2-methyl-p-phenylenediamine sulfate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 06, 2002 to June 27, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(according to German and OECD principles of GLP)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-methyl-p-phenylenediamine sulfate

EC Number:

210-431-8

EC Name:

2-methyl-p-phenylenediamine sulfate

Cas Number:

615-50-9

Molecular formula:

C7H10N2.H2O4S

IUPAC Name:

2-methylbenzene-1,4-diamine; sulfuric acid

Details on test material:

- Name of test material: SAT 010935 (Sample name)
- Substance type: Pure active substance
- Physical state: Beige solid
- Stability under test conditions: The test substance was prepared in the vehicle directly before application.
- Storage condition of test material: At room temperature, protected from light

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Cultures were seeded in Minimal Essential Medium supplemented with 10% fetal calf serum (complete medium). - Source of cell lines: Supplied by Laboratory for Mutagenicity Testing, Technical
University, Darmstadt
- Justification for the selection of cell line: The V79 cell line has been used successfully for many years in in vitro experiments
- Properly maintained: yes; Large stocks of the V79 cell line were stored in liquid nitrogen in the cell bank of RCC Cytotest Cell Research GmbH allowing the repeated use of the same cell culture batch in experiments.
- Propagation of stock culture: Thawed stock cultures (about 5 x 10(5) cells) were propagated at 37°C in 80 cm2 plastic culture flasks in 15 mL MEM medium and incubated at 4.5% carbon dioxide atmosphere (95.5% air). The cells were subcultured twice weekly.
- Periodically checked for Mycoplasma contamination: yes; before freezing, each batch was screened for contamination.
- Periodically checked for karyotype stability: yes; before freezing, each batch was checked for stability.

Additional strain / cell type characteristics:

other: The cell line has a high proliferation rate (doubling time of clone V79/T5 (sub-clone V79/D3) in stock cultrues: 12 hours), reasonable plating efficiency of untreated cells (>70 %) and a stable karyotype with a modal chromosome number of 22.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/B-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Without S9 mix: 0.6, 1.3, 2.5*, 5*, 10*, and 20 µg/mL
With S9 mix: 100*, 200*, 300*, 400*, 500 and 600 µg/mL

*=dose levels chosen for cytogenetic evaluation The cytogenetic evaluation of higher concentrations (with and without S9 mix) was impossible due to strong test substance induced toxic effects (reduced cell numbers and low metaphase numbers)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Minimal essential medium (with and without fetal calf serum)
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(Culture medium)

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

Prepared by dissolving in nutrient medium at a concentration of 0.7 µg/mL (2.5 µM)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(Culture medium)

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

ethylmethanesulphonate

Remarks:

Prepared by dissolving in nutrient medium at a concentration of 200 µg/mL (1.6 mM)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In the serum free Minimal essential medium (MEM) with 10% fetal calf serum (FCS).

EXPERIMENTAL PROCEDURE AND DURATION
- Seeding: Exponentially growing stock cultures (more than 50% confluent) were rinsed with Ca-Mg-free salt solution containing 200 mg/L EDTA (ethylene diamine tetraacetic acid) and treated with trypsin at 37°C for approx. 5 min. The enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration was 0.2 % in Ca-Mg-free salt solution. The Ca-Mg-free salt solution had the following constituents (per litre): NaCl - 8000 mg; KCl - 400 mg; Glucose - 1000 mg and NaHCO3 - 350 mg.
The cell suspension (approx. 4 x 10(4) was seeded in duplicates/dish/test group into Quadriperm dishes containing microscopic slides.
- Treatment (Exposure) duration: 4 h, After seeding the medium was replaced with serum-free medium (for treatment with 50 µL S9 mix) or complete medium (for treatment without S9 mix) with 10% FCS containing the test material and S9 mix. The pH was adjusted to 7.2.
- Expression time (cells in growth medium): 11.5 h, The culture were washed twice with saline G after treatment and then cultured in complete medium. 0.2 µg/mL colcemid was added to the cultures 15.5 h after the start of the treatment.
Two additional cultures per test material and negative control group (not treated with colcemid), were set up in parallel to determine microscopically the cell number within 10 defined fields per slide.
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h after the start of treatment, the cells on the slides were treated in the chambers with hypotonic solution (0.4% KCl) for 20 min at 37 ° C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts + 1 part) and stained by Giemsa.
SPINDLE INHIBITOR (cytogenetic assays): 15.5 h after the start of treatment Colcemid was added (0.2 µg/ml culture medium) to the cultures.
STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 well spread metaphases per culture were scored for cytogenetic damage using NIKON microscopes with 100x oil immersion objectives. Only metaphases with characteristic chromosome numbers of 22+/-1 were included in the analysis. Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates.

