Sodium 2,4,5-trichlorobenzenesulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In recent GLP guideline studies conducted to examine the genetic toxicity (mutagenicity and chromosome abberations) of the test substance in vitro in bacterial cells and mammalian cells (Chinese hamster V79 cells), both with and without S9 metabolic activation, the substance exhibited positive genotoxic effects only in the chomosome aberation test. In the mammalian Cell Gene Mutation Test (HPRT-Locus) in Chinese Hamster V79 Cells, with and without S9 metabolic activation, the substance showed no signs of mutagenicity up to and including doses that produced biologically relevant growth inhibition. In the mammalian cell chromosome abberation test in Chinese Hamster V79 cells the substance did not induce structural chromosomal aberrations in the short-term assay but did induce structural chromosomal aberrations in the long-term assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 28, 2017 - February 12, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Composition: Benzenesulfonic acid, 2,4,5-trichloro-, sodium salt (STB)
CAS #53423-65-7
Purity: 90.5%
Other ingredients - 9.5%
Physical Description: White powder
pH: 7.11
Stability: Test substance was expected to be stable for the duration of testing.
Expiration Date: October 2019

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

chemically-induced rat liver S9 mix

Test concentrations with justification for top dose:

0, 1.58, 5.5, 15.8, 50, 158, 500, 1580, and 5000 μg/plate, with the top dose considered the limit test for this model.

Vehicle / solvent:

Without metabolic activation: Sterile water
With metabolic activation: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Strains TA100 and TA1535 without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR 191 Acridine

Remarks:

TA1537 without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin

Remarks:

TA98 without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

All strains; with metabolic activation

Details on test system and experimental conditions:

The initial test followed the plate incorporation method, in which the following materials were mixed and poured over the surface of a minimal agar plate:
•100 µL of the prepared test substance solutions, negative (vehicle) control, or prepared positive control substance
•500 µL S9 mix or substitution buffer
•100 µL bacteria suspension (ST or EC)
•2000 µL overlay agar maintained at approximately 45°C
Plates were prepared in triplicate at each experimental point and uniquely identified. After pouring, plates were placed on a level surface until the agar gelled then inverted and incubated at approximately 37°C until growth was adequate for enumeration (approximately 65 hours). Appropriate sterility control check plates (treated with critical components in the absence of bacteria) were included as a standard procedural check.

Rationale for test conditions:

The background lawn for vehicle control plates should appear normal (i.e., slightly hazy with abundant microscopic non-revertant bacterial colonies). The mean revertant colony counts for each strain treated with the vehicle should lie close to or within the expected range taking into account the laboratory historical control range and/or published values (Mortelmans & Zeiger, 2000; Gatehouse, 2012).

Evaluation criteria:

Mutagenic activity of the test item was assessed by dividing the mean revertant colony count by the mean revertant colony count for the corresponding concurrent vehicle control group. Toxic effects of the test substance were indicated by the partial or complete absence of a background lawn of non-revertant bacteria, or a substantial dose-related reduction in revertant colony counts compared with lower dose levels and concurrent vehicle control taking into account the laboratory historical control range.

The results were considered positive (i.e., indicative of mutagenic potential) if:

- The results for the test item showed a substantial increase in revertant colony counts, i.e., response MF ≥ 2 for strains TA98, TA100, and WP2 uvrA or MF ≥ 3 for strains TA1535 and TA1537, with mean value(s) outside the laboratory historical control range. Otherwise, results were considered negative.

- The above increase must be dose related and/or reproducible, i.e., increases must be obtained at more than one experimental point (at least one strain, more than one dose level, more than one occasion or with different methodologies).

If the second criterion is not met, the results may be classified as equivocal, and further testing may be appropriate.

Statistics:

Product Safety Labs calculated means and standard deviations for all quantitative data collected.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

The mean revertant colony counts for each strain treated with the vehicle were close to or within the expected range, considering the laboratory historical control range and/or published values (Mortelmans & Zeiger, 2000; Gatehouse, 2012). The positive control substances caused the expected substantial increases in revertant colony counts in both the absence and presence of S9 in each phase of the test confirming the sensitivity of the test and the activity of the S9 mix. Therefore, each phase of the test is considered valid.

