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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Gene mutation in bacteria (Ames test according to OECD471): negative with and without metabolic activation Gene mutation in mammalian cells (Mouse Lymphoma Assay according to OECD476): negative without and positive with metabolic activation In vivo Mouse Micronucleus Test according to OECD474: negative
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
5 january 2000 - 23 March 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed under GLP and according to internationally accepted guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
The thymidine kinase (TK) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
The medium used for this study was RPMI 1640 supplemented with horse serum (10% by volume), Pluronic F68, L-glutamine, sodium pyruvate, penicillin and streptomycin.
Treatment medium was Fischer's medium with the same medium supplements used in the culture medium except that the horse serum concentration was reduced to 5% by volume.
Cloning medium consisted of the RPMI 1640 culture medium with up to 20% horse serum, without Pluronic F68 and with the addition of 0.24% BBL agar to achieve a semisolid state.
Selection medium was cloning medium that contained 3 µg/mL of TFT.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Dose range finding assay with and without S9-mix: 9.85, 19.7, 39.3, 78.5, 157, 313, 625, 1250, 2500, 5000 µg/L
1st Mutation assay with S9-mix: 125, 250, 500, 750, 1000, 1500, 2000, 2500, 3000, 4000 and 5000 µg/mL
2nd Mutation assay with S9-mix: 125, 250, 500, 750, 1000, 1500, 2000, 2250, 2500, 3000 and 4000 µg/mL
1st Mutation assay without S9-mix: 125, 250, 500, 750, 1000, 1500, 2000, 2500, 3000, 4000 and 5000 µg/mL
2nd Mutation assay without S9-mix: 31.3, 62.5, 125, 250, 500, 750, 1000, 1500, 2000, 2500 and 3000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test article, p-Toluenesulfonamide, was soluble in the vehicle (DMSO) at 500 mg/mL, the highest concentration prepared.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see "any other information on materials..."
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: not applicable
- Exposure duration:
1st mutation assay without S9-mix: 4 hours
2nd mutation assay without S9-mix: 24 hours
1st mutation assay with S9-mix: 4 hours
2nd mutation assay with S9-mix: 4 hours
- Expression time (cells in growth medium):
1st mutation assay without S9-mix: 2 days
2nd mutation assay without S9-mix: 2 days
1st mutation assay with S9-mix: 2 days
2nd mutation assay with S9-mix: 2 days
- Selection time (if incubation with a selection agent):
1st mutation assay without S9-mix: 13 days
2nd mutation assay without S9-mix: 13 days
1st mutation assay with S9-mix: 13 days
2nd mutation assay with S9-mix: 13 days
- Fixation time (start of exposure up to fixation or harvest of cells):
Not applicable

SELECTION AGENT (mutation assays): TFT (5-trifluorothymidin)
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable


NUMBER OF REPLICATIONS:
The assay conditions consisted of vehicle controls in triplicate, two positive controls and eleven different test article dose levels using one culture per dose level. Treated cultures may be eliminated during the expression period as long as four dose levels are left for analysis of mutant induction. After exposure and expression time cells from each selected tube was suspended in selection medium in soft agar to recover TFT-resistant mutants. Therfore for the selection of mutants the sample was distributed into three 100 mm dishes.

NUMBER OF CELLS EVALUATED: The total number colonies of cells were counted to assess mutant frequency and the number of large and small colonies was recorded since this is considered to reflect the types of genetic damage, with the large colonies derived from cells with intragenic mutations that affect only the TK gene and the small colonies the result of larger mutations that affect cell growth as well as the TK gene.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: not applicable

OTHER: not applicable
Evaluation criteria:
Assay Evaluation Criteria
The test article was evaluated as positive, negative, or equivocal in this assay.

Evaluation of a Positive Response.
The test article is evaluated as positive if dose-dependent increases of 2-fold or greater in mutant frequency are obtained over the concurrent background mutant frequency. The background mutant frequency is defined as the average mutant frequency of the vehicle control cultures. The 2-fold or greater increase is based on extensive experience which indicates such responses are repeatable in additional trials. It is desirable to obtain this
relationship for at least three doses, but this goal depends on the dose steps chosen for the assay and toxicity at which mutagenic activity appears.
The dose-dependent requirement is waived if a large increase in mutant frequency (4-fold or higher) is obtained for a single dose at or near the highest testable toxicity. However, for the test article to be evaluated as positive, any increases must be repeatable in a second trial.