DETERMINATION OF CYTOTOXICITY
- Method: To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid cells in 500 metaphase cells per culture was determined (% polyploid metaphases).
- Determination of endoreplication: yes

Evaluation criteria:

1) The test substance was classified as non-clastogenic if:
-The number of induced structural chromosome aberrations in all evaluated dose groups was in the range of the historical control data (0.0-4.0% aberration cells exclusive gaps).and/or
-No significant increase of the number of structural chromosome aberrations was observed.

2) The test substance was classified as clastogenic if:
-The number of induced structural chromosome aberrations was not in the range of the historical control data (0.0-4.0 % aberrant cells exclusive gaps).and-either a concentration
-Related or a significant increase of the number of structural chromosome aberrations was observed.

3) Although the inclusion of the structural chromosome aberrations was the purpose of the study, it was important to include the polyploid and endoreplications.

4) The following criteria was valid:
-The test substance could be classified as aneugenic if
The number of induced numerical aberrations was not in the range of the historical control data (0.0-8.5 % polyploid cells).

Statistics:

Statistical significance at the five per cent level was evaluated by means of the Fisher's exact test (p<0.05). Evaluation was performed only for cells carrying aberrations exclusive gaps.

However, both biological and statistical significance were considered together. If the evaluation criteria were not clearly met, the classification with regard to the historical data and the biological relevance was discussed and/or a confirmatory experiment was performed.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

(no cytotoxicity was observed at concentrations evaluated for chromsomechromosome aberrations)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Due to a slight pH shift observed in the pre-test the stock solutions were adjusted with 1 N NaOH to physiological values (vehicle control pH 7.4 versus pH 7.1 at 2210 µg/mL without S9).
- Effects of osmolality: No biologically relevant influence of the test substance on the osmolarity was observed (vehicle control 328 mOsm versus 346 mOsm at 2210 µg/mL) in the pre-test.
- Precipitation: In the pre-test, precipitation of the test substance after 4 h treatment was observed at 276.3 µg/mL and above in the absence and the presence of S9 mix. In the main experiment, no test substance precipitation was observed.

RANGE-FINDING/SCREENING STUDIES (PRE-TEST FOR TOXICITY): Using reduced cell numbers as an indicator for toxicity in the pre-test, clear toxic effects were observed after 4 h treatment with 17.3 µg/mL and above in the absence of S9 mix and with 552.5 µg/mL and above in the presence of S9 mix. In addition, 24 hours continuous treatment with 17.3 µg/mL and above in the absence of S9 mix induced strong toxic effects. Considering the toxicity data of the pre-test, 20 µg/mL (without S9 mix) and 600 µg/mL (with S9 mix) were chosen as top concentrations in the main experiment.

RESULTS OF MAIN TEST:
In the absence and the presence of S9 mix, statistically significant and biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test substance. In the absence of S9 mix a dose-related increase of aberrant cells was observed at all evaluated concentrations. After treatment with 10 µg/mL the aberration rate (14.5 % aberrant cells, exclusive gaps) was statistically significantly increased as compared to the corresponding vehicle control (1.0 % aberrant cells, exclusive gaps). Also, the number of cells carrying exchanges was distinctly increased (10.0 %) at this concentration as compared to the vehicle control (0.0%)
Additionally, in the presence of S9 mix, doses of 200, 300 and 400 µg/mL resulted in a dose-related statistically significant increase in the aberration rates (5.5, 7.5 and 18.5 % aberrant cells, exclusive gaps, respectively) compared to the corresponding control culture value (2.0 % aberrant cells, exclusive gaps). Also, the dose-related increase of cells carrying exchanges (1.0, 2.0, 3.0 and 10.5 %) observed at the concentrations evaluated (100, 200, 300 and 400 µg/ml, respectively) gave additional evidence for a clastogenic potential of the test substance. Therefore, these observations in the absence and the presence of S9 mix were regarded as being biologically relevant.
Since the test substance was considered to be clastogenic after 4 h treatment a second experiment was not performed.

RESULTS OF POSITIVE CONTROL: Ethylmethane sulfonate (without S9 mix) at 200 µg/mL and cyclophosphamide (with S9 mix) at 0.7 µg/mL were used as positive controls. They induced statistically significant increases (p<0.05) in cells with structural chromosome aberrations.

COMPARISON WITH HISTORICAL CONTROL DATA: All vehicle and positive control values were within historical data control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: At concentrations evaluated no clear toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed after treatment with the test substance. However, test substance concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage due to strongly reduced cell numbers and low metaphase numbers.

