2-amino-6-chloro-4-nitrophenol

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:

From Nov. 11, 2002 to Dec. 18, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

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)

Remarks:

according to OECD and German principles of GLP

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-amino-6-chloro-4-nitrophenol

EC Number:

228-762-1

EC Name:

2-amino-6-chloro-4-nitrophenol

Cas Number:

6358-09-4

Molecular formula:

C6H5ClN2O3

IUPAC Name:

2-amino-6-chloro-4-nitrophenol

Constituent 2

Reference substance name:

Chlororange base

IUPAC Name:

Chlororange base

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: 2-Amino-6-chloro-4-nitrophenol
- TSIN: WR23214
- Substance type: Pure active substance
- Physical state: Brown orange powder
- Storage and stability under test conditions: The substance on storage in dryness and darkness is considered to be stable more than 5 years.
- Solubility: 0.07-0.24% (pH 4.3) in water;8.7 weight% (pH 3.6) in acetone/water 1:1; > 10 weight% in DMSO
- Stability in solution: The stability was tested over a period of seven days.
DMSO solution (approx. 10% w/v): Very good stability (98.5-99.4%)
Acetone/water 1:1 solution (approx. 7% w/v): Very good stability (98.7-100.1%)
Water solution (approx. 0.05% w/v): Very good stability (101.3-104.1%)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Cosmital ; n°batch : GST009-01/30-07
- Expiration date of the lot/batch:
- Purity test date: may 8, 2007
- Purity : 99.9% (HPLC at 254 nm)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, light protected
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING No

Method

Target gene:

Thymidine kinase locus TK +/-

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: The complete culture medium was RPMI 1640 medium supplemented with 15% horse serum (HS), 100 U/100 µg/mL Penicillin/Streptomycin, 220 µg/mL Sodium-Pyruvate, and 1.25 U/mL Amphotericin used as Antifungal.

- Properly maintained: Yes, the cell culture are stored in liquid nitrogen in the cell bank of RCC Cytotest Cell Research.
- Propagation of stock culture: Thawed stock cultures were propagated in plastic culture flasks in RPMI 1640 complete culture medium and incubated at 37˚C in a 4.5% carbon dioxide atmosphere (95.5% air). The cells were subcultured thrice weekly.
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes, before freezing, each batch was screened for mycoplasma contamination and checked for karyotype stability.
- Periodically "cleansed" against high spontaneous background: Yes

Additional strain / cell type characteristics:

other: Heterozygous at the thymidine kinase locus; high proliferation rate (doubling time 10-12 h in stock cultures) and cloning efficiencies of untreated cells of usually more than 50%.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/B-Naphthoflavone induced rat liver S9 fractions

Test concentrations with justification for top dose:

Pre-test for toxicity:
With and without metabolic activation: 0, 14.8, 29.7, 59.4, 118.8, 237.5, 475, 950 and 1900 μg/mL.
Experiment I (4 h treatment):
Without metabolic activation: 0, 30, 60, 120, 240, 480 and 960 µg/mL
With metabolic activation: 0, 3.8, 7.5, 15, 30, 60 and 120 µg/mL
Experiment II (24 h treatment):
Without metabolic activation: 0, 15, 30, 60, 120, 180 and 240 µg/mL
Experiment II was not performed with metabolic activation.
- At Day 4, the lowest concentration (30 µg/mL) in experiment I without activation was not continued since a minimum of only four concentrations was required by the guidelines. The highest concentrations in experiment I (120 µg/mL) with activation and experiment II (240 µg/mL) without activation were not continued due to severe toxic effects.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen do to its solubility properties and its relative non-toxicity to the cell cultures.

Other information : on the day of the experiment (immediately before treatment), the test item was dissolved in DMSO (Merck, D-64293, purity 99,5%). The final concentration of DMSO in the culture medium did not exceed 0.5% (v/v).

Controls

Untreated negative controls:

yes

Remarks:

(medium)

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

True negative controls:

no

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Remarks:

Without S9 : Prepared by dissolving in nutrient medium at a final concentration of 13 µg/mL (0.12 mM) With S9 : Prepared by dissolving in DMSO at a final concentration of 3 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: In 10 ml RPMI 1640 medium with 3% (15% during 24 h treatment) horse serum (complete culture medium)

