(E)-3-(benzyl(4-((4-nitrophenyl)diazenyl)phenyl)amino)propanenitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 November 1998 to 14 December 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: In vitro Mammalian Cell Gene Mutation Test EEC Directive 87/302, L133, pp. 61 - 63, March 1987

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

429-530-4

EC Name:

-

Cas Number:

96662-24-7

Molecular formula:

Hill formula: C22H19N5O2 CAS formula: C22H19N5O2

IUPAC Name:

3-[benzyl({4-[2-(4-nitrophenyl)diazen-1-yl]phenyl})amino]propanenitrile

Test material form:

solid: particulate/powder

Details on test material:

Disperse Orange 288

Method

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The compound was suspended in DMSO and tested at the following concentrations:
without S9-mix; 5.0, 7.5, 12.5, 25.0, 50.0, 75.0. 125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml (main mutation experiment)
50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)
with S9-mix: 5.0, 7.5, 12.5, 25.0, 50.0, 75.0, 125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml (main mutation experiment)
50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without metabolic activation: EMS (Ethyl methane sulfonate), with metabolic activation: DMBA (9,10-dimethyl-1 ,2-benzanthracene)

Details on test system and experimental conditions:

Reference Compounds

Without metabolic activation
Name or number of compound (I.N.N. orU.SAN): ethyl methane sulfonate
Synonyms: EMS
Formula of the compound: C3Ha03S
CAS-Register number: 62-50-0
Product number / Code: 820774
Supplier of reference compound: Dr. Theodor Schuchardt & Co. Chemische Fabrik, Germany
Batch number: 40606721
Certificate of analysis: certificated by the supplier, Analytical Department, Dr. Bolkart dated December 18th, 1995

With metabolic activation
Name or number of compound (I.N.N. orU.S.A.N): 9,10-dimethyl-1,2-benzanthracene
Synonyms: DMBA
Chemical name: 7,12-dimethylbenz[a]anthracene
CAS-Register number: 57-97-6
Product number / Code: 21,626-7
Supplier of reference compound: Fa. Aldhch – Chemie, Germany
Batch number: TL 1825LL
Certificate of analysis: certificated by the supplier, Aldrich chemical company, David Swessel, dated April 3rd, 1996

Test system

Test groups
with and without metabolic activation: 5.0, 7.5, 12.5, 25.0, 50.0, 75.0, 125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml (main mutation experiment)
50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)

Control groups
negative controls: untreated control & cultures treated with the solvent

positive controls:
without metabolic activation: EMS (Ethyl methane sulfonate)
with metabolic activation: DMBA (9,10-dimethyl-1,2-benzanthracene)

Formulation of test compounds: suspended in DMSO 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

Source of biological material: cell bank of "Genetic Toxicology", HMR 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

Experimental conditions in vitro: approx. 37 °C and approx. 4 % CO2 in plastic flasks
Observations and Measurements

Preparation and storage of a liver homogenate fraction (S9)
The S9 fraction was prepared by the testing facility according to Ames et. al (1975). Male Sprague Dawley rats (200-300 g), supplied by Harlan Winkelmann, Gartenstrasse 27, 33178 Borchen, Germany, received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) 5 days before killing. The livers were removed from at least 5-6 animals at approx. 0 to 4 °C using cold sterile solutions and glassware, and were then pooled and washed in approx. 150 mM KCI (approximately 1 ml/g wet liver). The washed livers were cut into small pieces and homogenized in three volumes of KCI. The homogenate was cenfrifuged at approx. 9000g for 10 minutes. The supernatant, the S9 fraction, was divided into small portions, rapidly frozen and stored at approx. - 80 °C for not longer than six months. The protein content was determined for every batch. Also for every batch of S9 an independent validation was performed with a minimum of two different mutagens, e.g., 2-aminoanthracene and dimethylbenzanthracene to confirm metabolic activation by microsomal enzymes.

