Menadione

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:

07 August 2017 - 11 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 3 November 2015

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Menadione

EC Number:

200-372-6

EC Name:

Menadione

Cas Number:

58-27-5

Molecular formula:

C11H8O2

IUPAC Name:

2-methyl-1,4-dihydronaphthalene-1,4-dione

Test material form:

solid: particulate/powder

Details on test material:

- Physical appearance: yellow crystalline powder
- Storage conditions: at room temperature protected from light

Specific details on test material used for the study:

- No correction factor for purity required.

Method

Target gene:

Salmonella typhimurium: histidine gene
Escherichia coli: tryptophan gene

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate; 5 and 10%), prepared from male Sprague Dawley rats injected with Aroclor 1254 (500 mg/kg body weight).

Test concentrations with justification for top dose:

Dose range finding test (TA100 and WP2uvrA, with and without 5% S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (reported as part of experiment 1)
Based on the cytotoxicity observed in the dose-range finding test, the dose-range for the following experiments were selected.
First experiment (TA1535, TA 1537 and TA98, with and without 5% S9): 0.55, 1.7, 5.4, 17, 52 and 164 μg/plate

Second experiment (TA1535, TA1537, TA98 and TA100, without S9): 8.5, 15, 27, 48, 86, and 154 μg/plate
Second experiment (TA1535, TA1537, TA98 and TA100, with 10% S9): 15, 27, 48, 86, 154 and 275 μg/plate
Second experiment (WP2uvrA, without S9): 27, 48, 86, 154, 275 and 492 μg/plate
Second experiment (WP2uvrA, with 10% S9): 86, 154, 275, 492, 878 and 1568 μg/plate

Third experiment (WP2uvrA without S9): 275, 492, 878, 1568 and 2800 μg/plate
Third experiment (TA98 with 10% S9): 50, 75, 100, 125, 150, 175 and 200 μg/plate

Vehicle / solvent:

- Solvent used: dimethyl sulfoxide
- Justification for choice of solvent: A solubility test was performed based on visual assessment.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoanthracene

Remarks:

For more details on positive controls see section 'other information on materials and methods incl. tables'

Details on test system and experimental conditions:

Three experiments were performed. In the first experiment the test item was tested both in the presence and absence of 5% (v/v) S9-mix. In the second experiment the test item was tested both in the presence and absence of 10% (v/v) S9-mix. To verify and check the results from the second experiment, a third experiment was performed with strains TA98 (with 10% S9-mix) and WP2uvrA (without 10% S9-mix) only.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 +/- 4 hours

NUMBER OF REPLICATIONS: 3

METHOD: Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in Milli-Q water and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h.

CYTOTOXICITY:
- Cytotoxicity was examined by the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

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:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle 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 the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up 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.

Acceptability criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at this laboratory.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: all strains

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at all test concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at the dose levels of 86 and 154 μg/plate, resp. 2.9- and 6.2-fold increase.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 275 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 154 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

other: TA1535, TA1537 and TA100.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 154 μg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 275 μg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 878 μg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at the dose levels of 75 to 175 μg/plate; up to 7.6-fold

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

200 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

878 μg/plate and upwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

DOSE-RANGE FINDING STUDY AND EXPERIMENT 1:
- Precipitation: yes, at the start of the incubation period at concentrations of 1600 and 5000 μg/plate and at 5000 μg/plate (with and without metabolic activation) at the end of the incubation period.
- Cytotoxicity: yes, as evidenced by a decrease in the number of revertants and a reduction of the bacterial background lawn, cytotoxicity was observed in all tester strains in the absence and presence of S9-mix.
- Mutagenicity: no, there was no increase in the number of revertants upon treatment with the test item under all conditions tested.

EXPERIMENT 2:
- Precipitation: yes, at the start of the incubation period at the concentration of 1568 μg/plate (with metabolic activation) and no precipitate was observed at the end of the incubation period.
- Cytotoxicity: yes, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, cytotoxicity was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the absence of S9-mix.
- Mutagenicity: yes, in the presence of S9-mix, the test item induced increases in the number of revertant colonies in the tester strain TA98. The increases observed were above the laboratory historical data range at the dose levels of 86 and 154 μg/plate and were respectively 2.9- and 6.2-fold.

EXPERIMENT 3
- Precipitation: yes, at the start of the incubation period at the concentrations of 1568 and 2800 μg/plate (for tester strain WP2uvrA). No precipitate was observed at the end of the incubation period.
- Cytotoxicity: yes, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, cytotoxicity was observed in tester strain WP2uvrA at concentrations of 878 μg/plate (without metabolic activation) and upwards and in strain TA98 at the top dose of 200 μg/plate (with metabolic activation).
- Mutagenicity: yes, the test item induced increases in the number of revertant colonies in the tester strain TA98. The increases observed were above the laboratory historical data range at the dose levels of 75, 100, 125, 150 and 175 μg/plate (tested with metabolic activation) and were 3.8-, 5.5-, 7.6-, 6.2- and 5.3-fold respectively.

HISTORICAL CONTROL DATA (see table 2 in 'any other information on results incl. tables')
- The solvent and strain-specific positive control values were within the laboratory historical control data ranges. This indicated that the test conditions were adequate and the metabolic activation system functioned properly.

Remarks on result:

other: Experiment 1 (5% S9-mix used)

Any other information on results incl. tables

Table 2 Historical data for the solvent control

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	3 - 36	3 - 32	3 – 20	3 – 23	8 - 41	9 - 55	66 - 161	63 - 160	10 – 59	9 - 69
Mean	11	11	6	7	16	23	105	105	25	31
SD	4	4	3	3	5	7	19	20	7	8
n	2057	2039	1950	1931	2023	2083	2027	2033	1739	1745

SD = Standard deviation; n = Number of observations

Historical control data from experiments performed between May 2015 and May 2017. 

Table 3 Historical data for the positive control

	TA1535		TA1537		TA98
S9-mix	-	+	-	+	-	+
Range	125 - 1381	78 - 1058	55 – 1324	55 – 1051	410 – 1995	250 - 1977
Mean	839	220	736	382	1369	929
SD	153	112	331	150	310	345
n	2065	1967	1740	1933	1920	2014

	TA100		WP2uvrA
S9-mix	-	+	-	+
Range	537 – 1848	408 - 2651	93 – 1951	93 - 1359
Mean	908	1330	1128	422
SD	178	324	484	151
n	2007	2020	1679	1728

SD = Standard deviation; n = Number of observations

Historical control data from experiments performed between May 2015 and May 2017. 

Applicant's summary and conclusion

Conclusions:

An AMES test was performed according to OECD/EC guidelines and GLP principles. Based on the results of this Ames test it is concluded that Menadione is mutagenic in tester strain TA98 of the Salmonella typhimurium reverse mutation assay in the presence of S9-mix. The test item is not mutagenic in tester strain TA98 in the absence of S9-mix, in the other Salmonella typhimurium tester strains (TA1535, TA1537 or TA100) or the Escherichia coli reverse mutation assay using strain WP2uvrA.