(xylenes and 4-ethylbenzene)sulfonic acids

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:

other: Read across from analogue substance

Adequacy of study:

key study

Study period:

October 10-12, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Adopted May, 26, 1983

Deviations:

no

Principles of method if other than guideline:

In the test 200 cells were used respect 300 cells requested in the actual guideline. The test was perfomed before 2016 when the nubmer of cells was increased to determine a clearly negative repsonse.
The test performed in 1988 is considered valid and reliable to cover the endpoint.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

p-toluenesulfonic acid

IUPAC Name:

p-toluenesulfonic acid

Constituent 2

Reference substance name:

Toluene-4-sulphonic acid

EC Number:

203-180-0

EC Name:

Toluene-4-sulphonic acid

Cas Number:

104-15-4

Molecular formula:

C7H8O3S

IUPAC Name:

4-methylbenzene-1-sulfonic acid

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction following Aroclor 1254 exposure

Preparation and storage of a l iver homogenate fraction (S9)
Male Sprague Dawley rats (200-300 g) received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bodyweight) 5 days before sacrifice. Preparation is performed at 0 to 4 o c using cold sterile solution and glassware. The livers from at l east 5-6 animals are removed and pooled, washed in 150 mM KCI (approximately 1 ml/g wet livers) . The washed livers are cut into small pieces and homogenized in three volumes of KCI . The homogenate is centrifuged at 9000 g for 10 minutes. The supernatant is the S9 fraction. It is divided into small portions, rapidly frozen and stored at -80 o c for not longer than three months.

Preparation of S9-mix
Sufficient S9 fraction i s thawed immediately before each test at room temperature. One volume of S9 fraction is mixed with 9 volumes of the S9 cofactor sol ution and kept on ice until its use. This preparation i s termed S9-mix. The concentrations of the different compounds in the S9-mix are:
8 mM MgC12
33 mM KCI
5 mM glucose-6-phosphate
4 mM NADP+
100 mM phosphate buffer pH 7.4

Test concentrations with justification for top dose:

Main assay: 200, 600, 1902 µg/ml

In a preliminary experiment the substance was assayed with respect to its solubility in cell culture medium.
The concentrations in the toxicity test are: 50, 100, 200, 300, 500, 750, 1000, 1500 and 1902 µg/ml.
On the basis of the results of this preliminary test, the concentrations in the main assay were 200, 600, 1902 µg/ml.

Vehicle / solvent:

The test substance was dissolved in aqua bidest at the appropriate concentration.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Large stocks of the V 79 cell line stored in liquid nitrogen in the Laboratory of Genetic Toxicology of Hoechst AG are allowing the repeated use of the same cell culture batch in experiments. Consequently the cellular parameters of the experiments remain similar because of the reproducible characteristics of the cel ls.
The tawed stock cultures were progagated at 37 o c in 25 cm 2 plastic flasks . Seeding was done with about 1-3 x 10 5 cell s per flask in 5 ml of MEM-medium supplemented with 10 % fetal calf serum (FCS) . The cell s were subcultured twice a week.

Two days old exponentially growing stock cultures which were over 50 % confluent were trypsinised and a single cell suspension was prepared. The trypsin concentration was 0.5 % in Ca-Mg-free salt solution. 1-2 x 10 6 cell s/ flask were seeded into four 80 cm 2 plastic flasks containing 15 ml MEM with 10 % FCS (6 h preparation). 4-6 x 10 5 cells/ flask were seeded into four 25 cm 2 plastic flasks containing 5 ml MEM with 10 % FCS (18 h preparation) . 2-4 x 10 5 cell s/ flask were seeded into four 25 cm 2 plastic flasks containing 5 ml MEM with 10 % FCS (28 h preparation) .
After 24 h the medium was replaced with medium containing 5 % FCS and the test substance, both without S9-mix and with 40 ul/ml S9-mix.
After 2 h this medium was replaced with normal medium after rinsing once with physiological saline solution.
Treatment was performed with 3 concentrations of the test substance.