Preparation of S9-mix
Sufficient S9 fraction was thawed to room temperature immediately before each test. An appropriate quantity of S9 fraction (batch no. 98/1 for both mutation experiments, protein concentration 51.8 g/l) was mixed with S9 cofactor solution to yield a final protein concentration of 0.3 mg/ml in the cultures which was kept on ice until used. This preparation is termed S9-mix. The concentrations of the different components of the S9-mix were:
8 mM MgCI3
33 mM KCI
5 mM glucose-6-phosphate
5 mM NADP
100 mM phosphate buffer pH 7.4

Cell culture
Large stocks of the mycoplasma-free V79 cell line are stored in liquid nitrogen in the cell bank of "Genetic Toxicology", thus permitting repeated use of the same cell culture batch for numerous experiments. The identical characteristics of the cells ensure comparability of the experimental parameters.
Thawed stock cultures were kept at approx. 37 °C and approx. 4 % C03 in 175 cm2 plastic flasks. About 5 x 105 to 1 x 106 cells were seeded into each flask in 30 ml of MEM-medium supplement with approx. 10 % (v/v) FCS (fetal calf serum) containing approx. 2 mM L-glutamine and approx. 0.1 % (w/v) neomycinsulfate. The cells were subcultured twice a week.
For the selection of mutants the medium was supplemented with approx. 11 μg/ml thioguanine.

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 a single cell culture at each test point.

Test procedure
In preliminary toxicity experiments approximately 4500 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. Approx. 24 hours after treatment, 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. 4500 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 replaced with normal medium after rinsing twice with this. The cultures were stained with crystal violet and survival was determined after an incubation period of approx. 24 hours.

Rationale for dose selection
For non-toxic, freely soluble test compounds, the top dose should be 10 mM or 5000 ug/ml according to international testing guidelines.
For non-toxic, poorly soluble test compounds, the top dose should be the highest evaluable dose.
For toxic compounds the percentage survival relative to the solvent control should be calculated for each treatment. The dose level which resulted in a predicted survival of less than 30 % should be chosen as the highest dose level. At least eight respectively seven lower dose levels should be also included in each experiment.

Mutagenicity test
Two independent mutation tests were performed.
Two-day old, exponentially growing cultures which were more than 50 % confluent were trypsinated and a single cell suspension was prepared. The trypsin concentration was approx. 0.25 % (v/v) in Ca-Mg-free salt solution. The Ca-Mg-free salt solution was prepared as follows (per liter): NaCI 6.8 g; Ka 0.4 g; glucose 1 g; NaHC03 2.2 g; phenol red 5 mg; trypsin 2.5 g.
Subsequently the cells were replated to determine the mutation frequency and plating efficiency.

The treatment schedule of the mutagenicity test is described below:

Day 1: Subculturing of an exponentially growing culture
a) Approx. 4500 cells in each well of a microtiter plate for determination of the plating efficiency.
b) 6x 105 - 1 x 106 cells in 175 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. 0.3 mg/ml) for 4 hours.

Day 3: Fixation and staining of the cells in a) for the determination of the plating efficiency.

Day 5: Subculturing of b) in 175 cm2 flasks

Day 9: Subculturing of b) in five 75 cm1 flasks with culture medium containing 6-thioguanine:
Mutant selection (about 300 000 cells/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.

Evaluation criteria:

Evaluation of data

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 dose-related increase in the mutation frequency. Such an evaluation may be considered independently from the number 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

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility and toxicity
T-9601 was suspended in DMSO.
Evaluation of the solubility of that suspension in cell culture medium showed that 1285.0 μg/ml was the highest practicable concentration and produced precipitate.
Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 1285.0 μg/ml and a range of lower dose levels down to 10.0 pg/ml.
Following treatment in the absence of S9 metabolic activation, several toxicity was observed. Survival declined in a dose-related manner reaching 23.0 % of the solvent control value at the concentration of 750 μg/ml.
In the presence of S9 metabolic activation survival declined steeply up to the concentration of 500.0 μg/ml where survival was reduced to 64.8 % of the solvent control value.
Based on these results 1285 μg/ml was selected as the maximum dose level for the main mutation experiments in both the absence and in the presence of S9-mix. Ten lower concentrations down to 5 μg/ml were included in the main mutation experiment and seven lower concentrations down to 50 μg/ml in the repeat mutation experiment.

Mutagenicity
Experimental design
Two independent mutation assays to examine resistance to 6-thioguanine were performed.
In the absence and in the presence of S9 metabolic activation dose levels of 5.0, 7.5, 12.5, 25.0, 50.0, 75.0,125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml were used in the main mutation experiment, For the repeat mutation experiment a dose range of 50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml was selected in the absence and in the presence of S9-mix.
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 absence of S9 metabolic activation in both mutation experiments a dose-related decrease in survival was observed reaching 53.2 % respectively 39.4 % of the solvent control value in the microtiter plates at the highest dose level tested, 1285.0 μg/ml.
In the presence of S9 metabolic activation survival decreased in a dose-related manner reaching approximately 45.3 % respectively 53.8 % of the solvent control value in the microtiter plates after treatment at the highest dose level, 1285.0 μg/ml.