 

No signs of precipitation were noted in any of the strains. Contamination which did not obscure counts was seen for TA1537 at dose 5000 µg/plate with S9 in the pre-incubation method. Contamination did not affect mutagenic evaluation. For all strains, at least eight non-toxic doses were evaluated; hence bacterial mutagenicity was adequately assessed.

 

There was no concentration-related or substantial test substance related increases in the number of revertant colonies observed with strains TA1535, TA1537, TA98 , TA100 or E. coli WP2 uvrA in both the absence and presence of S9 using either the plate incorporation or the pre-incubation method.

Conclusions:

The test substance did not elicit evidence of bacterial mutagenicity in the Ames assay.

Executive summary:

In a GLP guideline bacterial reverse mutation assay using the plate incorporation and pre-incubation methods conducted according to OPPTS 870.5100, OECD 471, and EC method B.13/14, the substance did not elicit evidence of bacterial mutagenic activity with or without rat liver S9 metabolic activation with strains TA1535, TA1537, TA98 , TA100 or E. coli WP2 uvrA.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 February 2018 - 30 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Name: STB-FR
Batch No.: 1710-06
Chemical name Sodium Trichlorobenzene Sulfonate
CAS No 53423-65-7
Composition: 90.5% STB
9.5% other
Molecular Weight: 283.494 g/mol
Purity 90.5%
Physical State: solid powder
Color: white
Stability: stable
Storage Conditions: at room temperature
Expiry Date: October 2019

Target gene:

Entire Chromosome

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Pre-Experiment for Toxicity:
with and without: metabolic activation: 5, 10, 25, 50, 100, 250, 500, 1000, 2500, and 5000 µg/mL

Experiment I (exposure concentrations):
without metabolic activation: 1000, 1200, 1400, 1600, 1800, 2000, and 2250 µg/mL
with metabolic activation: 950, 1425, 1663, 1900, 2090, 2185, 2280, 2375, and 2613 µg/mL

Experiment II (exposure concentrations):
without metabolic activation: 100, 250, 500, 750, 1000, 1250, 1500, and 1750 µg/mL

Experiment I (microscopic analysis):
without metabolic activation: 1000, 1400, and 1800 µg/mL
with metabolic activation: 1900, 2185, and 2375 µg/mL

Experiment II (microscopic analysis):
without metabolic activation: 250, 500, 1000, and 1750 µg/mL

Vehicle / solvent:

None

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: Not Applicable

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

V79 cells in vitro are widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are chosen because of their relatively small number of chromosomes (diploid number, 2n = 22), their high proliferation rate (doubling time of the Eurofins Munich V79 in stock cultures: 12 - 14 h) and a high plating efficiency of untreated cells (normally more than 50%). These facts are necessary for the appropriate performance of the study.

The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich, as large stock cultures allowing the repeated use of the same cell culture batch in experiments. Routine checking of mycoplasma infections was carried out before freezing.

For the experiment thawed cultures were set up in 75 cm2 cell culture plastic flasks at 37 °C in a 5% carbon dioxide atmosphere (95% air). 5 x 105 cells per flask were seeded in 15 mL of MEM (minimum essential medium) supplemented with 10% FBS (fetal bovine serum) and subcultures were made 3-4 days after seeding.

Complete Culture Medium: MEM medium supplemented with:
- 10% (v/v) Fetal bovine serum (FBS)
- 100 U/100 µg/mL penicillin/streptomycin solution
- 2 mM L-glutamine
- 2.5 µg/mL amphotericin
- 25 mM HEPES
- Also used for the long-term treatment and the post incubation.

Treatment Medium (short-term exposure): Complete culture medium without FBS.

The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and beta-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route. The preparation was performed according to Ames et al.