Evaluation of a Negative Response.
The test article was evaluated as negative if a 2-fold increase in mutant frequency was not observed for (1) a range of doses that extended to toxicities causing 10% to 20% Relative Total Growth (RTG), or (2) for relatively nontoxic test articles, a range of doses extending to the maximum concentration of 5 mg/mL or 10 mM (whichever is lower), or (3) a range of doses that extended to a level approximately twice the solubility limit in culture medium, or (4) the increase(s) are not repeatable in a confirmatory trial.

Evaluation of an Equivocal Response.
The test article was evaluated as equivocal in this test system if there was no consistent evidence for either a positive or negative evaluation.
Statistics:
Not appicable
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
-S9 24-hrs: noncytotoxic up to 3 13 µg/mL, moderate cytotoxicity at 625 µg/mL, high cytotoxicity at 1250 µg/mL, excessive cytotoxicity at 2500 and 5000 µg/mL.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
A positive response was observed at the high dose only.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
+ S9 4-hrs: non- to weakly cytotoxic up to 2500 µg/ml, and excessively cytotoxic at 5000 µg/mL.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH at the top dose of test article prepared in treatment medium (5000 µg/mL) was the same as the pH of the vehicle control. In addition, for all doses there was no color change of the pH indicator in any of the treatments in the medium compared to the vehicle controls.
- Effects of osmolality: Osmolality of the treatment medium was also determined. However, due to the formation of a precipitate, the osmolality of the highest concentration of test article prepared could not be measured. Measurement of the second highest concentration prepared for the cytotoxicity assay (2500 µg/mL) indicated no change when compared to the vehicle control.
- Evaporation from medium: not applicable
- Water solubility: no data
- Precipitation: When the dosing stocks were added to treatment medium plus cells, a precipitate was observed at and above 3000 µg/mL.
- Other confounding effects: no data


RANGE-FINDING/SCREENING STUDIES:
The test article, p-Toluenesulfonamide, was tested in a preliminary dose rangefinding assay with a treatment period of approximately 4 hours both with and without S9 metabolic activation and a preliminary dose rangefinding nonactivation assay with a treatment period of approximately 24 hours. Ten dose levels were used in each case that ranged from 9.85 to 5000 µg/mL; a vehicle control was included under each activation condition.
In the presence and absence of rat liver S9 metabolic activity with a 4-hour treatment period, p-Toluenesulfonamide was noncytotoxic to weakly cytotoxic up to 2500 µg/ml, and excessively cytotoxic at 5000 µg/mL. In the nonactivation dose rangefinding assay using a 24-hour treatment period (Table 2), the test article induced no cytotoxicity up to 3 13 µg/mL, moderate cytotoxicity at 625 µg/mL, high cytotoxicity at 1250 µg/mL, and excessive cytotoxicity at 2500 and 5000 µg/mL. The top dose chosen was 5000 µg/mL, which was the testing limit for the assay.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Mutation Assays

Initial Nonactivation Mutation Assay.

Eleven concentrations of p-Toluenesulfonamide at 125, 250, 500, 750, 1000, 1500,2000, 2500, 3000,4000 and 5000 µg/mL were initiated. Treatments at and above 2500 µg/mL were terminated due to excessive cytotoxicity. The remaining seven treatments induced weak to high cytotoxicity (76.4% to 19.6% relative growths), In order for a treatment to be evaluated as mutagenic in the initial nonactivation assay, a mutant frequency greater than 94.0 % was required. This threshold value was equal to twice the average mutant frequency of the concurrent vehicle controls (background mutant frequency). None of the analyzed treatment induced this level of mutant action. A confirmatory assay was initiated.

Confirmatory Nonactivation Mutation Assay.

In the confirmatory nonactivation assay, the treatment period was approximately 24 hours. Eleven treatments at 31.3,62.5, 125, 250, 500,750, 1000, 1500,2000,2500 and 3000 µg/mL were initiated. Treatments at and above 1500 µg/mL were terminated due to excessive cytotoxicity.

The remaining seven treatments were selected for mutant analysis and induced weak to high cytotoxicity (70.6% to 11.7% relative growths). None of the analyzed treatments induced a mutant frequency that exceeded the minimum criterion of 122.3 x 10E-6.

The test article is, therefore, considered negative without metabolic activation.

Initial Activation Mutation Assay.

Eleven treatments at 125,250, 500, 750, 1000, 1500,2000, 2500,3000,4000 and 5000 µg/mL were initiated, and treatments at and above 2500 pg/mL were terminated due to excessive cytotoxicity. The remaining seven doses were cloned for mutant analysis and induced no cytotoxicity to moderately high cytotoxicity (133.9% to 29.0% relative growths). The minimum criterion for a positive response in this trial was 99.8 x 10E-6. Treatment at 2000 µg/mL induced this level of mutant action with a mutant frequency that was 2.1-times the average vehicle control mutant frequency. A confirmatory assay was performed.