Mutation results
The test compound T-9601 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 a statistically significant increase of the mutation frequency was observed only at a concentration of 7.5 μg/ml. This effect was not dose-dependent and not three fold higher than the corresponding controls and therefore of no biological relevance.
No relevant reproducible increases in the mutant colonies or mutant frequency over the range of the solvent control was found with any of the concentrations used, either with or without metabolic activation by S9-mix.
The sensitivity of the test system and efficacy of the S9-mix was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Toxicity Data (Main mutation experiment)

	Dose μg/ml	S9-mix	Extinction in microwell plates mean less blank values	Standard deviation	Relative survival*
Negative control	0.0	-	0.572	0.02	121.5
		+	0.451	0.05	125.3
Solvent control	0.0	-	0.471	0.04	100.0
		-	0.424	0.06	100.0
		+	0.360	0.06	100.0
		+	0.411	0.08	100.0
Positive control	1000.0	-	0.504	0.01	107.0
	7.7	+	0.361	0.04	100.3
T-9601	5.0	-	0.499	0.02	105.9
		+	0.461	0.03	128.0
	7.5	-	0.529	0.08	112.3
		+	0.462	0.03	128.3
	12.5	-	0.495	0.02	105.0
		+	0.448	0.04	124.4
	25.0	-	0.440	0.01	93.5
		+	0.437	0.03	121.4
	50.0	-	0.420	0.02	89.1 #
		+	0.378	0.03	105.2 #
	75.0	-	0.398	0.02	84.1 #
		+	0.413	0.03	114.7 #
	125.0	-	0.483	0.10	113.9 #
		+	0.387	0.03	94.2 #
	250.0	-	0.319	0.02	75.2 #§
		+	0.418	0.05	101.8 #§
	500.0	-	0.253	0.02	59.6 #§
		+	0.325	0.05	79.1 #§
	750.0	-	0.276	0.02	65.2 #§
		+	0.256	0.05	62.3 #§
	1285.0	-	0.226	0.03	63.2 #§
		+	0.186	0.04	45.3 #§

# microscopical visible precipitation

§ macroscopical visible precipitation

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

Solvent = DMSO

Positive control without S9-mix = EMS

Positive control with S9-mix = DMBA

 

Mutagenicity Data – Part 1 (Main experiment)

	Dose μg/ml	S9-mix	Number of cells per flask				Factor* calculated	Cells* seeded	Cells*** survived
			Seeded	Found		Mean
			I / II	I	II
Negative control	0.0	-	403	158.0	164.5	161.3	0.40	342900	137203
		+	401	101.0	98.0	99.5	0.25	337500	83744
Solvent control 1 (DMSO)	0.0	-	398	370.5	370.0	370.3	0.93	292350	271966
		+	399	370.0	372.0	371.0	0.93	298800	277832
Solvent control 2 (DMSO)	0.0	-	400	313.0	323.5	318.3	0.80	291150	231646
		+	402	201.5	200.5	201.0	0.50	287700	143850
Positive control (EMS)	1000.0	-	400	288.0	273.5	280.8	0.70	285000	200034
Positive control (DMBA)	7.7	+	398	281.0	299.0	290.0	0.73	276750	201652
T-9601	5.0	-	399	258.5	261.0	259.8	0.65	291900	190028
		+	400	368.0	386.5	377.3	0.94	258600	243892
	7.5	-	401	188.5	191.5	190.0	0.47	291000	137880
		+	401	284.0	274.0	279.0	0.70	326400	227096
	12.5	-	399	278.0	277.0	277.5	0.70	307800	214071
		+	394	271.0	278.5	274.8	0.70	331650	231271
	25.0	-	399	348.0	362.5	355.3	0.89	294300	262030
		+	402	361.5	380.0	370.8	0.92	276150	254683
	50.0	-	403	320.0	310.5	315.3	0.78	288150	225408
		+	402	292.5	296.0	294.3	0.73	280650	205426
	75.0	-	399	357.0	338.5	347.8	0.87	294300	256498
		+	405	276.5	272.5	274.5	0.68	299250	202825
	125.0	-	400	381.0	338.0	359.5	0.90	294300	264502
		+	399	340.0	351.0	345.5	0.87	269400	233277
	250.0	-	402	368.0	353.0	360.5	0.90	288750	258941
		+	401	450.5	449.0	449.8	1.12	258150	289534
	500.0	-	403	322.0	314.0	318.0	0.79	279750	220748
		+	402	302.0	314.5	308.3	0.77	288900	221526
	750.0	-	402	271.0	299.5	285.3	0.71	349200	247784
		+	399	313.0	314.0	313.5	0.79	336000	264000
	1285.0	-	400	156.0	144.0	150.0	0.38	337500	126563
		+	405	301.0	306.5	303.8	0.75	347250	260438

