3,4-dihydroxybenzaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria: Key study. Test method according to OECD 471, GLP study. The test item did not induce any mutagenic change in the bacterial reverse mutation test in any of the strains tested with and without metabolic activation up to 5000 μg/plate.

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:

4 July to 22 July 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

In test 1, error during preparation on Methyl methanesulfonate: 9.924 mg/mL instead of 10 mg/mL. In test 2, error during preparation on Amino 9: 1 mg/mL solution instead of 2.5 mg/mL. The results allow the test to be validated.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his D (S. typhimurium TA 98); his C (S. typhimurium TA 1537); his G (S. typhimurium TA 100 and
TA1535); tryp E (E. coli WP2 uvrA pKM101)

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: ΔuvrB and rfa mutated (TA 98 and TA 100: pKM 101)

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : S9 fraction prepared from Sprague Dawley rat liver homogenate and provided by MOLTOXTM (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 - USA).
- method of preparation of S9 mix : 10% S9 fraction, 8 mM MgCL2-6H2O, 33 mM KCl, 5 mM Glucose-6-Phosphate Na2, 4 mM NADP Na2 and 0.1 M Phosphate buffer pH 7.4.
- concentration or volume of S9 mix and S9 in the final culture medium: 500 μL of S9-mix.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Sterility test: 500 μL of S9-mix were added to 2 mL of top agar maintained at 45ºC, and poured after homogenization on the bottom agar (20 ml) onto a Petri plate (90 mm in diameter) (n = 3). Plates were incubated for 48 - 72 hours at 37°C and then examined.

Test concentrations with justification for top dose:

Initial mutation test (plate incorporation) / Confirmatory mutation test (pre-incubation +S9): 50, 150,
500, 1500 and 5000 μg/plate.
In the preliminary cytotoxicity test (strain TA100) no toxicity was found for doses up to 5000 μg/plate. Therefore, the test item was tested at the recommended maximum test concentration of 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test item was found soluble in DMSO at the highest
tested concentration.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO), acetone, NaCl 0.15M

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other:

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
1. Plate incorporation (initial mutation test): In a test tube, 0.1 mL of the bacterial suspension containing 1-9 E09 bacteria/mL and 0.1 mL of each dilution of the original solution and 0.5 mL of sterile phosphate buffer are successively added to 2 mL of overlay agar maintained super cooled at 45ºC containing 10% (v/v) of a L-Histidine-D-Biotine solution (0.5 mM) for Salmonella Typhimurium strains, or containing 5% (v/v) of nutrient broth nº2 to which are added 5 μL of a L-Tryptophane solution at 2 mg/mL for Escherichia coli strain. In the assay with metabolic activation, the protocol is similar to the described above, except that, 500 μL of S9-mix fraction is quickly added, before pouring the mixture onto the plates. After a 48-72 hour incubation period at 37ºC, revertant colonies are counted in each plate.
2. Pre-incubation (confirmatory mutation test +S9): The test item solution with the test strain, and 500μL of S9-mix fraction are preincubated with shaking for 30 min., at 37ºC prior to mixing with the overlay agar and pouring onto the minimal agar plate.. After a 48-72 hour incubation period at 37ºC, revertant colonies are counted in each plate.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 minutes (confirmatory mutation test)
- Exposure duration/duration of treatment: 48-72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Preliminary cytotoxicity test (Strain TA100): In a test tube 0.1 mL of the bacterial suspension (1-9 E03 bacteria /mL) and 0.1 mL of the stock solution and dilutions were successively added to 2mL of top agar at 45ºC, containing 10 % (v/v) of a solution of L-Histidine-D-Biotine (2.5 mM). After homogenization, the content of the tube was poured onto a Petri plate (90 mm in diameter) containing minimal agar (20 mL). 3 plates per concentration were incubated for 48-72 h at 37ºC, and the colonies counted. A negative control containing the blank alone was run in parallel. In case of bacteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75% or less. The precipitate, if present, should not interfere with the scoring.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
In the bacterial reverse mutation test, mutations are detected which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain. After a 48-72-hour incubation period at 37ºC, revertant colonies were manually counted in each plate.
The following ratio was calculated per plate: R = Number of revertant colonies in the presence of the test item / Number of revertant colonies in the absence of the test item.