Confirmatory Activation Mutation Assay.

In the confirmatory assay with metabolic activation, eleven treatments at 125,250,500, 750, 1000, 1500,2000,2250,2500,3000 and

4000 µg/mL were initiated. Treatments at and above 2250 µg/mL were terminated due to excessive cytotoxicity. The remaining seven doses induced no cytotoxicity to high cytotoxicity (116.7% to 14.5% relative growths). Treatment at 2000 µg/mL induced a mutant frequency that

exceeded the minimum criteria of 164.0 x 10E-6 with a mutant frequency that was 3.1 -times the average vehicle control mutant frequency. The test article was evaluated as positive with metabolic activation in this assay.

Control Values.

The average cloning efficiencies for the vehicle controis were 104.4% and 90.8% without activation and 83.8% and 95.8% with S9 metabolic activation, which demonstrated acceptable cloning conditions for the assays. The positive control cultures, MMS (nonactivation) and MCA (activation) induced large increases in mutant frequency that were greatly in excess of the minimum criteria.

Sizing Analysis.

The L5 178Y TK +/- mutation assay produces a bimodal distribution of large and small mutant colonies. The origin of the bimodal distribution of mutant colony sizes is considered to reflect the types of genetic damage, with the large colonies derived from cells with intragenic mutations that affect only the TK gene and the small colonies the result of larger mutations that affect cell growth as well as the TK gene. Colony sizing was performed on all cultures. Mutant colonies from all the cultures showed the expected bimodal distribution and mutant colonies from MMS and MCA treated cultures showed both small and large colonies. An increase in small colonies was observed in treatments which induced a positive response.

Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
positive with metabolic activation

The test article, p-Toluenesulfonamide, was evaluated as negative for inducing forward mutations at the TK locus in L5 178Y mouse lymphoma cells under nonactivation conditions. Under activation conditions, a positive response was observed at the high dose only.
Executive summary:

The objective of this in vitro assay was to evaluate the ability of p-Toluenesulfonamide to induce forward mutations at the thymidine kinase (TK) locus in the mouse lymphoma L5 178Y cell line. The study was performed according to OECD476 and under GLP. The test article, p-Toluenesulfonamide, was soluble in the vehicle (DMSO) at 500 mg/mL, which was the highest concentration prepared. When the dosing stocks were added to treatment medium plus cells, a precipitate was observed at and above 3000 µg/mL. A dose rangefinding assay was performed with and without metabolic activation using a treatment period of approximately 4 hours. In addition, a dose rangefinding assay without metabolic activation using a treatment period of approximately 24 hours was performed. The dose rangefinding assays were initiated with concentrations from 9.85 to 5000 µg/mL. In the presence and absence of rat liver S9 metabolic activity with a 4-hour treatment period, p-Toluenesulfonamide was noncytotoxic to weakly cytotoxic up to 2500 µg/mL, and excessively cytotoxic at 5000 µg/mL. In the nonactivation dose rangefinding assay using a 24 -hour treatment period, the test article induced no cytotoxicity up to 313 µg/mL, moderate cytotoxicity at 625 µg/mL, high cytotoxicity at 1250 µg/mL, and excessive cytotoxicity at 2500 and 5000 µg/mL. The top dose chosen for the mutation assays was 5000 µg/mL, which was the testing limit for the assay. In the nonactivation mutation assay with a treatment period of approximately 4 hours, seven doses ranging from 125 to 2000 µg/mL were analyzed for mutant induction and weak cytotoxicity to high cytotoxicity was induced. None of the analyzed treatments induced a mutant frequency that exceeded the minimum criteria for a positive response. A confirmatory assay was performed. In the confirmatory nonactivation mutation assay, which was performed with a 24-hour treatment period, seven treatments from 31.3 to 1000 µg/mL were analyzed and weak cytotoxicity to high cytotoxicity was induced. None of the treatments induced a mutant frequency that exceeded the minimum criteria for a positive response. The test article was, therefore, evaluated as negative without metabolic activation. In the initial mutation assay in the presence of S9 metabolic activation, seven treatments from 125 to 2000 µg/mL were analyzed and no cytotoxicity to moderately high cytotoxicity was induced. In the confirmatory assay, seven treatments from 125 to 2000 µg/mL were also analyzed, and no cytotoxicity to high cytotoxicity was induced. In both the initial and confirmatory assays, treatment at 2000 µg/mL induced a mutant frequency that exceeded the minimum criteria for a positive response with mutant frequencies 2.1- and 3.1 -times the average vehicle control values, respectively. The test article was evaluated as positive with metabolic activation. The test article, p-Toluenesulfonamide, was evaluated as negative for inducing forward mutations at the TK locus in L5178Y mouse lymphoma cells under nonactivation conditions. Under activation conditions, a positive response was observed at the high dose only.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vitro:

