(3-ammonio-4-methoxyphenyl)(2-hydroxyethyl)ammonium sulphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Salmonella Typhimurium Reverse Mutation Assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Dec. 13, 2004 to Jan. 10, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted : 21st July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

(3-ammonio-4-methoxyphenyl)(2-hydroxyethyl)ammonium sulphate

EC Number:

280-734-8

EC Name:

(3-ammonio-4-methoxyphenyl)(2-hydroxyethyl)ammonium sulphate

Cas Number:

83763-48-8

Molecular formula:

C9H14N2O2.H2O4S

IUPAC Name:

(3-ammonio-4-methoxyphenyl)(2-hydroxyethyl)ammonium sulphate

Constituent 2

Reference substance name:

Lehmann Blau

IUPAC Name:

Lehmann Blau

Constituent 3

Reference substance name:

2-amino-4-hydroxyethylaminoanisole sulfate

IUPAC Name:

2-amino-4-hydroxyethylaminoanisole sulfate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material : 2-amino-4-hydroxyethylaminoanisole sulfate; Lehmann Blau (Code # A000157)
-TSIN: WR 23081
- Substance type: Pure active substance
- Physical state: Pale grey powder
- Stability under test conditions: The substance on storage in dryness and darkness is considered to be stable till July, 2005.
- Stability in solution: The solutions of the test substance in water and DMSO solution over a total period of seven days can be regarded as stable with low degradation.
- Solubility: The solubility of test substance in different solvents is as follows:
10 g/L in water at pH 2.8 (>5 weight% pH 8)
1 weight% in acetone/water 1:1 (pH 2.1)
9-10 weight% in DMSO
0.2 weight% in ethanol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch # 57 (Sample no. R96000196)
- Expiration date of the lot/batch: July 2005
- Purity test date: 8 January 1996

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, moisture protected, light protected
- Stability under test conditions: The stability over a total period of seven days was tested by HPLC. The test stock solutions (approx. 5 weight %) were stored at room temperature and in a absence of light. Water solution : the results (t=0h: 100.0%; 6h: 93.6%; 2d : 95.1%; 7d : 79.7%) confirm a low degradation. DMSO solution: the results (t=0h: 100.0%; 6h: 98.8%; 2d : 92.0%; 7d : 80.5%) confirm a low degradation
- Solubility and stability of the test substance in the solvent/vehicle: 10g/l in the water pH 2.8 (>5 weight%, pH 8), 1 weight% in acetone water 1:1 (pH 2.1), 9-10 weight% in DMSO, 0.2 weight% in ethanol

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was dissolved in deionised water. The solvent was chosen because of its solubility properties. No precipitation occured up to the highest investigated dose.

Method

Target gene:

Histidine locus serves as a specific target gene of following Salmonella Typhimurium tester strains :
TA 1537: his C 3076 (Frame shift mutation)
TA98: his D 3052 (Frame shift mutation)
TA 1535 and TA 100: his G 46 (Base – pair substitution)
TA 102: his G 428 (Base – pair substitution)

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: Faulty lipopolysaccharide envelope, deficient in the excision-repair system, deletion in the nitrate reductase and biotin genes, R-factor plasmid (error-prone repair) and the ampicillin resistance marker (only for TA 98 and TA 100)

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: R-factor plasmid pKM (error-prone repair); faulty lipopolysaccharide envelope and multicopy plasmid pAQ1 (hisG428 mutation) and tetracycline resistance gene

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

The test substance was tested at following dose levels both with and without metabolic activation:
Pre-Experiment test: 0, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Mutagenicity test (Experiment I and II): 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

concurrent untreated

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

Sodium azide for TA1535 and TA100 (10 µg/plate); methyl methane sulfonate for TA102 (4 µL/plate); 4-nitro-o-phenylone-diamine, 4-NOPD for TA1537 and TA98 (10 µg/plate for strain TA 98; 50 µg/plate for strain TA 1537)

Controls 2

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

2-aminoanthracene (2-AA) for TA98, TA100, TA1535 and TA1537 (2.5 µg/plate) and for TA102 (10 µg/plate)

Details on test system and experimental conditions:

MAINTENANCE OF TESTER STRAIN: The bacterial strains TA 1535, TA 1537, TA 102, TA 100 and TA 98 were obtained from Trinova Biochem GmbH (35394 Gieβen, Germany). The strains were pre-cultured and stored as follows:
- Storage: The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5% DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.
- Precultures: From the thawed ampoules of the strains 0.5 mL bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 µL ampicillin (25 µg/mL) was added to the strains TA 98, TA 100, and TA 102. Additionally 20 µL tetracycline (2 µg/mL) was added to strain TA 102. The nutrient medium contained 8 g Nutrient Broth and 5 g NaCI per litre solution. The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C.