DETERMINATION OF POLYPLOIDY: No biologically relevant increase in the rate of polyploid metaphase was found after treatment with the test substance (1.5 -3.9%) as compared to the frequencies of the controls (1.3 -3.4%.)

Any other information on results incl. tables

Table 1: Structural chromosome aberrations (Main Experiment) with and without S9 mix

Slide Number	Cells Scored	% Aberrant cells			Aberrations
		incl. gaps	excl. Gaps*	with ex. Changes	Gaps		Chromatid type				Chromosome type				other
					g	ig	b	f	d	ex	ib	if	id	cx	ma	cd
Without S9 mix
Test material 2.5 µg/mL
1	100				1	0	0	0	0	0	0	0	0	0	0	0
2	100				0	0	0	0	0	0	0	0	0	0	0	0
1+2	200	0.5	0	0	1	0	0	0	0	0	0	0	0	0	0	0
Test material 5.0 µg/mL
1	100				0	0	0	1	0	0	0	0	0	0	1	0
2	100				0	0	1	1	0	1	0	0	0	0	0	0
1+2	200	2	2	0.5	0	0	1	2	0	1	0	0	0	0	1	0
Test material 10 µg/mL
1	100				7	0	5	3	0	13	1	0	0	0	0	0
2	100				3	0	4	1	0	13	1	0	0	0	0	0
1+2	200	18	4.5	10	10	0	9	4	0	26	2	0	0	0	0	0
With S9 mix
Test material 100 µg/mL
1	100				0	0	1	0	0	1	0	2	0	0	0	0
2	100				0	0	1	1	0	1	0	0	0	0	0	0
1+2	200	2.0	2.0	1.0	0	0	2	1	0	2	0	2	0	0	0	0
Test material 200 µg/mL
1	100				4	0	3	2	3	1	0	0	0	0	0	0
2	100				0	0	1	0	1	2	0	0	0	0	0	0
1+2	200	6.5	5.5	2.0	4	0	4	2	4	3	0	0	0	0	0	0
Test material 400 µg/mL
1	100				6	1	2	4	0	16	3	1	0	0	4	0
2	100				2	0	3	2	0	11	0	0	0	0	4	0
1+2	200	21.0	18.5	10.5	8	1	4	6	0	27	3	1	0	0	8	0

* =Inclusive cells carrying exchanges

g = gap

ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible).

b = break

ib = iso-break

f = fragment

if = iso fragment

d = deletion

id = iso deletion

ma = multiple aberration

ex = chromatid type exchange

cx = chromosome type exchange

cd = chromosomal disintegration

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
positive (with and without metabolic activation)

2 -methyl-p-phenylenediamine sulphate (SAT 010935) was considered clastogenic by the chromosome aberration assay in V79 cells (Chinese hamster cell line) in the presence and absence of S9 mix.

Executive summary:

The in-vitro chromosome aberration test of 2 -methyl-p-phenylenediamine sulphate (SAT 010935) was determined following OECD guideline 473 (In vitro Mammalian Chromosome Aberration Test).

The test material dissolved in minimal essential medium, was assessed for its potential to induce chromosome aberrations in V79 cells of the Chinese hamster in vitro both with and without metabolic activation. The cells were exposed to test material with serum free medium or complete medium for treatment with S9 mix and without S9 mix respectively for 4 h. A pre-test (with the same experimental conditions of main test) on cell growth inhibition with 4 and 24 h treatment was performed to determine maximum dose levels using concentrations separated by no more than a factor of 2 - 3.33.

Based on the pre-test results on toxicity and occurrence of precipitation, the following concentrations for main test were selected:

Without S9 mix: 0.6, 1.3, 2.5, 5.0, 10.0, and 20 µg/mL

With S9 mix: 100, 200, 300, 400, 500 and 600 µg/mL

2.5, 5.0 and 10.0 µg/mL (without S9 mix) and 100, 200, 300 and 400 µg/mL (with S9 mix) were used for cytogenetic evaluation. Concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage.

Each concentration, including the negative, positive and solvent controls was tested in duplicate. Per culture 100 metaphase plates were scored for breaks, fragments, deletions, exchanges and chromosome disintegrations indicative of structural chromosome aberrations.

No clear toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed in any of the test concentrations after treatment with the test material. Statistically significant and biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed in the absence and the presence of metabolic activation. No increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

The test material induced structural chromosome aberrations as determined by thein vitrochromosome aberration test in V79 cells (Chinese hamster cell line).

Based on above, 2 -methyl-p-phenylenediamine sulphate (SAT 010935) was considered clastogenic in the chromosome aberration assay in the presence and absence of S9 mix.

This in-vitro chromosome aberration assay is classified as acceptable, and satisfies the guideline requirements of the OECD 473 method.