The results from the genotoxicity testing of PTSA were read across to PTSI. This is considered justified because PTSI is extremely reactive with water and reacts instantaneously into the source substance PTSA. Moreover, in practice, exposure will be to PTSA rather than to PTSI.

PTSA was studied in two in vitro and one in vivo genotoxicity tests.

MLA. The objective of the in vitro Mouse Lymphoma Assay was to evaluate the ability of p-Toluenesulfonamide to induce forward mutations at the thymidine kinase (TK) locus in the mouse lymphoma L5 178Y cell line. The study was performed according to OECD476 and under GLP. The test article, p-Toluenesulfonamide, was soluble in the vehicle (DMSO) at 500 mg/mL, which was the highest concentration prepared. When the dosing stocks were added to treatment medium plus cells, a precipitate was observed at and above 3000 µg/mL. A dose rangefinding assay was performed with and without metabolic activation using a treatment period of approximately 4 hours. In addition, a dose rangefinding assay without metabolic activation using a treatment period of approximately 24 hours was performed. The dose rangefinding assays were initiated with concentrations from 9.85 to 5000 µg/mL. In the presence and absence of rat liver S9 metabolic activity with a 4-hour treatment period, p-Toluenesulfonamide was noncytotoxic to weakly cytotoxic up to 2500 µg/mL, and excessively cytotoxic at 5000 µg/mL. In the nonactivation dose rangefinding assay using a 24 -hour treatment period, the test article induced no cytotoxicity up to 313 µg/mL, moderate cytotoxicity at 625 µg/mL, high cytotoxicity at 1250 µg/mL, and excessive cytotoxicity at 2500 and 5000 µg/mL. The top dose chosen for the mutation assays was 5000 µg/mL, which was the testing limit for the assay. In the nonactivation mutation assay with a treatment period of approximately 4 hours, seven doses ranging from 125 to 2000 µg/mL were analysed for mutant induction and weak cytotoxicity to high cytotoxicity was induced. None of the analysed treatments induced a mutant frequency that exceeded the minimum criteria for a positive response. A confirmatory assay was performed. In the confirmatory nonactivation mutation assay, which was performed with a 24-hour treatment period, seven treatments from 31.3 to 1000 µg/mL were analysed and weak cytotoxicity to high cytotoxicity was induced. None of the treatments induced a mutant frequency that exceeded the minimum criteria for a positive response. The test article was, therefore, evaluated as negative without metabolic activation. In the initial mutation assay in the presence of S9 metabolic activation, seven treatments from 125 to 2000 µg/mL were analysed and no cytotoxicity to moderately high cytotoxicity was induced. In the confirmatory assay, seven treatments from 125 to 2000 µg/mL were also analysed, and no cytotoxicity to high cytotoxicity was induced. In both the initial and confirmatory assays, treatment at 2000 µg/mL induced a mutant frequency that exceeded the minimum criteria for a positive response with mutant frequencies 2.1- and 3.1 -times the average vehicle control values, respectively. The test article was evaluated as positive with metabolic activation. The test article, p-Toluenesulfonamide, was evaluated as negative for inducing forward mutations at the TK locus in L5178Y mouse lymphoma cells under nonactivation conditions. Under activation conditions, a positive response was observed at the high dose only.

Ames test.The ability of p-Toluenesulphonamide to induce mutations was investigated in the Bacterial Reverse Mutation Assay (Ames) by using 4 histidine-requiring strains of Salmonella typhimurium (TA100, TA1535, TA98 and TA1537), and a tryptophan-requiring strain of Escherichia coli (WP2 uvrA) in the presence and absence of a metabolic activation system (S9 mix). Two experiments were performed, Experiment 1: plate incorporation and Experiment 2: pre-incubation. The study procedures described in this reverse mutation assay were according to OECD guideline 471 and EU Method B.13/14. Test concentrations used in the main test were as follows: 50, 150, 500, 1500 and 5000 µg/plate per strain, with and without S-9 mix, for both Experiments. Each concentration was tested in triplicate.