PRE-EXPERIMENT: This experiment was performed to evaluate the toxicity of the test substance with all the tester strains. Eight concentrations (ranging between 3 - 5000 µg/plate) were tested for toxicity and mutation induction in triplicates by plate incorporation method. The pre-experiment was reported as part of Experiment I, since no relevant toxic effects were observed and evaluable plates (>0 colonies) were observed at five concentrations or more in all strains used. Therefore, 5000 µg/plate were chosen as maximal concentration for main mutagenicity experiment.

MAIN MUTAGENICITY EXPERIMENT:
- Experiment I: Plate incorporation method
- Experiment II: Pre-incubation method

EXPERIMENTAL DURATION AND PROCEDURE:
- In plate incorporation assay (Experiment I): 100 µL test solution/solvent /positive control, 500 µL S9 mix/S9 mix substitution buffer, 100 µL bacterial suspension and 2000 µL overlay agar were mixed in a test tube and poured onto the selective agar plate.
- In the pre-incubation assay (Experiment II): 100 µL test solution, 500 µL S9 mix/S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.

NUMBER OF REPLICATIONS: Triplicates/stain/dose level

DETERMINATION OF CYTOTOXICITY
- Cytotoxicity of test substance was detected as reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Evaluation criteria:

- A test substance was considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control was observed.
- A dose dependent increase was considered biologically relevant if the threshold is exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase was not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

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

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MUTAGENICITY EXPERIMENT RESULT (Experiment I and II):
- No substantial increase in revertant colony number was observed in the strains following treatment at any dose level in the presence or absence of S9 mix. There was also no tendency of higher mutation
rates with increasing concentrations in the range below the generally acknowledged border of biological relevance with the exception of strain TA 98 in Experiment I and II.

- In TA 98, a minor increase in revertant colony numbers was observed in Experiment I at 1000 and 2500 µg/plate without S9 mix and at 1000 µg/plate with S9 mix and in Experiment II at 1000 and 2500 µg/plate with S9 mix. However, the required threshold of two times the number of the corresponding solvent control was not reached. Therefore, this minor increase in revertant colony was considered to be based upon biologically irrelevant fluctuations in the number of colonies.

COMPARISON WITH HISTORICAL CONTROL DATA:
The historical solvent control range slightly exceeded in the following strains:

Experiment I (with S9 mix)
- In tester strain TA98 at 333 and 1000 µg/plate.

Experiment II (with S9 mix)
- In tester strain TA98 from 100 up to 2500 µg/plate.
- In tester strain TA1537 at 2500 and 5000 µg/plate.
- In tester strain TA102 at 333 µg/plate.
Due to the fact that a new image analysis system was used for the scoring of the revertant colonies, the historical data was based on 80 experiments. The number of experiment was rather small, therefore colony counts that exceeded the historical data range slightly was acceptable for the controls and test substance.

RESULTS OF POSITIVE AND NEGATIVE CONTROLS:
- The historical control range slightly exceeded in strains TA1535 (negative control) and TA102 (negative control) without S9 mix and in strains TA1537 (negative control) and TA102 (solvent and negative control) with S9 mix. Since the deviation was small, therefore the effect was considered to be based upon biologically irrelevant fluctuations in the number of colonies.
- Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY
Reduced background growth and minor toxic effects (evident as a reduction in the number of revertants) were observed at higher concentrations in nearly all the strains both in the presence and absence of metabolic activation.
Details on reduced background scores and toxic effects are provided in ‘Table 2’ and ‘Table 3’ in “any other information on results incl. tables” section.

Remarks on result:

other: strain/cell type: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 : Summary of results

