Sodium m-[[4-[[p-[bis(2-hydroxyethyl)amino]phenyl]azo]-1-naphthyl]azo]benzenesulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 05 to April 11, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

OECD Guideline For Testing Of Chemicals, 476 "Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Test". Adopted: July 21st, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

EEC-Direktive 2000/32/EC, L 136, Annex 4E, B.17: "Mutagenicity -In Vitro Mammalian Cell Gene Mutation Test". Adopted: May 19th, 2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

U.S. Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.5300, In Vitro Mammalian Cell Gene Mutation Test, August 1998

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

Sodium m-[[4-[[p-[bis(2-hydroxyethyl)amino]phenyl]azo]-1-naphthyl]azo]benzenesulphonate

EC Number:

266-867-4

EC Name:

Sodium m-[[4-[[p-[bis(2-hydroxyethyl)amino]phenyl]azo]-1-naphthyl]azo]benzenesulphonate

Cas Number:

67674-28-6

Molecular formula:

C26H25N5O5S.Na

IUPAC Name:

sodium m-[[4-[[p-[bis(2-hydroxyethyl)amino]phenyl]azo]-1-naphthyl]azo]benzenesulphonate

Test material form:

solid: particulate/powder

Details on test material:

Acid Red 299

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Source of cells: cell bank of "Genetic Toxicology", Aventis Pharma Deutschland GmbH, ProTox
Test organism: cell line V79 of Chinese hamster lung fibroblasts
Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The compound was suspended in cell culture medium and tested at the following concentrations:
without S9-mix: 160, 500, 1600 and 5000 μg/ml
with S9-mix: 160, 500, 1600 and 5000 μg/ml
In a preliminary experiment for solubility Telon Rubin A5B 01 was studied with respect to its solubility in hepatocyte attachment medium. The highest evaluable concentration was found to be 5000 ug/ml. Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 5000 ug/ml and a range of lower dose levels down to 10 ug/ml. Following treatment in the presence and in the absence of S9 metabolic activation, moderate toxicity was observed at the highest concentration of 5000 ug/ml. On the basis of these results, a concentration of 5000 ug/ml was selected for the main assays and three lower dose levels down to 160 ug/ml were included in the treatment series.

Vehicle / solvent:

Formulation of test compound: suspended in cell culture medium at appropriate concentrations immediately before use.

Formulation of reference compounds:
EMS dissolved in cell culture medium on the day of treatment, final concentration: 1.0 mg/ml = 8 mM.
DMBA dissolved in DMSO and frozen in small portions. Aliquot thawed on the day of treatment,
final concentration in cell culture medium: 7.7 μg/ml = 30 μM

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Toxicity experiments and dose range finding
A preliminary toxicity test was undertaken in order to select appropriate dose levels for the mutation assay. In this test a wide range of dose levels of test compound was used.
Cell cultures were subjected to the same treatment conditions as in mutation assays, and the survival of the cells was subsequently determined.
The test included the following treatments:
Solvent control: the maximum final concentration of organic solvents will not exceed approx. 1 % (v/v).
Test compound: the highest dose level for the preliminary toxicity test was determined by the solubility of the test compound up to the maximum of 10 mM or 5000 μg/ml.
Treatments were performed both in the presence and absence of S9 metabolic activation system using duplicate doses at each test point.

Test procedure
In preliminary toxicity experiments approximately 1500 cells were seeded in each well of a microliter plate, allowed to attach overnight and then exposed to the test and control compound for four hours.
For each concentration at least 6 wells were used. After 4 or 5 days, the cells were fixed and stained with crystal violet.
Survival was determined by measurement of the crystal violet extinction.
In the main mutation experiments the cultures for assessing toxicity were prepared and treated with the test compound in the same way as for the preliminary experiment. 24 hours after seeding of approx. 1500 cells per well in a microtiter plate, the medium was replaced with serum-reduced (5 % v/v) medium containing the test compound to which either buffer or S9-mix was added as appropriate. After 4 hours the treatment medium was removed and the cells were rinsed twice with normal medium. Thereafter normal medium was added to the wells. The cultures were stained with crystal violet and survival was determined after an incubation period of 4 or 5 days.