- OTHER:
- Sterility test: Test item and the corresponding dilutions are added to 2 mL of top agar maintained at 45ºC, and poured after homogenization on the bottom agar (20 mL) onto a Petri plate (90 mm in diameter) (n=3). Plates are incubated for 48-72 hours at 37ºC and then examined. There should be no bacterial growth on any plate. S9-mix sterility is checked using the same protocol.

Rationale for test conditions:

Results of sterility controls show the absence of any bacterial growth in the presence of test item and S9-mix. Results of the bacteriostatic activity control show a toxicity in presence of the test item at 5000μg/plate compatible with the maximum tolerate . Values and frequency are within the laboratory's historical control ranges.

Evaluation criteria:

The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of sp ontaneous reversions for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101) strains without and with metabolic activation.

The result of the test is considered positive if a dependent relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being t
aken into account for a given dilution of test item if the number of revertant colonies is at least two fold that of spontaneous revertant colonies for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101), and three fold for TA 1535 and TA 1537. All results must be confirmed in an independent experiment.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS : None observed.

RANGE-FINDING/SCREENING STUDIES: In the preliminary cytotoxicity test (strain TA100) no toxicity was found for doses up to 5000 μg/plate. Therefore, the test item was tested at the recommended maximum test concentration of 5000 μg/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: see tables below.

Ames test:
- Signs of toxicity: see table 5 below.
- Individual plate counts: see table 5 below.
- Mean number of revertant colonies per plate and standard deviation: see table 5 below.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see table 6 below
- Negative (solvent/vehicle) historical control data: see table 6 below
- There is no significant difference between the number of spontaneous reversions, the number of
reversions obtained in the positive controls (without and with metabolic activation), and the mean of
corresponding experimental “historical” values obtained in the laboratory.

Table 3. Sterility control.

 

Serie	Doses	Colony number/plate
Control n° 1		1	2	3
Solution of   RFL-1 BATCH M21736C (Identification code: PH-22/0351)	5000 µg /plate	0	0	0
	1500 µg /plate	0	0	0
	500 µg /plate	0	0	0
	150 µg /plate	0	0	0
	50 µg /plate	0	0	0
S9-mix	500 µL/plate	0	0	0
Control n° 2		1	2	3
Solution of   RFL-1 BATCH M21736C (Identification code: PH-22/0351)	5000 µg /plate	0	0	0
	1500 µg /plate	0	0	0
	500 µg /plate	0	0	0
	150 µg /plate	0	0	0
	50 µg /plate	0	0	0
S9-mix	500 µL/plate	0	0	0

 

Table 4. Bacteriostatic activity controls.

 

		Doses (/plate)
		0 (negative control)			DMSO			50 µg			150 µg			500 µg			1 500 µg	5000 µg
	N1		768			789			805			806			698		796		303
Solution of	N2		818			697			768			819			753		818		431
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	N3		740			766			746			780			760		813		330
	N	775	±	40	751	±	48	773	±	30	802	±	20	737	±	34	778±16	355	±	67
	%		-			97%			100%			103%			95%		104%		46%

 

		Doses (/plate)
		0 (negative control)			DMSO			50 µg			150 µg			500 µg			1 500 µg	5 000 µg
	N1		812			795			785			820			775		745		452
Solution of	N2		823			786			769			786			810		852		362
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	N3		825			796			845			810			753		793		398
	N	820	±	7	792	±	6	800	±	40	805	±	17	779	±	29	797±54	404	±	45
	%		-			97%			98%			98%			102%		97%		100%

 

Table 5. Result tables.

 

TA1535 Assay n°1 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	4	17	11	10.67	6.51	_
Positive control solvent	5 µL	11	7	14	10.67	3.51	_
Positive control : Sodium azide	5 µg in 5 µL	764	842	887	831.00	62.23	77.91
Vehicle	50µL	15	11	9	11.67	3.06	_
	5000 µg	0	2	0	0.67	1.15	0.06
Solution of	1500 µg	11	5	13	9.67	4.16	0.83
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	16	11	10	12.33	3.21	1.06
	150 µg	5	15	11	10.33	5.03	0.89
	50 µg	10	14	18	14.00	4.00	1.20

 

TA1535 Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	13	7	10	10.00	3.00	_
Positive control solvent	20 µL	7	12	6	8.33	3.21	_
Positive control : 2-Anthramine	2 µg in 20 µL	43	63	48	51.33	10.41	6.16
Vehicle	50µL	15	13	12	13.33	1.53	_
	5000 µg	1	3	2	2.00	1.00	0.15
Solution of	1500 µg	11	11	12	11.33	0.58	0.85
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	10	9	7	8.67	1.53	0.65
	150 µg	18	11	14	14.33	3.51	1.08
	50 µg	13	19	11	14.33	4.16	1.08