The highest concentration did not reduce the number of scorable metaphases more than 20 % of the negative controls. The mitotic index was determined in samples of 1000 cells. The toxicity of the test substance was determined in a preliminary experiment by establishing the concentration-related plating efficiency. According to these data the concentration range was chosen.
3. 5, 15.5 and 25.5 h after the start of the treatment colcemide was added (0.04 ug/ml culture medium) to the cultures. 2.5 h later (6 h, 18 h and 28 h preparation) the cells were trypsinised.
For hypotonic treatment, approximately 5 ml of 0.075 M potassium chloride solut ion at 37 o c was quickly added and suspended. This suspension was then allowed to incubate for 10 minutes in a water bath at 37 o c. Addition of 1 .5 ml fixative and flow through with air.
After re-centrifuging for five minutes at 1000 rpm, al l but one drop of the supernatant was drawn off by pipette. The sediment was carefully covered with a layer composed of 2.5 ml fixative (methanol glacial acetic acid 3 + l) . After 20 minutes the fixation was removed carefully with a pipette and suspended in 2.5 ml fixative. After another 30 minutes, the mixture was centrifuged, after which the liquid was removed by pipette and fresh fixative added. The tubes were covered and kept for at least 12 hours (overnight) in a refrigerator at 4 o c.
After re-centrifuging for 5 minutes at 1000 rpm, al l but one drop of the liquid was removed by pipette and a new suspension formed with a small quantity of freshly prepared fixative. A few drops of this suspension were placed with a pasteur pipette onto clean microscopic slides which had been stored in distill ed water at 4 o c, the drops were then briefly passed through a Bunsen flame and air-dried for 24 hours. Staining was performed as follows:
- staining for 10 minutes in 2 % orcein solutio
- rinsing 3 times in destilled water rinsing twice in acetone
- brief rinsing in acetone/xylene
- 2 minutes in acetone/xylene
- 5 minutes in xylene
- 10 minutes in xylene
- embedding in Entellan R or Eukitt R
2-5 slides were prepared from each flask.
In the same way both negative and positive control s were prepared 16 h after medium change or treatment, respectively.

Analysis of metaphases
After the slides had been coded (Coding Scheme 1115/88) , 100 metaphases per experimental group were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed. The chromosomal aberrations were classified as shown in chapter 6. 1 . Only metaphases with 22 +/- 1 chromosomes are included in the analysis . The metaphases were examined for the following aberrations: gap (g) , break (b) , fragment (f) , minute (m) , deletion (d) , exchanges including intrachanges (ex) , dicentrics (di ) , chromosome disintegration (cd) and ring formation (r i ) . In addition, metaphases with 5 and more aberrations were classified separately as multiple aberrations (ma) .
After the metaphases had been evaluated, the code was l ifted. The values for the control group were compared with the results from the dose group and the positive control at each preparation time.

Evaluation criteria:

The evaluation of the results was performed as follows:
- The test substance is cl assified as mutagenic if i t induces a significantly increased aberration rate as compared with the negative control s with one of the concentrations tested The significance is obvious either by an enhancement of the rate clearly exceeding the control range or it is proven by adequate biometry (Binomial statistic with Fisher' s exact test) .
- the test substance is classified as mutagenic i f there is a reproducible concentration rel ated increase in the aberration rate.
- the test substance is classi fied as not mutagenic when it tests negatively both with and without metabol ic activation .