Mutagenicity test
Two independent mutation tests were performed.
Exponentially growing cultures which were more than 50% confluent were trypsinated by an approx. 0.25 % (v/v) trypsin ready for use (mfr. Gibco).
A single cell suspension was prepared.
Subsequently the cells were replated to determine the mutation frequency and plating efficiency. The procedures used were as described in Test Procedure
The treatment schedule of the mutagenicity test is described below:
Day 1: Subculturing of an exponentially growing culture
a) Approx. 1500 cells in each well of a micro titer plate for determination of the plating efficiency.
b) 6E+5 – 1E+6 cells in 182 cm2 flasks with 30 ml medium for the mutagenicity test, one flask per experimental point.
Day 2: Treatment of a) and b) with the test compound in the presence and absence of S9-mix (final protein concentration: approx. 0.15 mg/ml) for 4 hours.
Day 5 or 6: Fixation and staining of the cells in a) for the determination of the plating efficiency Subculturing of b) in 182 cm2 flasks
Day 9: Subculturing of b) in five 75 cm2 flasks with culture medium containing 6-thioguanine:
Mutant selection (about 300 000 cellss/flask); subculturing of b) in two 25 cm2 flasks for plating efficiency (about 400 cells per flask)
Day 16: Fixation and staining of colonies of b) - from subcultures seeded on day 9.
All incubations were carried out at approx. 37 °C and 4 % CO2.
Staining was performed with approx. 10 % (v/v) methylene blue in approx. 0.01 % (w/v) KOH solution.
Only colonies with more than 50 cells were counted.

Rationale for test conditions:

In accordance with test guidelines.

Evaluation criteria:

Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range
- the plating efficacy for the solvent control was greater than 50 %

Criteria for a positive response
The test compound is classified as mutagenic if:
- it reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.
- there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from the enhancement factor for induced mutants.
- survival of the responding dose group is at least 30 %.
However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.

Statistics:

The biometry of the results for the test compound is performed off-line with the MANN WHITNEY-U-TEST

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

Solubility and toxicity
In a preliminary experiment for solubility Acid Red 299 was studied with respect to its solubility in hepatocyte attachment medium. The highest evaluable concentration was found to be 5000 μg/ml.
Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 5000 μg/ml and a range of lower dose levels down to 10 μg/ml.
Following treatment in the presence and in the absence of S9 metabolic activation, moderate toxicity was observed at the highest concentration of 5000 μg/ml.
On the basis of these results, a concentration of 5000 μg/ml was selected for the main assays and three lower dose levels down to 160 μg/ml were included in the treatment series.

Mutagenicity
Experimental design
Two independent mutation assays to examine resistance to 6-thioguanine were performed.
In the presence and in the absence of S9 metabolic activation dose levels of 160, 500, 1600 and 5000 μg/ml were used.
Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test compound solutions did not have any effect on these parameters.

Survival after treatment
In the presence and in the absence of 89 metabolic activation survival was slightly reduced in the second mutation experiment with the concentration of 5000 μg/ml.
In the presence of S9-mix a decrease in the survival rate was also observed with the concentration of 500 μg/ml in both mutation experiments.

Mutation results
The test compound was assessed for its mutagenic potential in vitro in the HPRT-test in two independent experiments without metabolic activation and two independent experiments with metabolic activation.
In the presence of metabolic activation with the toxic concentration of 500 μg/ml a slight enhancement of the mutation rate over the range of the solvent control was induced, which did not fulfil the criteria for a positive response.
In addition, this effect was based mainly on one of the duplicate cultures. Beside this no concentration-related increases were observed and therefore the effect is considered to be without biological relevance.
No relevant reproducible increases in the mutant colonies or mutant frequency over the range of the solvent control was found with any other of the investigated concentrations, either with or without metabolic activation by S9-mix.
The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

Any other information on results incl. tables

Toxicity Data (Preliminary experiment)

	Dose μg/ml	S9-mix	Extinction in microwell plates mean less blank values	Standard deviation	Relative survival*
Solvent control	0	-	2.403	0.10	100.0
Telon Rubin	10 50 100 250 500 1000 2500 5000	- - - - - - - -	2.315 2.248 2.295 2.396 2.325 2.452 2.349 1.512	0.15 0.12 0.09 0.05 0.06 0.09 0.10 0.17	96.3 § 93.5 #§ 95.5 #§ 99.7 #§ 96.6 #§ 102.0 #§ 97.8 #§ 62.9 #§

 

	Dose μg/ml	S9-mix	Extinction in microwell plates mean less blank values	Standard deviation	Relative survival*
Solvent control	0	+	2.416	0.06	100.0
Telon Rubin	10 50 100 250 500 1000 2500 5000	+ + + + + + + +	2.415 2.360 2.364 2.076 1.973 2.355 2.357 1.777	0.04 0.06 0.07 0.20 0.33 0.06 0.06 0.10	99.9 § 97.7 #§ 97.8 #§ 85.9 #§ 81.7 #§ 97.5 #§ 97.5 #§ 73.5 #§