Three or four days old stock cultures (in exponential growth) more than 50% confluent were rinsed with Ca-Mg-free PBS solution prior to the trypsin treatment. Cells subsequently were trypsinised with a solution of 0.05% trypsin in Ca-Mg-free PBS at 37 °C for about 5 min. By adding complete culture medium the detachment was stopped and a single cell suspension was prepared. About 1 x 104 cells/mL were seeded into cell culture flasks with complete culture medium.

Treatment parameters/conditions were as follows:

Exp. I (without S9 mix):
- Treatment Period: 4 hours
- Recovery Time: 17 hours
- Preparation interval: 21 hours
Exp. II (without S9 mix):
- Treatment Period: 21 hours
- Recovery Time: not applicable
- Preparation interval: 21 hours
Exp. I (with S9 mix):
- Treatment Period: 4 hours
- Recovery Time: 17 hours
- Preparation interval: 21 hours

Rationale for test conditions:

A pre-experiment was conducted under identical conditions as described for the main experiment. The following concentrations were tested without and with S9 mix: 5, 10, 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/mL. The results of the first main experiment then prompted a need for a second experiment with a later sampling time.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:

a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the 95% control limits of the historical negative control data.

When all of these criteria are met, the test chemical is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined

a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the 95% control limits of the historical negative control data.

The test chemical is then considered unable to induce chromosomal aberrations in cultured mammalian cells in this test system.

Statistics:

Statistical significance at the 5% level (p < 0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding negative control. Aberrant cells without gaps were only used for the calculation. Gaps are recorded separately and reported but generally not included in the total aberration frequency calculation according to the guideline. For the trend test, Statistical significance at the 5% level (p < 0.05) was evaluated by the x² test. The p value was used as a limit in judging for significance levels

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity: In experiment I without metabolic activation, a biologically relevant decrease in cell count (decrease below 70% RICC) was noted at 1800 µg/mL and higher (40% at 1800 µg/mL, 24% at 2000 µg/mL and 5% at 2250 µg/mL). With metabolic activation, a biologically relevant decrease of the relative increase in cell count (decrease below 70% RICC) was noted at 2090 µg/mL (68%) and higher concentrations (58% at 2185 µg/mL, 41% at 2280 µg/mL, 46% at 2375 µg/mL and 28% at 2613 µg/mL). In experiment II without metabolic activation, a biologically relevant decrease in cell count (decrease below 70% RICC) was noted at 500 µg/mL (68%) and higher (60% at 750 µg/mL, 44% at 1000 µg/mL, 47% at 1250 µg/mL, 50% at 1500 µg/mL and 52% at 1750 µg/mL). The number of viable cells was lowest at a concentration of 1000 µg/mL, but increased slightly at higher tested concentrations.

Clastogenicity: In experiment I without metabolic activation, the aberration rate of the negative control (1.3%) and all test item concentrations were within the historical control data of the testing facility (-0.28 to 3.70% aberrant cells exclusive gaps). With metabolic activation, the aberration rates of the negative control (1.7%) and all concentrations of the test item were within the historical control data of the testing facility (-0.23 to 3.95% aberrant cells exclusive gaps) in the short-term experiement. In experiment II (long-term) without metabolic activation, the aberration rate of the negative control (1.0%) and the lowest evaluated test item concentration of 250 µg/mL were within the historical control data of the testing facility (-0.20 to 2.71% aberrant cells exclusive gaps). The higher concentrations were all above the historic control range. The number of cells with chromosomal aberrations was increased at these concentrations. The Fisher´s exact test was performed to verify the results in the experiment. No statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted in the experiment I without and with metabolic activation. However, a statistically significant induction was noted in the evaluated concentrations 1000 µg/mL and 1750 µg/mL of experiment II without metabolic activation. The χ² Test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations. A statistically significant concentration-related increase was observed in experiment I and II without metabolic activation. In experiment I without metabolic activation, the significance was not regarded as biologically relevant as the values were all within the historic control range. In experiment I with metabolic activation, no concentration-related increase was observed. EMS (400 and 600 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.

