[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In conclusion, based on the results of this study it is concluded that Red HF2 is mutagenic in the Salmonella typhimurium reverse mutation assay and is not mutagenic in the Escherichia coli reverse mutation assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 September - 16 September 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

GLP study to current guidelines

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Specific details on test material used for the study:

No further details in the study report

Target gene:

his operon (S. typhimurium strains)
trp operon ( E. coli strains)

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA)]
Stock cultures of the five strains were stored in the ultra-low freezer set to maintain -150°C.

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Trinova Biochem GmbH, Germany [Master culture from Dr.
Bruce N. Ames (TA1535, TA1537, TA98, TA100;
Stock cultures of the five strains were stored in the ultra-low freezer set to maintain -150°C.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

Preparation of S9-Mix
S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6 phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 μm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.

Test concentrations with justification for top dose:

1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate

Vehicle / solvent:

Vehicle
The vehicle of the test material was acetone (Merck, Darmstadt, Germany).

The test material formed a red liquid in acetone at a concentration of 100 mg/mL. Due to the dark red colour, it was not possible to determine if the liquid was a solution or a suspension. At a concentration of 1.04 mg/mL and lower, the test material formed a clear red solution. The stock solution was treated with ultrasonic waves to obtain a homogeneous formulation. Test material concentrations were used within 2.5 hours after preparation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other:

Details on test system and experimental conditions:

Test System Salmonella typhimurium bacteria and Escherichia coli bacteria Rationale Recommended test system in international guidelines (e.g. OECD, EC).
Source Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535, TA1537, TA98, TA100; and Master culture from The National Collections of Industrial and Marine
Bacteria, Aberdeen, UK (WP2uvrA)]
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Salmonella typhimurium strains were checked at least every year to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment. The strain was checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants at least every year.
Stock cultures of the five strains were stored in the ultra-low freezer set to maintain -150°C.

Cell Culture
Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/mL).
Freshly grown cultures of each strain were used for a test.

Agar plates
Agar plates (ø 9 cm) contained 25 mL glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin (Merck) and 15 μg/plate histidine (Sigma) and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan (Sigma).

Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 mL top agar were transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

Environmental conditions
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 33.3 - 38.6°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Metabolic Activation System
S9-Fraction
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

Preparation of S9-Mix
S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 μm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.

Mutation Experiment
Eight different doses (increasing with approximately half-log steps) of the test material were tested in triplicate both in the absence and presence of S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. 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 (109 cells/mL) of one of the tester strains, 0.05 ml of a dilution of the test material in acetone or control solution 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 highest concentration of the test material used was 5000 μg/plate.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
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. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Colony Counting
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material 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.

Rationale for test conditions:

Recommended test system in international guidelines (e.g. OECD, EC).

Evaluation criteria:

6. INTERPRETATION
No formal hypothesis testing was done.
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 material 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 material 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.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test material was tested in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix.

Precipitate
The test material was tested up to or beyond a precipitating dose level. The observations of precipitate varied between strains and between exposure conditions, the lowest dose level where precipitate was observed as at 52 ug/plate in TA1535 without activation.

Toxicity
To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed except for strain TA100 where a reduction in mean revertants was observed at the high dose level such that the mean count was below the 95%
lower limit of the historical control data.

Mutagenicity

In tester strain TA1537, in the presence of S9-mix, the test item induced dose-related increases in the number of revertant colonies compared to the solvent control. The increases observed were up to 10- fold the concurrent control. The increases were outside the historical control data range and the increases were more than three-fold the concurrent control.
In tester strain TA98, both in the absence and presence of S9-mix, the test item induced dose related increases in the number of revertant colonies compared to the solvent control. The increases observed were up to 21- fold and 37- fold the concurrent control, in the absence and presence of S9-mix, respectively. The increases were outside the historical control data range and the increases were more than three-fold the concurrent control.
The test material did not induce a dose-related increase in the number of revertant (His+) colonies in tester strains TA1535 and TA100 and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation.

Mutagenic Response of Red HF2 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 different strains of Salmonella typhimurium and one Escherichia coli strain.
	TA1535		TA1537		TA98		TA100		WP2uvrA
	Without S9-mix
Positive control	866	± 25	959	± 24	1125	± 68	911	± 46	1311	± 56
Solvent control	9	± 6	4	± 1	11	± 5	105	± 10	20	± 7
1.7	11	± 1	5	± 2	14	± 4	99	± 10	18	± 7
5.4	12	± 6	4	± 1	15	± 4	103	± 12	21	± 4
17	10	± 4 ^NP	6	± 3	20	± 2	100	± 21	17	± 6
52	8	± 2 ^SP	6	± 2	42	± 6 ^NP	82	± 10 ^NP	21	± 6
164	11	± 5 ^SP	5	± 4 ^NP	95	± 11 ^SP	79	± 4 ^SP	23	± 9 ^NP
512	8	± 3 ^SP	9	± 6 ^SP	182	± 16 ^SP	96	± 10 ^SP	18	± 1 ^SP
1600	11	± 1 ^SP	7	± 4 ^SP	236	± 21 ^SP	78	± 7 ^SP	15	± 1 ^SP
5000	11	± 5 ^n MP	9	± 6 ^n SP	224	± 43 ^n SP	62	± 15 ^n SP	12	± 4 ^n SP
	With S9-mix
Positive control	312	± 29	394	± 98	1038	± 117	1349	± 98	278	± 30
Solvent control	12	± 6	4	± 1	22	± 4	96	± 7	22	± 4
1.7	11	± 1	5	± 4	21	± 8	94	± 16	31	± 5
5.4	13	± 3	7	± 4	24	± 5	102	± 14	23	± 6
17	11	± 1	10	± 2	39	± 3	111	± 9	26	± 4
52	5	± 5	4	± 1	122	± 13	117	± 23	31	± 4^NP
164	11	± 1 ^NP	12	± 3 ^NP	396	± 13 ^NP	139	± 18 ^NP	34	± 6 ^SP
512	9	± 2 ^SP	26	± 10 ^SP	746	± 156 ^SP	142	± 27 ^SP	26	± 3 ^SP
1600	17	± 2 ^SP	39	± 3 ^SP	700	± 73^SP	131	± 9 ^SP	24	± 9^SP
5000	10	± 6 ^n SP	40	± 8 ^n SP	814	± 75 ^n SP	101	± 7 ^n SP	26	± 5 ^n SP

MP Moderate Precipitate
NP No precipitate
SP Slight Precipitate
n Normal bacterial background lawn

Conclusions:

In conclusion, based on the results of this study it is concluded that Red HF2 is mutagenic in the Salmonella typhimurium reverse mutation assay and is not mutagenic in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of Red HF2 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). A direct plate assay was performed.
The study procedures described in this report were based on the most recent OECD and EC guidelines.
Batch SR164:1 of the test material was a dark red solid. The vehicle of the test material was acetone.
In the mutation experiment, the test material was tested up to concentrations of 5000 μg/plate in the strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the direct plate assay. The test material was tested up to or beyond a precipitating 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 except for strain TA100 where a reduction in mean revertants was observed at the high dose level such that the mean count was below the 95% lower limit of the historical control data.
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.
In tester strain TA1537, in the presence of S9-mix, the test item induced dose-related increases in the number of revertant colonies compared to the solvent control. The increases observed were up to 10- fold the concurrent control. The increases were outside the historical control data range and the increases were more than three-fold the concurrent control.
In tester strain TA98, both in the absence and presence of S9-mix, the test item induced dose related increases in the number of revertant colonies compared to the solvent control. The increases observed were up to 21- fold and 37- fold the concurrent control, in the absence and
presence of S9-mix, respectively. The increases were outside the historical control data range and the increases were more than three-fold the concurrent control.
The test material did not induce a dose-related increase in the number of revertant (His+) colonies in tester strains TA1535 and TA100 and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation.
In conclusion, based on the results of this study it is concluded that Red HF2 is mutagenic in the Salmonella typhimurium reverse mutation assay and is not mutagenic in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)