Statistics:

Binomial statistic with Fisher' s exact 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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test substance was assessed for its mutagenic potential in vitro in the chromosome-aberration-test with two independent cell cultures without metabolic activation and two independent cell cultures with metabolic activation. No significant reproducible enhancement of the chromosome aberration rate over the range of the solvent control was found with any of the concentrations used, neither with, nor without metabolic activation by S9-mix. A small increase of phases with aberrations was observed at 1902 µg/ml 6 and 28 h, and 200 µg/ml 18 h after treatment in the absence of metabolic activation, as well as 200 µg/ml and 600 µg/ml, 18 h after treatment in the presence of metabolic activation. This effect is statistical not significant and not dose dependent and therefore of no genotoxic relevance. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.
The results lead to the conclusion that the test compound is not mutagenic in the chromosome aberration test system in vitro with cell s of the V 79 Chinese hamster cell line.

Mitotic Index: Concentration related plating efficiency was established in 1000 cells from each of two slides per test group. No influence on mitotic index was observed.

Any other information on results incl. tables

Results are given in the following tables

Preliminary Toxicity test
Doses (µg/ml)	Survival rate of cells (without S9)	Survival rate of cells (with S9)
Negative control	69,7 %	64,2 %
Solvent control	66,5 % = 100 %	62,9 % = 100 %
50	81,50%	74,30%
100	77 %	71,7 %
200	64,4 %	83,5 %
300	73,1 %	74,7 %
500	83,6 %	79,3 %
750	87,8 %	81,1 %
1000	74,2 %	76,3 %
1500	84,4 %	76,7 %
1902	80,6 %	65,3 %

I experiment

DUPLICATE A
Exposure: 2 h	Fixation: 6 h
	S9 mix	solvent (μg a.i./ml)	1902 (μg a.i./ml)
Mitotic index (%)	without	13,9	14,4
Number of cells scored	without	100	100
No. of cells with aberration (+ gaps)	without	0	1
Type of aberration	without	-	exchanges including intrachanges
No. of cells with aberration (- gaps)	without	0	1
DUPLICATE B
Exposure: 2 h	Fixation: 6 h
	S9 mix	solvent (μg a.i./ml)	1902 (μg a.i./ml)
Mitotic index (%)	without	15,8	14,8
Number of cells scored	without	100	100
No. of cells with aberration (+ gaps)	without	0	3
Type of aberration	without	-	gap, fragments, dicentrics
No. of cells with aberration (- gaps)	without	0	2
DUPLICATE A
Exposure: 2 h	Fixation: 6 h
	S9 mix	solvent (μg a.i./ml)	1902 (μg a.i./ml)
Mitotic index (%)	with	10,6	10,8
Number of cells scored	with	100	100
No. of cells with aberration (+ gaps)	with	2	4
Type of aberration	with	gap	gap, dicentrics
No. of cells with aberration (- gaps)	with	0	2
DUPLICATE B
Exposure: 2 h	Fixation: 6 h
	S9 mix	solvent (μg a.i./ml)	1902 (μg a.i./ml)
Mitotic index (%)	with	12,4	10,3
Number of cells scored	with	100	100
No. of cells with aberration (+ gaps)	with	3	2
Type of aberration	with	gap, fragments	gap, dicentrics
No. of cells with aberration (- gaps)	with	1	1