# macroscopic precipitate§ microscopic precipitate

*= relative survival (mean value/mean value corresponding control x 100)

Solvent = MEM

 

Toxicity Data (First main mutation experiment)

	Dose μg/ml	S9-mix	Extinction in microwell plates mean less blank values	Standard deviation	Relative survival*
Solvent control	0	-	2.143	0.07	100.0
Positive control	1000	-	1.784	0.06	83.2
Telon Rubin	160 500 1600 5000	- - - -	2.158 2.135 2.002 1.717	0.12 0.12 0.08 0.13	100.7 #§ 99.7 #§ 93.4 #§ 80.1 #§

 

	Dose μg/ml	S9-mix	Extinction in microwell plates mean less blank values	Standard deviation	Relative survival*
Solvent control	0	+	2.081	0.19	100.0
Positive control	7.7	+	1.882	0.07	90.4
Telon Rubin	160 500 1600 5000	+ + + +	2.146 1.67 1.731 1.688	0.08 0.19 0.07 0.09	103.1 #§ 56.1 #§ 83.2 #§ 81.1 #§

# macroscopic precipitate§ microscopic precipitate

*= relative survival (mean value/mean value corresponding control x 100)

Solvent = MEM

Positive control without S9-mix = EMS

Positive control with S9-mix = DMBA

 

Toxicity Data (Second main mutation experiment)

	Dose μg/ml	S9-mix	Extinction in microwell plates mean less blank values	Standard deviation	Relative survival*
Solvent control	0	-	2.210	0.10	100.0
Positive control	1000	-	1.694	0.147	76.6
Telon Rubin A5B 01	160 500 1600 5000	- - - -	2.235 2.197 2.091 1.625	0.15 0.07 0.15 0.15	101.1 #§ 99.4 #§ 94.6 #§ 73.6 #§

 

	Dose μg/ml	S9-mix	Extinction in microwell plates mean less blank values	Standard deviation	Relative survival*
Solvent control	0	+	2.030	0.34	100.0
Positive control	7.7	+	2.000	0.12	98.5
Telon Rubin A5B 01	160 500 1600 5000	+ + + +	0.1940 0.246 2.149 1.295	0.12 0.04 0.12 0.27	95.6 #§ 12.1 #§ 105.8 #§ 63.8 #§

# macroscopic precipitate§ microscopic precipitate

*= relative survival (mean value/mean value corresponding control x 100)

Solvent = MEM

Positive control without S9-mix = EMS

Positive control with S9-mix = DMBA

 

Mutagenicity Data – Part 1 (First main mutation experiment)

	Dose μg/ml	S9-mix	Number of cells per flask				Factor* calculated	Cells** seeded	Cells*** survived
			Seeded	Found		Mean
			I / II	I	II
Solvent control 1 (MEM)	0.0	-	396	254.0	257.5	255.8	0.65	302250	195203
Solvent control 2 (MEM)	0.0	-	399	254.0	259.5	256.8	0.64	330150	212446
Positive control (EMS)	1000.0	-	404	239.0	247.0	243.0	0.60	315000	189468
Telon Rubin A5B 01	160.0	-	403	328.3	316.5	322.4	0.80	280800	224640
	160.0	-	402	217.5	218.0	217.8	0.54	327150	177206
	500.0	-	399	279.0	280.5	279.8	0.70	318900	223590
	500.0	-	398	278.5	267.0	272.8	0.69	290400	199012
	1600.0	-	400	263.0	271.7	267.4	0.67	299700	200312
	1660.0	-	399	293.0	283.0	288.0	0.72	321150	231808
	5000.0	-	395	253.5	248.0	250.8	0.63	278100	176541
	5000.0	-	393	251.5	259.5	255.5	0.65	351900	228780
Solvent control 1 (MEM)	0.0	+	400	246.7	245.7	246.2	0.62	328350	202099
Solvent control 2 (MEM)	0.0	+	407	223.0	222.0	222.5	0.55	345900	189098
Positive control (DMBA)	7.7	+	401	226.0	213.5	219.8	0.55	311400	170649
Telon Rubin A5B 01	160.0	+	404	298.0	296.8	297.4	0.74	342600	252201
	160.0	+	399	273.0	275.5	274.3	0.69	252500	242289
	500.0	+	399	258.0	252.5	255.3	0.64	336600	215331
	500.0	+	397	280.3	282.0	281.2	0.71	353250	250167
	1600.0	+	397	271.5	265.0	268.3	0.68	318150	214972
	1600.0	+	402	269.5	285.7	277.6	0.69	327300	226016
	5000.0	+	400	283.0	294.0	288.5	0.72	351750	253700
	5000.0	+	402	291.0	293.0	292.0	0.73	342900	249072