Polyploid Cells: A biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item in the highest evaluated concentration of experiment II.

Experiment I (short-term)- Summary of Cytotoxicity Data

Dose Group	Concentration [µg/mL]	Cell Count				Precipitate (+/-)
		Culture			RICC
		1	2	Mean	[%]
without metabolic activation
C	0	176.79	161.17	168.98	100	-
1	1000	134.81	151.41	143.11	84	-
2	1200	156.29	126.03	141.16	83	-
3	1400	136.77	113.34	125.05	72	-
4	1600	139.70	130.91	135.30	79	-
5	1800	77.13	70.30	73.71	40	-
6	2000	49.80	47.84	48.82	24	-
7	2250	16.61	20.51	18.56	5	-
EMS	600	129.93	158.24	144.09	84	-
with metabolic activation
C	0	188.50	163.12	175.81	100	-
1	950	148.48	131.89	140.18	79	-
2	1425	171.91	157.27	164.59	93	-
3	1663	113.34	148.48	130.91	73	-
4	1900	148.48	127.00	137.74	77	-
5	2090	123.10	123.10	123.10	68	-
6	2185	88.84	123.10	105.97	58	-
7	2280	85.91	71.27	78.59	41	-
8	2375	90.80	82.99	86.89	46	-
9	2613	66.39	47.84	57.12	28	-
CPA	0.83	168.00	157.27	162.64	92	-

RICC: Relative Increase in Cell Count, calculated by the increase in cell number of the test groups compared to the negative control. The cell count was determined by a cell counter per culture for each test group

C: Negative Control (Culture Medium)

EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)

CPA: Positive Control (with metabolic activation: Cyclophosphamide)

Experiment I (short-term) - Summary of Abberation Rates

Dose Group	Concentration (µg/mL)	Treatment Time (hour)	Fixation Interval	Mean % Abeerrant Cells		Precipitation (+/-)
				incl. Gaps	excl. Gaps
without metabolic activation
C	0	4	21	4	1.3	-
1	1000	4	21	2.7	0.3	-
3	1400	4	21	4.3	2.3	-
5	1800	4	21	5.0	3.0	-
EMS	600	4	21	10.3	8.3	-
with metabolic activation
C	0	4	21	2.7	1.7	-
4	1900	4	21	5.7	2.3	-
6	2185	4	21	1.3	1.0	-
8	2375	4	21	2.0	1.7	-
CPA	0.83	4	21	9.0	9.0	-

300 cells evaluated for each concentration

C: Negative control (culture medium)

EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)

CPA: Positive Control (with metabolic activation: Cyclophosphamide)

Experiment II (long-term)- Summary of Cytotoxicity Data

Dose Group	Concentration [µg/mL]	Cell Count				Precipitate (+/-)
		Culture			RICC
		1	2	Mean	[%]
without metabolic activation
C	0	208	225	216.5	100	-
1	100	236	214	225.0	104	-
2	250	182	161	171.5	78	-
3	500	160	141	150.5	68	-
4	750	133	136	134.5	60	-
5	1000	103	99	101.0	44	-
6	1250	104	111	107.5	47	-
7	1500	120	107	113.5	50	-
8	1750	101	133	117.0	52	-
EMS	400	180	158	169.0	77	-

RICC: Relative Increase in Cell Count, calculated by the increase in cell number of the test groups compared to thenegative control. The cell count was determined by a cell counter per culture for each test group

C: Negative Control (Culture Medium)

EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)

Experiment II (long-term) - Summary of Abberation Rates

Dose Group	Concentration (µg/mL)	Treatment Time (hour)	Fixation Interval	Mean % Abeerrant Cells		Precipitation (+/-)
				incl. Gaps	excl. Gaps
without metabolic activation
C	0	21	21	1.3	1.0	-
2	250	21	21	1.3	0.7	-
3	500	21	21	4.3	3.3	-
5	1000	21	21	6.7	5.7	-
8	1750	21	21	9.0	7.3	-
EMS	400	21	21	10.3	9.0	-

300 cells evaluated for each concentration

C: Negative control (culture medium)

EMS:Positive Control (without metabolic activation: Ethylmethanesulfonate)

Conclusions:

In the short-term assays (Experiment I), the substance did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. In the long-term assay (Experiment II), the substance did induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, the test item is considered to be clastogenic in this chromosome aberration test.