II experiment

DUPLICATE A
Exposure: 2 h	Fixation: 18 h
	S9 mix	Solvent	0 (control) (μg a.i./ml)	200 (μg a.i./ml)	600 (μg a.i./ml)	1902 (μg a.i./ml)	Positive control (EMS)
Mitotic index (%)	without	15,6	13,7	14,6	13,9	13,8	14,3
Number of cells scored	without	100	100	100	100	100	100
No. of cells with aberration (+ gaps)	without	0	1	5	0	0	14
Type of aberration	without	-	gap	gap, fragment, dicentrics, exchanges including intrachanges	-	-	different aberration types
No. of cells with aberration (- gaps)	without	0	0	3	0	0	10
	S9 mix	Solvent	0 (control) (μg a.i./ml)	200 (μg a.i./ml)	600 (μg a.i./ml)	1902 (μg a.i./ml)	Positive control (CPA)
Mitotic index (%)	with	10,5	7,4	14	14,9	14,9	11
Number of cells scored	with	100	100	100	100	100	100
No. of cells with aberration (+ gaps)	with	1	1	4	3	1	17
Type of aberration	with	other aberration	gap	break, dicentrics	gap	fragment	gap, break, exchanges including intrachanges
No. of cells with aberration (- gaps)	with	1	0	3	2	1	9
DUPLICATE B
Exposure: 2 h	Fixation: 18 h
	S9 mix	Solvent	0 (control) (μg a.i./ml)	200 (μg a.i./ml)	600 (μg a.i./ml)	1902 (μg a.i./ml)	Positive control (EMS)
Mitotic index (%)	without	16	15,9	16,1	13,5	14	11,8
Number of cells scored	without	100	100	100	100	100	100
No. of cells with aberration (+ gaps)	without	1	3	1	3	0	28
Type of aberration	without	gap	gap, dicentrics	dicentrics	gap	-	different aberration types
No. of cells with aberration (- gaps)	without	0	2	1	1	0	28
	S9 mix	Solvent	0 (control) (μg a.i./ml)	200 (μg a.i./ml)	600 (μg a.i./ml)	1902 (μg a.i./ml)	Positive control (CPA)
Mitotic index (%)	with	8,2	10,2	14,1	13,4	13,5	10,1
Number of cells scored	with	100	100	100	100	100	100
No. of cells with aberration (+ gaps)	with	3	2	3	5	1	11
Type of aberration	with	gap, fragments	gap, break	break, multiple aberration	gap, dicentrics	gap	gap, break, exchanges including intrachanges
No. of cells with aberration (- gaps)	with	1	1	2	2	0	5

III experiment

DUPLICATE A
Exposure: 2 h	Fixation: 28 h
	S9 mix	solvent (μg a.i./ml)	\
Mitotic index (%)	without	15,4	8,7
Number of cells scored	without	100	100
No. of cells with aberration (+ gaps)	without	2	2
Type of aberration	without	gap, dicentrics	fragments
No. of cells with aberration (- gaps)	without	1	2
DUPLICATE B
Exposure: 2 h	Fixation: 28 h
	S9 mix	solvent (μg a.i./ml)	1902 (μg a.i./ml)
Mitotic index (%)	without	13,4	10,5
Number of cells scored	without	100	100
No. of cells with aberration (+ gaps)	without	0	2
Type of aberration	without	-	fragments
No. of cells with aberration (- gaps)	without	0	2
DUPLICATE A
Exposure: 2 h	Fixation: 28 h
	S9 mix	solvent (μg a.i./ml)	1902 (μg a.i./ml)
Mitotic index (%)	with	8,7	12,5
Number of cells scored	with	100	100
No. of cells with aberration (+ gaps)	with	2	0
Type of aberration	with	fragments	-
No. of cells with aberration (- gaps)	with	0	0
DUPLICATE B
Exposure: 2 h	Fixation: 28 h
	S9 mix	solvent (μg a.i./ml)	1902 (μg a.i./ml)
Mitotic index (%)	with	7,1	12
Number of cells scored	with	100	100
No. of cells with aberration (+ gaps)	with	0	1
Type of aberration	with	-	minute
No. of cells with aberration (- gaps)	with	0	1

Applicant's summary and conclusion

Conclusions:

The test substance does not induce chromosome mutations (aberrations) in V79 Chinese Hamster cells. The substance is neither mutagenic nor cytotoxic under the conditions of the study.

Executive summary:

The potential to cause structural chromosome aberrations in cultured mammalian somatic cells of Toluene 4-sulphonic acid was assessed following official guideline OECD 473, In vitro Mammalian Chromosome Aberration Test. The test compound did not induce a significant increase in the number of chromosome aberrations at any preparation time and dose level of the test substance. No relevant cytotoxic effect (reduction of mitotic index) of the compound was observed in the main experiments.