	Metabolic activation	Test item	Dose level(µg/plate)	Revertant Colony Count (Mean�±SD)
				TA 98	TA 100	TA 1535	TA 1537	TA 102
Pre experiment and Experiment I	Without	Dionisedwater		30�±5	115±1	17±3	10±3	357±22
		Untreated		30±7	119±13	16±6	9±3	314±41
		WR 23081	3	28±6	120±12
			10	35±5	133±6
			33	34±10	128±16	14±6	11±5	336±32
			100	36±9	129±3	16±4	7±1	367±11
			333	41±16	127±8	14±1	21±6	393±20
			1000	51±5	127±8	15±5	9±1	329±20
			2500	51±8	141±20	14±2	12±3	229±30
			5000	18±5	92±9	7±3	3±2	43±10
		Positive Control	10	415±28	2901±71	1477±77	68±15	5588±151
	With	Dionisedwater		41±7	112±18	15±1	16±3	430±19
		Untreated		47±4	110±18	15±2	18±5	428±34
		WR 23081	3	47±7	121±11
			10	50±10	116±3
			33	48±12	112±24	20±11	20±11	492±57
			100	42±8	108±10	16±2	16±2	503±37
			333	63±9	102±21	15±2	15±2	462±28
			1000	74±10	119±4	15±1	15±1	321±33
			2500	58±3	101±23	13±2	13±2	208±15
			5000	19±1	37±9	15±4	15±4	57±15
		Positive Control	10	3528±219	3569±134	584±29	584±29	2445±241
ExperimentII	Without	Dionisedwater		43±8	138±14	26±4	17±2	411±25
		Untreated		37±8	136±3	30±4	16±1	399±4
		WR 23081	33	32±4	158±27	28±2	19±4	378±3
			100	42±12	170±18	29±4	18±3	412±40
			333	38±7	161±6	29±2	21±6	425±24
			1000	43±6	145±19	28±7	20±4	364±19
			2500	39±6	20±2	26±5	25±10	293±30
			5000	0±0	0±0	0±0	na	106±8
		Positive Control	10	317±23	1472±167	1423±13	101±3	2483±232
	With	Dionised water		55±3	196±9	32±7	31±4	547±43
		Untreated		52±9	186±23	34±3	31±4	557±7
		WR 23081	33	54±10	182±9	34±9	34±8	589±32
			100	65±8	180±10	32±3	33±3	515±35
			333	64±3	198±15	29±2	32±5	533±52
			1000	80±7	208±8	37±6	29±4	430±37
			2500	87±11	166±17	36±7	53±7	307±15
			5000	51±11	82±17	37±6	64±13	172±21
		Positive Control	10	2261±297	3060±636	312±25	280±27	2519±109

 

Table 2: Reduced background growth were observed at the following concentrations (µg/plate) (Study # 83827):

Tester strains	Experiment I (µg/plate)		Experiment II (µg/plate)
	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix
TA1535	5000	-	5000	-
TA1537	5000	5000	5000	-
TA98	5000	-	2500-5000	-
TA100	5000	-	2500-5000	-
TA102	5000	5000	5000	5000

Table 3: Minor toxic effectswere observed at the following concentrations (µg/plate) (Study # 83827):

Tester strains	Experiment I (µg/plate)		Experiment II (µg/plate)
	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix
TA1535	5000	-	5000	-
TA1537	5000	-	-	-
TA98	-	-	5000	-
TA100	-	5000	2500-5000	5000
TA102	5000	5000	5000	5000

‘-‘denotes no reduced background growth observed.

Applicant's summary and conclusion

Conclusions:

2-amino-4-hydroxyethylaminoanisole sulfate (Lehmann Blau) when evaluated up to 5000 µg/plate was considered non-mutagenic in the Salmonella typhimurium reverse mutation assay, in the presence or absence of metabolic activation.

Executive summary:

The bacterial reverse mutation test of 2-amino-4-hydroxyethylaminoanisole (Lehmann Blau) was performed by following OECD 471 guideline (Bacterial Reverse Mutation Test) and EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria).

2-Amino-hydroxyethylamino-anisole sulfate dissolved in de-ionised water was tested for mutagenicity in the bacterial gene mutation assay (experiment 1: plate incorporation method, experiment 2: pre-incubation method). The Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 were exposed to the test substance at concentrations ranging from 33 μg/plate to 5000 μg/plate with and without S9-mix (S9-mix from the liver of phenobarbital/ß-naphthoflavone induced male Wistar Hanlbm rats). Test concentrations were selected based on the results obtained in a pre-experiment with strains TA98 and

TA100. For control purposes, untreated, solvent (deionised water), and positive controls (without S9-mix: 4-nitro-o-phenylene-diamine for strains TA98 and TA1537, sodium azide for strains

TA100 and TA1535; methyl methane sulfonate for strain TA102; with S9-mix: 2 - aminoanthracene for all tester strains) were evaluated in parallel.

Results

Reduced background growth was observed at higher concentrations (2500 and 5000 μg/ml) in the presence and absence of S9-mix in almost all strains investigated. No biologically relevant increase in revertant colony numbers was observed in any of the five tester strains following treatment with 2-Amino-hydroxyethylamino-anisole sulfate at any dose level, neither in presence nor in absence of metabolic activation.

Reference mutagens revealed a distinct increase in revertant colonies and demonstrated the sensitivity of the assay.

In conclusion, it can be stated that under the experimental conditions reported 2-Aminohydroxyethylamino- anisole sulfate did not induce gene mutations in Salmonella typhimurium in any of the tester strains in the presence or absence of S9-mix.

This bacterial reverse mutation test is classified as acceptable, and satisfies the guideline requirements of the OECD 471 method.