The mutagenicity of the test compound was judged negative because the number of revertant colonies in the test compound treatment groups was not significantly higher than that of each vehicle control in all test strains. The number of revertant colonies in the positive control groups were more than twice that of vehicle control groups. The test results showed that the number of revertant colonies for the negative control and the positive controls were within the range of the historical data at Harlan laboratories Ltd, indicating that the test can be considered valid.

It is therefore concluded that p-Toluenesulphonamide has no ability to induce mutations in bacteria under the present test conditions.

In vivo Micronucleus test.The objective of this study was to evaluate the test article, p-Toluenesulfonamide, for in vivo clastogenic activity and/or disruption of the mitotic apparatus by quantifying micronuclei in polychromatic erythrocyte (PCE) cells in Crl:CD-1 (ICR) BR mouse bone marrow. The assay design was based on OECD Guideline 474, updated and adopted July 21, 1997 and OPPTS Guideline 870.5395. The study was performed under GLP.

In the dose range-finding study, the test article was suspended in corn oil and administered by oral gavage at dose levels of 0,250,500, 1000, 1500, and 2000 mg/kg to five male and five female mice per dose level. The animals were observed for signs of clinical toxicity immediately and 1 hour post dose and at least once daily for up to 2 days after dosing for toxic signs and/or mortality. Based on the dose range-finding study and discussions with the Sponsor, doses of 0 (vehicle control), 187.5, 375,750, and 1500 mg/kg were selected. Since differences in toxicity between the sexes were observed in the dose range-finding assay, both male and female mice were tested in the micronucleus assay.

The animals were observed for signs of clinical toxicity immediately and 1 and 6 hours post dose and at least once daily and body weights were collected daily until the respective harvest time point. Five animals dosed with the test article at the 187.5, 375, and 750 mg/kg dose levels and five animals dosed with the positive control article were euthanized approximately 24 hours after dosing for extraction of the bone marrow. Five animals dosed with the test article at the1500 mg/kg bw dose level and five animals dosed with the vehicle control article were euthanized approximately 24 and 48 hours after dosing for extraction of the bone marrow. At least 2000 PCEs per animal were analysed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 500 erythrocytes for each animal.

Death occurred in p-Toluenesulfonamide-treated animals at the 750 mg/kg dose level (1 of 6 females) and 1500 mg/kg dose level (4 of 18 females and 1 of 18 males). There were no signs of toxicity at the 187.5 mg/kg dose level. At 375 mg/kg, hypoactivity and/or squinted eyes were noted in one animalhex. At 750 mg/kg, animals were noted with hypoactivity, flattened posture, ataxia, squinted eyes, and/or recumbency. At 1500 mg/kg, findings included hypoactivity, cold to touch, flattened posture, recumbency, ataxia, squinted and/or sealed-shut eyes, laboured breathing, and/or rolling over to the right. No significant or biologically relevant consistent body weight losses were observed in the surviving male or female mice in any test-article-treated group.

No statistically significant increase in micronucleus frequencies occurred in polychromatic erythrocytes (PCEs) in either male or female mice treated with up to 1500 mg/kg of p-Toluenesulfonamide. However, a statistically significant decrease was observed in the 1PCE:normochromatic erythrocytes (NCE) ratio in the 750 mg/kg females at the 24-hour harvest time point. No other statistically significant decreases were observed in the PCE:NCE ratios at any other test article dose level examined at either bone marrow sampling time of 24 or 48 hours post dose. When observed, a significant decrease in the PCE:NCE ratio is direct evidence of test article exposure to the bone marrow resulting in cytotoxicity. In conclusion, p-Toluenesulfonamide was evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.

Based on the results of the MLA, PTSA shows a positive effect with metabolic activation and at the highest dose level only. Ames and in vivo Micronucleus tests showed negative results.

PTSI does not need to be classified as mutagenic in accordance with the criteria outlined in Annex VI of 67/548/EEC and Annex I of 1272/2002/EC, until the positive mutagenic result is reproduced in an in vivo gene mutation test. The UDS test is proposed for this purpose.

Justification for selection of genetic toxicity endpoint
Reliable GLP genotoxicity studies according to OECD Guidelines 471, 474 and 476

Justification for classification or non-classification

PTSI does not need to be classified as mutagenic in accordance with the criteria outlined in Annex VI of 67/548/EEC and Annex I of 1272/2002/EC, until the positive mutagenic result is reproduced in an in vivo gene mutation test. The UDS test is proposed for this purpose.