* 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 (Main experiment)

	Dose μg/ml	S9-mix	Number of mutant colonies						Standard deviation	Mutation frequency	Stat. sig.
			I	II	III	IV	V	Mean
Negative control	0.0	-	1	4	1	3	2	2.2	1.30	16.0
		+	2	1	2	3	0	1.6	1.14	19.1
Solvent control 1 (DMSO)	0.0	-	3	3	1	2	1	2.0	1.00	7.4
		+	1	1	1	1	3	1.4	0.89	5.0
Solvent control 2 (DMSO)	0.0	-	9	11	6	3	6	7.0	3.08	30.2
		+	3	1	2	7	2	3.0	2.35	20.9
Positive control (EMS)	1000.0	-	166	147	161	156	148	155.8	8.20	777.9	*
Positive control (DMBA)	7.7	+	35	35	45	40	36	38.2	4.32	189.4	*
T-9601	5.0	-	1	0	0	1	3	1.0	1.22	5.3
		+	5	3	1	1	1	2.2	1.79	9.0
	7.5	-	1	1	1	2	0	1.0	0.71	7.3
		+	8	5	12	10	6	8.2	2.88	35.1	*
	12.5	-	3	3	0	4	3	2.6	1.52	12.1
		+	3	2	1	3	1	2.0	1.00	8.6
	25.0	-	5	5	1	2	3	3.2	1.79	12.2
		+	1	3	1	4	4	2.6	1.52	10.2
	50.0	-	0	0	4	1	1	1.2	1.64	5.3
		+	1	1	1	3	0	1.2	1.10	5.8
	75.0	-	1	4	5	5	2	3.4	1.82	13.3
		+	5	1	2	7	8	4.6	3.05	22.7
	125.0	-	1	4	2	0	2	1.8	1.48	6.8
		+	2	1	2	7	3	3.0	2.35	12.9
	250.0	-	5	6	2	4	2	3.8	1.79	14.7
		+	5	0	2	2	4	2.6	1.95	9.0
	500.0	-	4	6	2	1	2	3.0	2.00	13.6
		+	4	1	8	3	5	4.2	2.59	19.0
	750.0	-	6	7	3	5	3	4.8	1.79	19.4
		+	11	8	2	7	11	7.8	3.70	29.5
	1285.0	-	1	6	5	5	3	4.0	2.00	31.6
		+	1	7	2	2	3	3.0	2.35	11.5

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:

T-9601 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.

Executive summary:

The present study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 476 "Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Test". Adopted: July 21, 1997 and U.S. Environmental Protection Agency (EPA) Health Effect Test Guidelines, OPPTS 870.5300, In Vitro Mammalian Cell Gene Mutation Test, august 1998 and In vitro Mammalian Cell Gene Mutation Test EEC Directive 87/302, L133, pp. 61 - 63, March 1987. This study was conducted in compliance with the Principles of Good Laboratory Practice (GLP).

 

The study was performed to investigate the potential of T-9601 to induce gene mutations at the HPRT locus in V79 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 DMSO and tested at the following concentrations:

without S9-mix; 5.0, 7.5, 12.5, 25.0, 50.0, 75.0. 125.0, 250.0, 500.0, 750.0 and1285.0 μg/ml (main mutation experiment)

50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)

With S9 -mix: 5.0, 7.5, 12.5, 25.0, 50.0, 75.0, 125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml (main mutaation experiment)

50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)

 

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

In the presence of metabolic activation a significant increase of the mutation frequency was observed only at a concentration of 7.5 μg/ml. This effect was not dose-dependent and not three fold higher than the corresponding controls and therefore of no biological relevance.

Up to the highest investigated dose no further 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, T-9601 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.

T-9601 is therefore considered to be non-mutagenic in this HPRT assay.