EXPERIMENTAL PROCEDURE AND DURATION: The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without metabolic activation and a treatment period of 4 h. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 h.
- Preparation of cell cultures: Prior to mutagenicity testing, the amount of spontaneous mutants was reduced by growing the cells for one day in RPMI1640-HAT medium supplemented with hypoxanthine (1.0 x 10-4 M), aminopterin (2.0 x 10-7 M) and thymidine (1.6 x10-5 M). The incubation of the cells in HAT-medium was followed by a recovery period of 2 days in RPMI 1640 medium containing hypoxanthine (1.0 x 10-4 M) and thymidine (1.6 x10-5 M). Post incubation, the cells were returned to normal RPMI1640 medium (complete culture medium).
- Seeding and exposure: The cell suspension (1 x 10(7) cells/flask (80 cm2 flasks)) suspended in 10 mL RPMI medium with 3% (4 h exposure) or 15% (24 h exposure) horse serum were exposed to various concentrations of the test material either in the presence or absence of metabolic activation.
- Expression and growth:
After 4 h (after 24 h in case of second experiment) the test substance was removed by centrifugation (425 g, 10 min) by washing twice in "saline G”. Subsequently, the cells were resuspended in 30 mL complete culture medium and incubated for an expression and growth period of 72 h. In the second experiment, the expression time without metabolic activation was 48 hours (RPMI medium with 15 % horse serum).
The cell density was determined each day and adjusted to 3x10(5) cells/mL, if necessary. Relative suspension and total growth (RSG and RTG) of the treated cell cultures were calculated after 48 h (72 h following continuous treatment). One sample of the cells was taken at the end of the treatment (4 h and 24 h, respectively), diluted and seeded into microtiter plates (about 2.5 cells/well), to determine the survival of the cells after treatment (cloning efficiency 1)
- Selection of mutant: After the expression period the cultures were seeded into microtiter plates. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4x10(3) cells in selective medium with Triflurothymidine (TFT). The viability (cloning efficiency 2) was determined by seeding about 2.5 cells per well into 2 microtiter plates (same medium without TFT). The plates are incubated at 37°C in 4.5% CO2/95.5 % humidified air for 10 - 15 days to determine the cloning efficiency and to evaluate mutagenicity. Then the plates were evaluated manually.

SELECTION AGENT: 5 μg/mL of Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
Method: Relative suspension growth (RSG) and relative total growth (RTG)

DETERMINATION OF MUTATION FREQUENCY:
- The mutation frequency was derived from the cloning efficiency under selective conditions compared to the corresponding viability under non-selective conditions.
- Cloning efficiency 1 (survival): Cloning efficiency determined immediately after treatment to measure toxicity.
- Cloning efficiency 2 (viability): Cloning efficiency determined after the expression period to measure viability of the cells without selective agent.

SIZE DISTRIBUTION OF COLONIES:
The numbers of colonies were counted manually. The colony size distribution was determined in the controls and at all concentrations of the test substance. Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the density of the small colonies were considerably higher).

Evaluation criteria:

- A test material is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible positive response for at least one of the test points.
- A test material producing neither a reproducible concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non mutagenic in this system.
- The test material is classified as mutagenic if it reproducibly induces a mutation frequency that is at least two times higher than the spontaneous mutation frequency (negative or solvent control) in the experiment.
- The test material is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently of the enhancement factor for induced mutants.
- Results of test groups are rejected if the relative total growth, the relative suspension growth and/or cloning efficiency 1 is less than 10 % of the solvent control or the cloning efficiency 2 after the expression period is less than 20 %.
- Whenever a test material is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations and clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

Statistics:

A statistical evaluation of the results is not necessary according to the OECD 476 guidelines. However, the survival rate and viability was determined based on the Poisson distribution method.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

TEST-SPECIFIC CONFOUNDING FACTORS:
- The osmolarity in the solvent control and the maximum concentration in the pre-experiment without metabolic activation was 343 and 454 respectively.
- The pH-value in the solvent control and the maximum concentration in the pre-experiment without metabolic activation was 7.3 and 7 respectively.
- No precipitation of the test substance was observed up to the maximum concentration in the pre-experiment.

PRE-EXPERIMENTAL TOXICITY STUDIES:
The toxicity was observed at 475 µg/mL and above in the absence of metabolic activation (relative cell growth of 37.2% to 12.3%) and at 59.4 µg/mL and above in the presence of metabolic activation (relative cell growth 38.2 % to 1.4 %) following 4 h treatment. After 24 h of treatment in the absence of S9 mix, strong toxic effects (relative cell growth of 9.4% to 4.2%) occurred at 237.5 µg/mL and above. Based on these results, 6 concentrations covering a range of 30 to 960 μg/mL without and of 3.8 to 120 μg/mL with S9 mix were chosen for the main experiments, from which 4 concentrations each were evaluated.