* factor calculated: mean value / number of cells per flask seeded

** cells seeded in 6-thioguanine (TG) containing medium

*** cells survived after plating in (TG) containing medium (cells seeded x factor calculated)

 

Mutagenicity Data – Part 1 (Second main mutation experiment)

	Dose μg/ml	S9-mix	Number of cells per flask				Factor* calculated	Cells** seeded	Cells*** survived
			Seeded	Found		Mean
			I / II	I	II
Solvent control 1 (MEM)	0.0	-	400	308.8	308.0	308.4	0.77	356850	275131
Solvent control 2 (MEM)	0.0	-	397	310.8	292.0	301.4	0.76	289650	219901
Positive control (EMS)	1000.0	-	400	280.0	282.0	281.0	0.70	315150	221393
Telon Rubin A5B 01	160.0	-	400	266.5	285.0	275.8	0.69	330750	228011
	160.0	-	399	307.3	308.0	307.7	0.77	351600	271102
	500.0	-	401	411.0	397.5	404.3	1.01	320850	323450
	500.0	-	402	314.8	306.0	310.4	0.77	326850	252374
	1600.0	-	398	271.5	268.0	269.8	0.68	340200	230575
	1660.0	-	395	230.0	227.0	228.5	0.58	296100	171288
	5000.0	-	407	285.0	273.0	279.0	0.69	368500	184058
	5000.0	-	399	286.5	288.0	287.3	0.72	340200	244918
Solvent control 1 (MEM)	0.0	+	397	225.5	213.0	219.3	0.55	326850	180508
Solvent control 2 (MEM)	0.0	+	404	325.0	314.0	319.5	0.79	332250	262757
Positive control (DMBA)	7.7	+	399	270.0	274.0	272.0	0.68	350700	239074
Telon Rubin A5B 01	160.0	+	402	287.0	287.0	287.0	0.71	325200	232170
	160.0	+	395	267.0	265.0	266.0	0.67	309750	208591
	500.0	+	402	292.3	304.0	298.2	0.74	337350	250201
	500.0	+	402	268.0	271.0	269.5	0.67	344850	231197
	1600.0	+	402	317.5	315.5	316.5	0.79	328650	258751
	1600.0	+	400	294.5	274.7	284.6	0.71	325350	231487
	5000.0	+	403	264.5	254.0	259.3	0.64	332100	213640
	5000.0	+	407	299.9	295.0	297.5	0.73	297750	217606

* factor calculated: mean value / number of cells per flask seeded

** cells seeded in 6-thioguanine (TG) containing medium

*** cells survived after plating in (TG) containing medium (cells seeded x factor calculated)

 

Mutagenicity Data – Part 2 (First main mutation experiment)

	Dose μg/ml	S9-mix	Number of mutant colonies						Standard deviation	Mutation frequency		Stat. sig.
			I	II	III	IV	V	Mean			ẍ
Solvent control 1 (MEM)	0	-	4	3	3	3	5	3.6	0.89	18.4	21.5
Solvent control 2 (MEM)	0	-	6	3	4	4	9	5.2	2.39	24.5
Positive control (EMS)	1000	-	213	195	209	203	215	207.0	8.12	1092.5		*
Telon Rubin A5B 01	160	-	2	7	5	5	1	4.0	2.45	17.8	28.7
	160	-	7	4	8	3	13	7.0	3.94	39.5
	500	-	3	3	3	1	5	3.0	1.41	13.4	11.7
	500	-	1	4	2	1	2	2.0	1.22	10.0
	1600	-	0	3	3	7	2	3.0	2.55	15.0	22.2
	1600	-	6	10	7	4	7	6.8	2.17	29.3
	5000	-	3	5	4	6	7	5.0	1.58	28.3	23.4
	5000	-	4	4	4	7	2	4.2	1.79	18.4
Solvent control 1 (MEM)	0	+	7	4	6	4	6	5.4	1.34	26.7	24.5
Solvent control 2 (MEM)	0	+	5	3	4	4	5	4.2	0.84	22.2
Positive control (DMBA)	7.7	+	28	24	20	36	28	27.2	5.93	159.4		*
Telon Rubin A5B 01	160	+	4	5	1	6	4	4.0	1.87	15.9	22.0
	160	+	6	7	6	9	6	6.8	1.30	28.1
	500	+	10	15	10	16	15	13.2	2.95	61.3	41.5
	500	+	5	7	6	6	3	5.4	1.52	21.6
	1600	+	3	8	3	4	2	4.0	2.35	18.6	17.3
	1600	+	5	4	5	3	1	3.6	1.67	15.9
	5000	+	4	6	5	5	5	5.0	0.71	19.7	17.5
	5000	+	5	6	3	3	2	3.8	1.64	15.3

Mutation frequency (mutant colonies per 1 million cells): mean value / cells surviving

*Statistical significant (p<0.05) Mann-Whitney-U-Test

 

Mutagenicity Data – Part 2 (Second main mutation experiment)

	Dose μg/ml	S9-mix	Number of mutant colonies						Standard deviation	Mutation frequency		Stat. sig.
			I	II	III	IV	V	Mean			ẍ
Solvent control 1 (MEM)	0	-	6	4	7	8	1	5.2	2.77	18.9	21.3
Solvent control 2 (MEM)	0	-	6	6	3	5	6	5.2	1.30	23.6
Positive control (EMS)	1000	-	110	97	118	131	114	114.0	12.35	514.9		*
Telon Rubin A5B 01	160	-	7	2	5	4	5	4.6	1.82	20.2	13.4
	160	-	0	2	1	3	3	1.8	1.30	6.6
	500	-	1	1	6	4	1	2.6	2.30	8.0	10.4
	500	-	1	3	5	4	3	3.2	1.48	12.7
	1600	-	8	4	4	9	5	6.0	2.35	26.0	18.3
	1600	-	2	1	1	2	3	1.8	0.84	10.5
	5000	-	1	3	3	6	1	2.8	2.05	15.2	13.7
	5000	-	3	2	4	3	3	3.0	0.71	12.2
Solvent control 1 (MEM)	0	+	5	4	3	8	5	5.0	1.87	27.7	20.0
Solvent control 2 (MEM)	0	+	4	4	3	1	4	3.2	1.30	12.2
Positive control (DMBA)	7.7	+	17	25	28	25	20	23.0	4.42	96.2		*
Telon Rubin A5B 01	160	+	5	6	4	0	9	4.8	3.27	20.7	16.1
	160	+	3	1	1	3	4	2.4	1.34	11.5
	500	+	13	23	23	26	16	20.2	5.45	80.7	46.4
	500	+	1	0	6	5	2	2.8	2.59	12.1
	1600	+	3	3	1	2	3	2.4	0.89	9.3	15.9
	1600	+	9	5	3	5	4	5.2	2.28	22.5
	5000	+	5	2	2	2	2	2.6	1.34	12.2	15.8
	5000	+	2	5	8	0	6	4.2	3.19	19.3

Mutation frequency (mutant colonies per 1 million cells): mean value / cells surviving

*Statistical significant (p<0.05) Mann-Whitney-U-Test

Applicant's summary and conclusion

Conclusions:

Acid Red 299 did not induce gene mutation, i.e. was not mutagenic, in this HPRT-test with V79 Chinese hamster cells, either in the presence or in the absence of metabolic activation.
The substance is not classified in accordance with CLP criteria.

Executive summary:

The study was performed to investigate the potential of Acid Red 299 (Batch No. 0481 09300) to induce gene mutations at the HPRT locus in V 79 cells of the Chinese hamster in vitro.

 

Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix).

 

The compound was suspended in cell culture medium and tested at the following concentrations:

without S9-mix: 160, 500, 1600 and 5000 μg/ml

with S9-mix: 160, 500, 1600 and 5000 μg/ml

 

With all investigated concentrations microscopically and macroscopically precipitation was observed.

 

The concentration ranges were based on the results of preliminary tests for solubility and toxicity. The highest concentration showed slight toxic effects with and without metabolic activation.

 

Up to the highest investigated dose no statistically significant and reproducible increase in mutant colony numbers was obtained in two independent experiments.

 

Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, thus indicating the sensitivity of the assay and the efficacy of the S9-mix.

 

In conclusion, Acid Red 299 does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described.

 

Acid Red 299 (Batch No. 048109300) is therefore considered to be non-mutagenic in this HPRT assay.