Executive summary:

In this GLP guideline study, conducted according to OECD 472, EC method B.10, and US EPA Method OPPTS 870.5375, to investigate in vitro the potential for the test substance to induce structural chromosome abberations in chinese hamster V79 cells the substance did not induce structural chromosomal aberrations in the short-term assay (Experiment I) but did induced structural chromosomal aberrations in the long-term assay (Experiment II).

In the short-term assay (experiment I) without metabolic activation, a biologically relevant decrease in cell count (decrease below 70% RICC) was noted at 1800 µg/mL and higher (40% at 1800 µg/mL, 24% at 2000 µg/mL and 5% at 2250 µg/mL) and the aberration rate of the negative control (1.3%) and all test item concentrations were within the historical control data of the testing facility (-0.28 to 3.70% aberrant cells exclusive gaps). With metabolic activation, a biologically relevant decrease of the relative increase in cell count (decrease below 70% RICC) was noted at 2090 µg/mL (68%) and higher concentrations (58% at 2185 µg/mL, 41% at 2280 µg/mL, 46% at 2375 µg/mL and 28% at 2613 µg/mL) and the aberration rates of the negative control (1.7%) and all concentrations of the test item were within the historical control data of the testing facility (-0.23 to 3.95% aberrant cells exclusive gaps). The Fisher´s exact test was performed to verify the results in the experiment. No statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted. The χ² Test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations. A statistically significant concentration-related increase was observed without metabolic activation but the significance was not regarded as biologically relevant as the values were all within the historic control range. No statistically significant concentration-related increase was observed with metabolic activation.

In the long-term assay (experiment II) without metabolic activation, a biologically relevant decrease in cell count (decrease below 70% RICC) was noted at 500 µg/mL (68%) and higher (60% at 750 µg/mL, 44% at 1000 µg/mL, 47% at 1250 µg/mL, 50% at 1500 µg/mL and 52% at 1750 µg/mL). The number of viable cells was lowest at a concentration of 1000 µg/mL, but increased slightly at higher tested concentrations. The aberration rate of the negative control (1.0%) and the lowest evaluated test item concentration of 250 µg/mL were within the historical control data of the testing facility (-0.20 to 2.71% aberrant cells exclusive gaps). The higher concentrations were all above the historic control range. The number of cells with chromosomal aberrations was increased at these concentrations. The Fisher´s exact test was performed to verify the results in the experiment. A statistically significant induction was noted in the evaluated concentrations 1000 µg/mL and 1750 µg/mL. The χ² Test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations. A statistically significant concentration-related increase was observed. A biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item in the highest evaluated concentration.

EMS (400 and 600 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.

In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item did induce structural chromosomal aberrations in the V79 Chinese hamster cell line in the long-term experiment. Therefore, the test item is considered to be clastogenic in this chromosome aberration test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 January 2018 - 6 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Name: STB-FR
Batch No.: 1710-06
Chemical name Sodium Trichlorobenzene Sulfonate
CAS No 53423-65-7
Composition: 90.5% STB
9.5% other
Molecular Weight: 283.494 g/mol
Purity 90.5%
Physical State: solid powder
Color: white
Stability: stable
Storage Conditions: at room temperature
Expiry Date: October 2019

Target gene:

Hprt and xprt genes

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

exogenous metabolic

Test concentrations with justification for top dose:

Without Metabolic activation: 1000, 1400, 1600, 1800 and 2200 µg/mL
With metabolic activation: 250, 500, 1250, 2500 and 3250 µg/mL

Vehicle / solvent:

No

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: Not Applicable

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins BioPharma Product Testing Munich GmbH. This allows the repeated use of the same cell culture batch in experiments. Each cell batch was routinely checked for mycoplasma infections (via PCR), stable spontaneous mutant frequency as well as stability of the modal chromosome number. Freshly thawed cells from stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM) and cultured at a humidified atmosphere of 5% CO2 and at 37°C incubation temperature. For purifying the cell population of pre-existing HPRT- mutants cells were exposed to HAT medium containing 10 µM hypoxanthine, 3.2 µM aminopterin, 5 µM thymidine and 10 µM glycine for several cell doublings (2-3 days) with a subsequent recovery period in medium supplemented with 10 µM hypoxanthine and 5 µM thymidine.

Approx. 5 million cells per treatment group were seeded in complete culture medium in a 175 cm2 culture flask. Approx. 24 h after seeding, the cells were exposed to designated concentrations of the test item either in the presence or absence of metabolic activation. After 4 h the cultures were checked for precipitation and the treatment medium containing the test item (MEM without FBS) was removed. The cells were washed twice with PBS, trypsinised and counted with a cell counter. During the following expression period most of the cells were subcultured in complete culture medium (MEM supplemented with 10% FBS) in a sufficient number of cells (at least 2 x 106 cells per treatment group). In addition, for determination of the relative survival (RS) two 25 cm2 flasks were seeded with approx. 200 cells in complete culture medium for each treatment group. After incubation for an appropriate time (6 - 7 days) colonies were fixed with methanol, stained with Giemsa and counted. Cytotoxicity (relative survival) was calculated based on the cloning efficiency of cells plated immediately after treatment adjusted by any loss of cells during treatment.

Rationale for test conditions:

V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are characterized by their high proliferation rate (12 - 14 h doubling time of the Eurofins BioPharma Product Testing Munich GmbH stock cultures) and their high cloning efficiency of untreated cells, usually more than 50%. These facts are necessary for the appropriate performance of the study.

Evaluation criteria:

A test chemical is considered to be clearly negative if, in all experimental conditions examined
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test
- all results are inside the distribution of the historical negative control data

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control, and
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the negative/solvent controls were used as reference.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Main Experiment – Toxicity, without metabolic activation

Dose Group	Concen-tration [µg/mL]	Number of cells at the		Number of colonies per flask			CE[%]	Adjusted CE [%]	Relative Survival (RS) [%]
		beginning of treatment	end of treatment	I	II	mean
NC1	0	10000000	12818000	197	201	199	100	128	100
NC2		10000000	13328000	190	195	193	96	128
1	500	10000000	12121000	192	169	181	90	109	86
2	1000	10000000	11407000	152	169	161	80	92	72
3	1200	10000000	10421000	179	154	167	83	87	68
4	1400	10000000	8772000	185	176	181	90	79	62
5	1600	10000000	5661000	147	133	140	70	40	31
6	1800	10000000	2652000	135	132	134	67	18	14
7	2000	10000000	3910000	139	135	137	69	27	21
8	2200	20000000	6698000	151	140	146	73	24	19
9	2400	20000000	1570800	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
EMS	300	10000000	13583000	207	183	195	98	132	104

NC:negative control; CE:Cloning efficiency; EMS:Ethylmethanesulfonate; n.d.:no data

Main Experiment – Mutagenicity, without metabolic activation

		CE in non-selective medium				CE in selective medium
Dose Group	Concen-tration [µg/mL]	Number of colonies per flask			CE[%]	Number of colonies per flask							CE[%]	Mutant Frequency per 10^6cells
		I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	166	175	171	85	5	11	11	10	5	8.4	2.8	0.0021	24.6
NC2		157	167	162	81	7	3	9	5	10	6.8	2.6	0.0017	21.0
2	1000	154	145	150	75	11	4	4	5	6	6.0	2.6	0.0015	20.1
4	1400	166	149	158	79	2	11	9	8	11	8.2	3.3	0.0021	26.0
5	1600	150	156	153	77	8	4	8	8	13	8.2	2.9	0.0021	26.8
6	1800	174	160	167	84	8	7	9	4	5	6.6	1.9	0.0017	19.8
8	2200	151	154	153	76	3	4	4	3	4	3.6	0.5	0.0009	11.8
EMS	300	147	147	147	74	100	111	93	114	108	105.2	7.7	0.0263	357.8