 

TA1535 Assay n°2 – without metabolic activation (-S9 mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	19	16	21	18.67	2.52	_
Positive control solvent	5 µL	15	14	21	16.67	3.79	_
Positive control : Sodium azide	5 µg in 5 µL	671	722	620	671.00	51.00	40.26
Vehicle	50µL	20	16	17	17.67	2.08	_
	5000 µg	1	3	0	1.33	1.53	0.08
Solution of	1500 µg	8	11	10	9.67	1.53	0.55
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	13	12	8	11.00	2.65	0.62
	150 µg	18	15	16	16.33	1.53	0.92
	50 µg	20	16	17	17.67	2.08	1.00

 

TA1535 Assay n°2 – with metabolic activation (10 % S9-mix) – with pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	7	16	13	12.00	4.58	_
Positive control solvent	10 µL	10	8	10	9.33	1.15	_
Positive control : 2-Anthramine	1 µg in 10 µL	105	112	92	103.00	10.15	11.04
Vehicle	50µL	15	12	18	15.00	3.00	_
	5000 µg	1	2	1	1.33	0.58	0.09
Solution of	1500 µg	15	10	5	10.00	5.00	0.67
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	17	15	16	16.00	1.00	1.07
	150 µg	18	20	21	19.67	1.53	1.31
	50 µg	24	15	20	19.67	4.51	1.31

 

TA1537 Assay n°1 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	8	8	6	7.33	1.15	_
Positive control solvent	20 µL	11	12	5	9.33	3.79	_
Positive control : 9-Aminoacridine	50 µg	813	620	923	785.33	153.38	217.29
Vehicle	50µL	10	13	9	10.67	2.08	_
	5000 µg	1	2	4	2.33	1.53	0.29
Solution of	1500 µg	10	10	8	9.33	1.15	0.67
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	8	8	6	7.33	1.15	0.75
	150 µg	7	7	14	9.33	4.04	0.67
	50 µg	7	11	10	9.33	2.08	0.75

 

TA1537 Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	8	11	5	8.00	3.00	_
Positive control solvent	20 µL	8	16	13	9.00	3.61	_
Positive control : 2-Anthramine	2 µg	531	603	432	522.00	85.85	58.00
Vehicle	50µL	7	6	811	8.00	2.65	_
	5000 µg	5	1	3	3.00	2.00	0.38
Solution of	1500 µg	7	11	9	9.00	2.00	1.13
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	11	18	5	8.00	3.00	1.00
	150 µg	8	10	10	9.33	1.15	1.17
	50 µg	9	8	141	9.33	1.53	1.17

 

TA1537 Assay n°2 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	6	7	5	6.00	1.00	_
Positive control solvent	20 µL	8	6	6	6.67	1.15	_
Positive control : 9-Aminoacridine	20 µL	862	1280	1480	1207.33	315.34	181.10
Vehicle	50µL	7	5	9	7.00	2.00	_
	5000 µg	3	2	2	2.33	0.58	0.33
Solution of	1500 µg	4	6	5	5.00	1.00	0.71
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	5	4	7	5.33	1.53	0.76
	150 µg	6	7	5	6.00	1.00	0.86
	50 µg	5	3	6	4.67	1.53	0.67

 

TA1537 Assay n°2 – with metabolic activation (10% S9-mix) – with pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	10	12	10	10.67	1.15	_
Positive control solvent	10 µL	12	8	11	10.33	2.08	_
Positive control : 2-Anthramine	1 µg	42	39	51	44.00	6.24	4.26
Vehicle	50µL	10	10	6	8.67	2.31	_
	5000 µg	5	7	12	8.00	3.61	0.92
Solution of	1500 µg	9	9	8	8.67	0.58	1.00
RFL-1 BATCH (Identification code: PH-22/0351)	500 µg	13	10	10	11.00	1.73	1.27
	150 µg	12	16	12	13.33	2.31	1.54
LEMI code : 20/0151-250520-S1	50 µg	14	16	10	13.33	3.06	1.54

 

