Reactive Orange 143

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

L5178Y Mouse Lymphoma Cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 May 2012 to 14 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): FAT 40854/A TE
- Substance type: Reddish-brown powder
- Physical state: Solid
- Storage condition of test material: At room temperature in the dark

Specific details on test material used for the study:

Identification: FAT 40854/A TE
Description: Reddish-brown powder (determined at WIL Research Europe)
Batch: TZ 5719 / BOP 02-11
Content: 46.2 % (4 main constituents)
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 01 April 2016
Stability in vehicle: Dimethyl sulfoxide
Solubility in vehicle: Dimethyl sulfoxide

Method

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 33, 100, 333, 1000 and 3330 µg/mL

Mutation experiment:
Without S9-mix, 3 hours treatment: 0.3, 1, 3, 10, 33, 100 and 200 µg/mL
With S9-mix, 3 hours treatment: 30, 100, 500, 600, 700, 800 and 850 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
A homogeneous suspension could be obtained in DMSO and DMSO is accepted and approved by authorities and international guidelines

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Details on test system and experimental conditions:

Test System: L5178Y/TK+/--3.7.2C mouse lymphoma cells.
Rationale: Recommended test system in international guidelines (e.g. OECD, EC) and literature (see chapter 9 for references).
Source: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
Stock cultures of the cells were stored in liquid nitrogen (-196 °C). The cultures were checked for mycoplasma contamination. Cell density was preferably kept below 1 E+6 cells/ml.

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: With and without S9-mix: 3 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120 %. An acceptable number of surviving cells (10E6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10E6 survivors and ≤ 170 per 10E6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32.
d) The mutation frequency of MMS should not be below 500 per 10E6 survivors, and for CP not below 700 per 10E6 survivors.

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Statistics:

The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: Test system L5178Y/TK+/-3.7.2C

Any other information on results incl. tables

Dose range finding test

In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 33.3 to 3330 μg/ml in the absence and presence of S9-mix with a 3 hour treatment period. In the absence of S9-mix, the relative suspension growth was 14 % at the test substance concentration of 333 μg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at test substance concentrations of 1000 μg/ml and above. In the presence of S9-mix, the relative suspension growth was 8 % at the test substance concentration of 1000 μg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 3330 μg/ml.

 

Mutagenicity test

Based on the results of the dose range finding test, the following dose range was selected for the first mutagenicity test:

Without S9-mix: 0.3, 1, 3, 10, 33, 100, 200, 250, 300, 325, 350, 400 and 450 μg/ml exposure medium.

 

With 8 % (v/v) S9-mix: 10, 30, 100, 300, 500, 600, 700, 800, 900, 1000, 1100 and 1200 μg/ml exposure medium.

 

Evaluation of toxicity

In the absence of S9-mix, the dose levels of 250 μg/ml and above were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. In the presence of S9-mix, too many dose levels showed severe cytotoxicity, this part of the experiment was repeated (experiment 1A): the following dose range was selected: 0.3, 1, 3, 10, 33, 100, 300, 400, 500, 600, 700, 750 and 800 μg/ml. In this repeat, no dose level with a cell survival below 23 % was reached, therefore this experiment was rejected. In the repeat experiment (1B), the following dose range was selected: 30, 100, 300, 500, 600, 700, 750, 800, 850, 900 and 1000 μg/ml. The dose levels of 750, 900 and 1000 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The dose levels selected to measure mutation frequencies at the TK-locus were:

Without S9-mix: 0.3, 1, 3, 10, 33, 100 and 200 μg/ml exposure medium.

 

With S9-mix: 30, 100, 300, 500, 600, 700, 800 and 850 μg/ml exposure medium.

 

In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 88 % compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 94 % compared to the total growth of the solvent controls.

 

Evaluation of the mutagenicity

In the absence of S9-mix, FAT 40854/A induced an up to 5.7-fold increase in the mutation frequency at the TK locus. FAT 40854/A showed up to 5.1- and 5.4-fold increases in the mutation frequency of the small and large colonies, respectively, compared to the mean mutation frequency of the small and large colonies of the solvent controls.

In the presence of S9-mix, FAT 40854/A induced an up to 11-fold increase in the mutation frequency at the TK locus. FAT 40854/A showed up to 13- and 6.3-fold increases in the mutation frequency of the small and large colonies, respectively, compared to the mean mutation frequency of the small and large colonies of the solvent controls.

Applicant's summary and conclusion

Conclusions:

FAT 40854/A is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions.

Executive summary:

In a GLP-compliant study, evaluation of the mutagenic activity of FAT 40854/A in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells was conducted according to OECD guideline 476 and EU method B.17. This report describes the effects of FAT 40854/A on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). FAT 40854/A was tested up to concentrations of 200 and 850 μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. FAT 40854/A was tested up to cytotoxic levels of 88 and 94 in the absence and presence of S9-mix, respectively.

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Except the response of one of the solvent control cultures in the absence of S9-mix. However, since this response was just below the lower limit of the range and clear positive results were obtained, the validity of the test was considered to be not affected. Mutation frequencies in cultures treated with positive control chemicals were increased by 15-fold for MMS in the absence of S9-mix, and by 35-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, FAT 40854/A TE induced an up to 5.7-fold increase in the mutation frequency. The mutation frequencies were above the GEF + MF(controls)(174 per 106survivors), more than three-fold, outside the historical control data range and in a dose dependent manner, therefore, this increase is considered relevant and FAT 40854/A is considered mutagenic in the absence of S9-mix. In the presence of S9-mix, FAT 40854/A induced an up to 11-fold increase in the mutation frequency. The mutation frequencies were above the GEF + MF(controls) (207 per 10E6 survivors), more than three-fold, outside the historical control data range and in a dose dependent manner, therefore, this increase is considered relevant and FAT 40854/A is considered mutagenic in the presence of S9-mix.

It is concluded that FAT 40854/A is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions.