, TA1537, TA100 and WP2uvrA); 1.7, 5.4, 17, 52, 164, 512 and 1600 μg/plate (TA98)
- With S9-mix: 52, 164, 512, 1600 and 5000 μg/plate (TA1535, TA1537, TA100 and WP2uvrA); 5.4, 17, 52, 164, 512 and 1600 μg/plate (TA98)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10^9 cells/mL
- Test substance added in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration: 48 ± 4 h (direct plate assay); 30 ± 2 minutes + 48 ± 4 h after solidification of the top agar (pre-incubation assay)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Colony counting: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Evaluation criteria:

In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

No cytotoxicity observed in experiment 1. Cytotoxicity observed in experiment 2 (without S9-mix).

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

No cytotoxicity observed in experiment 1. Cytotoxicity observed in experiment 2 (with and without S9-mix).

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

No cytotoxicity observed in experiment 1. Cytotoxicity observed in experiment 2 (with and without S9-mix).

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

No cytotoxicity observed in experiment 1. Cytotoxicity observed in experiment 2 (with and without S9-mix).

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

No cytotoxicity observed in experiment 1. Cytotoxicity observed in experiment 2 (with and without S9-mix).

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

First Experiment: Direct Plate Assay (dose-range finding study is reported as part of the first experiment):
- Precipitate: Precipitation of the test item on the plates was not observed in any tester strain.
- Toxicity: In strain TA1537 (presence of S9-mix), a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed. However, since no doserelationship was observed, the reduction was not considered to be caused by toxicity of the
test item. It is more likely the reduction was caused by an incidental fluctuation in the number of revertant colonies.
- Mutagenicity: In the direct plate test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Second Experiment: Pre-Incubation Assay:
- Precipitate: The test item precipitated on the plates at dose levels of 512 and/or 1600 μg/plate and above.
- Toxicity: Cytotoxicity, as evidenced by a reduction in the number of revertants, a reduction in the
bacterial lawn and/or the presence of microcolonies was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the presence of S9-mix.
- Mutagenicity: In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Any other information on results incl. tables

Table 1. Dose-Range Finding Test:  Mutagenic Response of EMETAM in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

Direct Plate Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with one Salmonella typhimurium and one Escherichia coli strain.
	TA100	WP2uvrA

 

Without S9-mix

 

Positive control	931	±	91		1504	±	20
Solvent control	97	±	14		21	±	3
1.7	100	±	15		13	±	3
5.4	94	±	3		15	±	2
17	99	±	9		17	±	5
52	103	±	9		15	±	4
164	108	±	6		21	±	4
512	91	±	13		17	±	4
1600	92	±	17		14	±	6
5000	86	±	6	^n NP	18	±	6	^n NP

 

With S9-mix

 

Positive control	1906	±	214		195	±	17
Solvent control	98	±	5		25	±	4
1.7	81	±	5		33	±	12
5.4	83	±	5		27	±	3
17	96	±	26		17	±	5
52	96	±	5		23	±	4
164	99	±	6		23	±	4
512	97	±	21		17	±	4
1600	97	±	18		20	±	3
5000	85	±	23	^n NP	12	±	4	^n NP

 

NP	No precipitate
n	Normal bacterial background lawn

 

Table 2. Experiment 1:  Mutagenic Response of EMETAM in the Salmonella typhimurium Reverse Mutation Assay 

Direct Plate Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium.
	TA1535	TA1537	TA98

 

Without S9-mix

 

Positive control	876	±	56		449	±	71		1252	±	25
Solvent control	9	±	3		2	±	1		13	±	4
52	12	±	2		3	±	2		15	±	4
164	12	±	6		2	±	2		16	±	5
512	11	±	4		5	±	2		15	±	4
1600	5	±	1		4	±	4		14	±	4
5000	5	±	2	^n NP	2	±	1	^n NP	10	±	2	^n NP

 

With S9-mix

 