TA98 Assay n°1 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	25	23	21	23.00	2.00	_
Positive control solvent	20 µL	18	19	20	19.00	1.00	_
Positive control : 2-Nitrofluorene	2 µg	608	545	703	618.67	79.54	32.56
Vehicle	50µL	20	16	24	20.00	4.00	_
	5000 µg	7	6	6	6.33	0.58	0.32
Solution of	1500 µg	17	22	21	20.00	2.65	1.00
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	23	16	14	17.67	4.73	0.88
	150 µg	17	19	25	20.33	4.16	1.02
	50 µg	24	19	18	20.33	3.21	1.02

 

TA98 Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	30	31	34	31.67	2.08	_
Positive control solvent	20 µL	29	32	27	29.33	2.52	_
Positive control : 2-Anthramine	2 µg	988	964	997	983.00	17.06	33.51
Vehicle	50µL	32	32	33	32.33	0.58	_
	5000 µg	18	11	18	15.67	4.04	0.48
Solution of	1500 µg	23	32	18	24.33	7.09	0.75
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	24	34	33	30.33	5.51	0.94
	150 µg	22	29	23	24.67	3.79	0.76
	50 µg	29	32	28	29.67	2.08	0.92

 

TA98 Assay n°2 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	28	25	29	27.33	2.08	_
Positive control solvent	20 µL	25	23	26	24.67	1.53	_
Positive control : 2-Nitrofluorene	2 µg	575	336	436	449.00	120.03	18.20
Vehicle	50µL	24	22	24	23.33	1.15	_
	5000 µg	10	10	8	9.33	1.15	0.40
Solution of	1500 µg	21	18	23	20.67	2.52	0.89
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	21	25	19	21.67	3.06	0.93
	150 µg	26	24	20	23.33	3.06	1.00
	50 µg	19	20	25	21.33	3.21	0.91

 

TA98 Assay n°2 – with metabolic activation (10 % S9-mix) – with pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	34	36	31	33.67	2.52	_
Positive control solvent	10 µL	35	25	28	29.33	5.13	_
Positive control : 2-Anthramine	1 µg	337	391	582	436.67	128.73	14.89
Vehicle	50µL	32	36	35	34.33	2.08	_
	5000 µg	10	20	8	12.67	6.43	0.37
Solution of	1500 µg	26	28	30	28.00	2.00	0.82
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	30	34	28	30.67	3.06	0.89
	150 µg	37	33	30	33.33	3.51	0.97
	50 µg	30	35	34	33.00	2.65	0.96

 

TA100 Assay n°1 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	69	61	87	72.33	13.32	_
Positive control solvent	20 µL	70	75	81	75.33	5.51	_
Positive control : Sodium azide	20 µg	1603	1341	1350	1431.33	148.74	19.00
Vehicle	50µL	89	95	70	84.67	13.05	_
	5000 µg	72	76	7	5.33	2.08	0.06
Solution of	1500 µg	93	75	65	65.67	4.04	0.78
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	73	79	96	75.67	17.79	0.89
	150 µg	71	85	89	87.33	6.66	1.03
	50 µg	82	80	84	77.00	6.08	0.91

 

TA100 Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	68	96	102	88.67	18.15	_
Positive control solvent	20 µL	81	94	98	91.00	8.89	_
Positive control : 2-Anthramine	2 µg	893	840	905	879.33	34.59	9.66
Vehicle	50µL	120	69	110	99.67	27.02	_
	5000 µg	11	8	8	9.00	1.73	0.09
Solution of	1500 µg	57	57	55	56.33	1.15	0.57
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	57	63	62	60.67	3.21	0.61
	150 µg	72	77	80	76.33	4.04	0.77
	50 µg	62	85	91	79.33	15.31	0.80

 

TA100 Assay n°2 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	71	60	91	74.00	15.72	_
Positive control solvent	20 µL	63	71	69	67.67	4.16	_
Positive control : Sodium azide	20 µg	934	946	1045	975.00	60.92	14.41
Vehicle	50µL	61	59	70	63.33	5.86	_
	5000 µg	5	8	9	7.33	2.08	0.12
Solution of	1500 µg	63	75	64	67.33	6.66	1.06
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	66	62	84	70.67	11.72	1.12
	150 µg	80	79	78	79.00	1.00	1.25
	50 µg	73	75	83	77.00	5.29	1.22

 