NC:negative control;CE:Cloning efficiency;EMS:Ethylmethanesulfonate

Main Experiment – Toxicity, with metabolic activation

Dose Group	Concen-tration [µg/mL]	Number of cells at the		Number of colonies per flask			CE[%]	Adjusted CE [%]	Relative Survival (RS) [%]
		beginning of treatment	end of treatment	I	II	mean
NC1	0	10000000	10557000	129	138	134	67	70	100
NC2		10000000	10659000	85	103	94	47	50
1	100	10000000	10591000	105	106	106	53	56	93
2	250	10000000	11135000	105	114	110	55	61	101
3	500	10000000	10200000	115	120	118	59	60	99
4	1250	10000000	7871000	107	94	101	50	40	66
5	2500	10000000	7259000	53	73	63	32	23	38
6	3250	10000000	3383000	58	63	61	30	10	17
7	3500	20000000	6494000	84	77	81	40	13	22
8	4000	20000000	1394000	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
9	4250	20000000	843200	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
10	4500	20000000	411400	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
DMBA	1.0	10000000	11560000	97	99	98	49	57	94

NC:negative control; CE: cloning efficiency; DMBA: 7,12-dimethylbenz(a)anthracene; n.d.: no data

Main Experiment – Mutagenicity, with metabolic activation

		CE in non-selective medium				CE in selective medium
Dose Group	Concen-tration [µg/mL]	Number of colonies per flask			CE[%]	Number of colonies per flask							CE[%]	Mutant Frequency per 10^6cells
		I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	162	143	153	76	16	10	7	10	12	11.0	3.0	0.0028	36.1
NC2		167	173	170	85	12	12	10	15	9	11.6	2.1	0.0029	34.1
2	250	177	170	174	87	13	4	4	6	10	7.4	3.6	0.0019	21.3
3	500	172	180	176	88	4	8	14	9	15	10.0	4.0	0.0025	28.4
4	1250	159	163	161	81	8	5	9	7	6	7.0	1.4	0.0018	21.7
5	2500	158	161	160	80	14	13	12	7	8	10.8	2.8	0.0027	33.9
6	3250	164	186	175	88	14	13	15	5	7	10.8	4.0	0.0027	30.9
DMBA	1.0	134	138	136	68	104	94	88	96	99	96.2	5.3	0.0241	353.7

NC:negative control;CE:cloning efficiency;DMBA: 7,12-dimethylbenz(a)anthracene

Conclusions:

The test item was considered to be non-mutagenic in the in vitro Mammalian Cell Gene Mutation Test (HPRT-Locus) in Chinese Hamster V79 Cells.

Executive summary:

In this GLP guideline study conducted according to OECD 476, EU Method B.17 and OPPTS 870.5300 to assess the test item's potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster, with and without S9 metabolic activation, the substance showed no signs of mutagenicity with or without metabolic activation up to and including doses that produced biologically relevant growth inhibition. In addition, positive controls showed the expected biologically relevant effects in mutation frequency, thus supporting the validity of the study. Given these results, the test item can be considered to be non-mutagenic in the in vitro Mammalian Cell Gene Mutation Test (HPRT-Locus) in Chinese Hamster V79 Cells.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

The relevance of these in vitro findings to humans is unknown.

Justification for classification or non-classification

Despite the positive findings in vitro, the substance does not meet the GHS criterial for classification as a Germ Cell Mutagen on the basis that there is no positive evidence in vivo in either germ cells or somatic cells and there is no known chemical structure activity relationship to known germ cell mutagens.