MAIN EXPERIMENT RESULTS:
Cytotoxicity:
- In experiment I severe toxic effects occurred in the absence of metabolic activation at 960.0 µg/mL in both cultures (relative cloning efficiency 1: 12.8 to 15.3%). In the presence of metabolic activation both, the relative cloning efficiency 1 and the relative total growth were reduced to less than 50 % of the corresponding solvent control at the highest analysable concentration of 60.0 µg/mL (both cultures).
- In experiment II, a steep toxicity gradient was noted with severe toxicity noted at 180 μg/mL and above for both parallel cultures (relative cloning efficiency 1: 1.5 and 1.9%) and only a minor effect at 120 μg/mL. Values obtained at 180 μg/mL and above were rejected as the acceptance criteria (cloning efficiency 1 ≥ 10 %) were not fulfilled at these severely toxic concentrations.
Mutation frequency :
- In experiment I no substantial and reproducible increase of the mutation frequency was observed in both cultures.
- In Experiment II, the mutation frequency just reached the threshold of twice the corresponding solvent control at 120 µg/mL in the first culture. However, the absolute value of mutation frequency remained well within the range of negative and solvent controls and no comparable effects occurred in the second culture under identical conditions. Therefore, the isolated increase in the first culture was judged as based upon biologically irrelevant fluctuations.
- No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test substance.

RESULTS OF NEGATIVE AND POSITIVE CONTROLS:
- In this study, the range of the negative and solvent controls was from 50 up to 200 mutant colonies per 10(6) cells; the range of the acceptable groups treated with the test substance was from 50 up to 259 mutant colonies per 10(6) cells.
- The positive controls showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: TK locus in cultured L5178Y Mouse Lymphoma cells

Applicant's summary and conclusion

Conclusions:

2-amino-6-chloro-4-nitrophenol was considered non-mutagenic in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y, in the absence and presence of metabolic activation.

Executive summary:

The in vitro mammalian gene mutation test of 2-amino-6-chloro-4-nitrophenolwas determined following OECD guideline 476 (In vitro Mammalian Cell Gene Mutation Test).The assay was performed in two independent experiments (both with and without liver microsomal activation) using two parallel cultures each. The cells were exposed to test substance for 4 h in Experiment I (with and without metabolic activation) and for a treatment period of 24 h in Experiment II (without metabolic activation only). Based on the pre-test toxicity (conducted in the range 14.8 – 1900 µg/mL) results, the following concentrationsof the test substance, which was dissolved in DMSO, were selected for the main test:

Experiment I (4 h treatment):

• Without metabolic activation: 0, 30, 60, 120, 240, 480 and 960 µg/mL
• With metabolic activation: 0, 3.8, 7.5, 15, 30, 60 and 120 µg/mL

Experiment II (24 h treatment):

• Without metabolic activation: 0, 15, 30, 60, 120, 180 and 240 µg/mL

DMSO served as the vehicle control. Methylmethane sulfonate (in the presence of metabolic activation) and 3-Methylcholanthrene (3 -Methylcholanthrene) served as positive controls. Each concentration, including the negative, positive and solvent controls was tested in duplicate.

In experiment I severe toxic effects occurred in the absence of metabolic activation at 960.0 µg/mL in both cultures (relative cloning efficiency 1: 12.8 to 15.3%). In the presence of metabolic activation both the relative cloning efficiency and the relative total growth were reduced to less than 50 % of the corresponding solvent control at the highest analysable concentration of 60.0 µg/mL (both cultures).

In experiment II, a steep toxicity gradient was noted with severe toxicity noted at 180 μg/mL and above for both parallel cultures (relative cloning efficiency: 1.5 and 1.9%) and only a minor effect at 120 μg/mL. Values obtained at 180 μg/mL and above were rejected as the acceptance criteria (cloning efficiency ≥ 10 %) were not fulfilled at these severely toxic concentrations.

In experiment I, no substantial and reproducible increase of the mutation frequency was observed in either culture. In Experiment II, the mutation frequency just reached the threshold of twice the corresponding solvent control at 120 µg/mL in the first culture. However, the absolute value of mutation frequency remained well within the range of negative and solvent controls; and no comparable effects occurred in the second culture under identical conditions. Therefore, the isolated increase in the first culture was judged as biologically irrelevant fluctuation.

No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test substance.

The positive controls showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

Based on above, 2-amino-6-chloro-4-nitrophenol was considered non-mutagenic in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y, in the absence and presence of metabolic activation.

This mammalian gene mutation test is classified as acceptable, and satisfies the guideline requirements of the OECD 476 method.