TA100 Assay n°2 – with metabolic activation (10 % S9-mix) – with pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	68	72	65	68.33	3.51	_
Positive control solvent	10 µL	52	52	51	51.67	0.58	_
Positive control : 2-Anthramine	1 µg	570	507	599	558.67	47.04	10.81
Vehicle	50µL	63	57	77	65.67	10.26	_
	5000 µg	9	21	1	10.33	10.07	0.16
Solution of	1500 µg	52	68	73	64.33	10.97	0.98
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	81	74	76	77.00	3.61	1.17
	150 µg	71	75	84	76.67	6.66	1.17
	50 µg	99	96	80	91.67	10.21	1.40

 

E. COLI Assay n°1 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	107	82	76	84.33	16.44	_
Positive control solvent	10 µL	98	87	81	88.67	8.62	_
Positive control : cis-Platinum (II)	99.24 µg	343	456	511	436.67	85.65	4.92
Vehicle	50µL	76	46	75	65.67	17.04	_
	5000 µg	37	25	39	33.67	7.57	0.51
Solution of	1500 µg	60	47	59	55.33	7.23	0.84
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	65	81	80	75.33	8.96	1.15
	150 µg	87	70	81	79.33	8.62	1.21
	50 µg	71	73	83	75.67	6.43	1.15

 

E. COLI Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	91	101	95	95.67	5.03	_
Positive control solvent	5 µL	97	119	96	104.00	13.00	_
Positive control : 2-Anthramine	50 µg	799	896	904	866.33	58.45	8.33
Vehicle	50µL	98	77	67	80.67	15.82	_
	5000 µg	12	19	19	16.67	4.04	0.21
Solution of	1500 µg	45	42	42	43.00	1.73	0.53
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	65	63	74	67.33	5.86	0.83
	150 µg	71	98	110	93.00	19.97	1.15
	50 µg	81	85	97	87.67	8.33	1.09

 

E. COLI Assay n°2 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	83	66	86	78.33	10.79	_
Positive control solvent	10 µL	88	81	74	81.00	7.00	_
Positive control : MMS	100 µg	476	518	486	493.33	21.94	6.09
Vehicle	50µL	70	79	71	73.33	4.93	_
	5000 µg	25	31	26	27.33	3.21	0.37
Solution of	1500 µg	65	69	46	60.00	12.29	0.82
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	69	72	78	71.67	2.52	0.98
	150 µg	69	72	78	73.00	4.58	1.00
	50 µg	68	70	82	73.33	7.57	1.00

 

E. COLI Assay n°2 – with metabolic activation (10 % S9-mix) – with pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	95	91	81	89.00	7.21	_
Positive control solvent	12.5 µL	97	101	91	96.33	5.03	_
Positive control : 2-Anthramine	12.5 µg	1001	965	1034	1000.00	34.51	10.38
Vehicle	50µL	105	97	76	92.67	14.98	_
	5000 µg	13	22	15	16.67	4.73	0.18
Solution of	1500 µg	61	62	63	62.00	1.00	0.67
RFL-1 BATCH M21736C (Identification code: PH-22/0351)	500 µg	65	64	69	66.00	2.65	0.71
	150 µg	91	96	62	83.00	18.36	0.90
	50 µg	98	91	90	93.00	4.36	1.00

Conclusions:

The test item did not induce any mutagenic change in the bacterial reverse mutation test in any of the strains tested with and without metabolic activation up to 5000 μg/plate.

Executive summary:

A bacterial reverse mutation test was conducted on the test substance according to OECD guideline 471 under GLP conditions. Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA were exposed to concentrations of the test substance ranging from 50 to 5000 μg/plate in DMSO, with and without metabolic activation, based on preliminary solubility and cytotoxicity tests. The metabolic activation system (S9 fraction) was prepared from Sprague Dawley rat liver homogenate. Two independent assays were performed in all strains: an initial mutation test (plate incorporation method) and a confirmatory mutation test (plate incorporation method without S9 and pre-incubation method with S9). Untreated, solvent controls and strain specific positive controls were included in the assays and the values obtained were within ranges of the historical control values of the laboratory in all strains. All validity criteria were fulfilled. The test item did not induce any significant increase in the number of revertants in any of the strains tested, with and without metabolic activation, up to 5000 μg/plate. Based on these results, the test item can be considered as not mutagenic according to the OECD TG 471.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data (negative Ames test), the test substance is not classified for mutagenecity in accordance with CLP Regulation (EC) No. 1272/2008.