Positive control	270	±	12		766	±	47		1291	±	95
Solvent control	13	±	5		3	±	2		11	±	1
52	12	±	2		4	±	3		16	±	9
164	14	±	5		7	±	5		17	±	3
512	9	±	5		6	±	3		22	±	6
1600	6	±	2		1	±	1		12	±	3
5000	6	±	1	^n NP	2	±	1	^n NP	12	±	2	^n NP

 

NP	No precipitate
n	Normal bacterial background lawn

 

Table 3. Experiment 2:  Mutagenic Response of EMETAM in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

Pre-incubation Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain.
	TA1535	TA1537	TA98	TA100	WP2uvrA

 

Without S9-mix

 

Positive control	1045	±	63		197	±	27		1724	±	144		628	±	34		1143	±	125
Solvent control	9	±	2		3	±	2		13	±	6		86	±	19		18	±	4
Positive control^1	999	±	89		165	±	22			-			679	±	48			-
Solvent control^1	10	±	2		4	±	4			-			94	±	9			-
0.17^1	11	±	0		8	±	4			-			101	±	12			-
5.4^1	12	±	3		6	±	5		12	±	4		112	±	7	^NP		-
17^1	12	±	4	^n NP	5	±	2	^n NP	17	±	5		110	±	15	^n NP		-
52	9	±	4		5	±	3		19	±	3		89	±	19	^i	14	±	2
164	10	±	5	^n	7	±	4	^n	18	±	5	^n NP	96	±	17	^n	17	±	1
512	4	±	2	^m NP				^e NP MC	11	±	6	^m SP				^e NP MC	12	±	2	^NP
1600				^e SP MC				^e SP MC	12	±	4	^e MP				^e SP MC	9	±	2	^MP
5000				^e HP				^e HP		-						^e HP				^n HP

 

With S9-mix

 

Positive control			294	±	37		382	±	19		991	±	71		782	±	242		385	±	13
Solvent control			8	±	4		5	±	2		19	±	10		96	±	6		24	±	7
5.4			-				-				23	±	1		-				-
17			-				-				14	±	2		-				-
52			6	±	2		7	±	4		15	±	4		101	±	1		20	±	3
164			9	±	5		5	±	3	^n	18	±	6		99			^n ii	21	±	3
512			6	±	5	^n NP	5	±	2	^s NP	18	±	2	^n NP	75	±	13	^m NP	20	±	4	^NP
1600			4	±	3	^s SP	1	±	2	^s SP	14	±	8	^s SP	60	±	11	^m SP	25	±	5	^SP
5000			3	±	1	^m SP	3	±	1	^s MP	-							^m HP	16	±	3	^n MP
	^1 - HP	Data from additional experiment, except for tester strain TA98 Not tested Heavy Precipitate
	MC	Microcolonies
	MP	Moderate Precipitate
	NP	No precipitate
	SP	Slight Precipitate
	e	Bacterial background lawn extremely reduced
	i	One plate infected, mean of two plates
	ii	Two plates infected
	m	Bacterial background lawn moderately reduced
	n	Normal bacterial background lawn
	s	Bacterial background lawn slightly reduced

Applicant's summary and conclusion

Conclusions:

In an AMES test, performed according to OECD guideline 471 and in accordance with GLP principles., EMETAM is not mutagenic with or without metabolic activation.

Executive summary:

An AMES test was performed according to OECD guideline 471 and in accordance with GLP principles. The objective of this study was to determine the potential of EMETAM and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).
The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose-range finding test were reported as part of the first mutation assay.
In the first mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the strains TA1535, TA1537 and TA98. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
In the second mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the tester strains TA1535, TA1537, TA100 and WP2uvrA and up to 1600 μg/plate in the tester strain TA98 in the pre-incubation assay. The test item was tested up to or beyond a precipitating dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the presence of S9-mix.
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
The test item did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.
In conclusion, based on the results of this study it is concluded that EMETAM is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.