Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay: negative (BASF, 1996)
Chromosome aberration test: weakly positive (BASF, 1996)

HPRT gene mutation assay: negative (BASF, 2018)

Link to relevant study records

Referenceopen allclose all

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:
1995-05-22 to 1995-10-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted at a concentration of ca. 39.5 % test substance in an aqueous solution but limit dose of 10 mM was achieved.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
May 1983/Draft September 1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
December 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)
Version / remarks:
May 1987
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
testing lab.
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- ot/batch No.of test material: 80-9159

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELL LINE, STORAGE
The V79 cell line derived from the Chinese hamster has a
- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (>= 90 %)
- stable karyotype (modal number of 22 chromosomes).

Stocks of the V79 cell line (1 mL portions) were maintained at -196 °C in liquid nitrogen using 7 % DMSO in culture medium as a cryoprotectant. Each batch used for the cytogenetic experiments was checked for
- mycoplasma contamination
- karyotype stability
- plating efficiency (incl. vital staining).

Deep-frozen cell suspensions were thawn at 37 °C in a water bath, and volumes of 0.5 mL were transferred into 25 cm2 plastic flasks containing about 5.0 mL MEM (minimal essential medium incl. glutamine), supplemented with 10% FCS (fetal calf serum) and antibiotics. Cells were grown with 5 % CO2 at 37 °C and >= 90% humidity and subcultured twice weekly. Cell monolayers were suspended in culture medium after dispersion with 2.5 % trypsin solution (about 0.1 mL).
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S-9 fraction
Test concentrations with justification for top dose:
1st experiment: without S-9 mix 0, 500, 1000, 2000 µg/mL (i.e. 0, 197.5, 395, 790 µg/ml of active compund); with S-9 mix 0, 3500, 5000, 6500 µg/mL (i.e. 0, 1382.5, 1975, 2567.5 µg/ml of active compund)
2nd experiment: without S-9 mix, 18 hours harvest time: 0, 500, 1000, 2000 µg/mL (i.e. 0, 197.5, 395, 790 µg/ml of active compund); with S-9 mix, 18 hours harvest time 0, 3500, 5000, 6500 µg/mL (i.e. 0, 1382.5, 1975, 2567.5 µg/ml of active compund);without S-9 mix, 28 hours harvest time 0, 2000, 3000 µg/mL; (i.e. 0, 790, 1382.5, µg/ml of active compund) with S-9 mix; 28 hours harvest time 0, 5000, 6500 µg/mL (i.e. 1975, 2567.5 µg/ml of active compund)
3rd experiment: without S-9 mix 0, 1000, 2000, 3000 µg/mL (i.e. 0, 395, 790, 1185 µg/ml of active compund); with S-9 mix 0, 6000, 7000 µg/mL (i.e. 0, 2370, 2765, µg/ml of active compund)
Vehicle / solvent:
Due to the good solubility of the test substance in water, the aqueous culture medium (MEM) was selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation (S-9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
with metabolic activation (S-9 mix)
Details on test system and experimental conditions:
DOSES PER EXPERIMENT
- 1st experiment
The following doses were selected for the 1st experiment using an exposure time of 4 hours with S-9 mix and of 18 hours without S-9 mix:

Doses without S-9 mix; 18 hours harvest time
250 µg/mL aqueous test substance = 98.75 µg/mL active component
500 µg/mL aqueous test substance = 197.5 µg/mL active component
1000 µg/mL aqueous test substance = 395 µg/mL active component
2000 µg/mL aqueous test substance = 790 µg/mL active component
5000 µg/mL aqueous test substance = 1970 µg/mL active component

Doses with S-9 mix; 18 hours harvest time
2000 µg/mL aqueous test substance = 790 µg/mL active component
3500 µg/mL aqueous test substance = 1382.5 µg/mL active component
5000 µg/mL aqueous test substance = 1970 µg/mL active component
6500 µg/mL aqueous test substance = 2567.5 µg/mL active component

In general, depending on the toxicity actually found three dose levels were assessed.

- 2nd experiment
The following doses were selected for the 2nd experiment again with an exposure time of 4 hours using S-9 mix and with a continuous treatment of
18 hours without S-9 mix:

Doses without S-9 mix; 18 hours harvest time
500 µg/mL aqueous test substance = 197.5 µg/mL active component
1000 µg/mL aqueous test substance = 395 µg/mL active component
2000 µg/mL aqueous test substance = 790 µg/mL active component
3000 µg/mL aqueous test substance = 1185 µg/mL active component

Doses without S-9 mix; 28 hours harvest time
2000 µg/mL aqueous test substance = 790 µg/mL active component
3000 µg/mL aqueous test substance = 1185 µg/mL active component

Doses with S-9 mix; 18 hours harvest time
3500 µg/mL aqueous test substance = 1382.5 µg/mL active component
5000 µg/mL aqueous test substance = 1970 µg/mL active component
6500 µg/mL aqueous test substance = 2567.5 µg/mL active component

Doses with S-98 mix; 28 hours harvest time
5000 µg/mL aqueous test substance = 1970 µg/mL active component
6500 µg/mL aqueous test substance = 2567.5 µg/mL active component

This selection was based on the findings from the 1st cytogenetic experiment. Again, three dose levels were assessed at a sampling time of 18 hours. At the additional later harvest time of 28 hours two doses were evaluated both with and without metabolic activation.

- 3rd experiment
The following doses were selected for the 3rd experiment with an exposure time of 4 hours with S-9 mix and with a continuous treatment of 18 hours
without S-9 mix:

Doses without S-9 mix; 28 hours harvest time
1000 µg/mL aqueous test substance = 395 µg/mL active component
2000 µg/mL aqueous test substance = 790 µg/mL active component
3000 µg/mL aqueous test substance = 1185 µg/mL active component
4000 µg/mL aqueous test substance = 1580 µg/mL active component

Doses with S-9 mix; 28 hours harvest time
4000 µg/mL aqueous test substance = 1580 µg/mL active component
5000 µg/mL aqueous test substance =1970 µg/mL active component
6000 µg/mL aqueous test substance = 2370 µg/mL active component
7000 µg/mL aqueous test substance = 2765 µg/mL active component

This selection was based on the findings from the 2nd experiment. Two doses with metabolic activation and 3 doses without S-9 mix were evaluated.

CELL CYCLE TIME
The cell cycle of the untreated V79 cells lasted for about 13 - 14 hours [last measurement based on the BrdU method of SPEIT, G. et al.: December 1995] under the selected culture conditions. Thus, the selected 1st sampling time of 18 hours was within the 1 - 1.5 x the normal cell cycle time, as recommended in an "EEC Guidance Note - The practical interpretation of Ames V Test Method B 10, the in vitro mammalian cell cytogenetic test".
The later sampling time of 28 hours was chosen to cover a possible cell cycle delay.

SAMPLING TIMES
Chromosomal aberrations were generally analyzed in the first metaphase after they had formed to avoid loss during mitoses or conversion of the initial aberrations into more complex derivatives during subsequent cell cycles. Since aberrations are induced by the majority of chemical clastogens during DNA replication, the harvest time must allow cells to progress through the 5-phase after treatment to convert initial DNA damage
into chromosome alterations visible at mitosis. Since V79 cells are asynchronous and different chemicals may affect different stages of the cell
cycle, more than one sampling time is necessary. Furthermore, mitotic delay may result from clastogen exposure and thus considerably delay the first post-treatment mitosis. Thus, samples were taken at 18 hours and 28 hours after the beginning of a 4-hour treatment (with S-9 mix) or of an 18-hour treatment (without S-9 mix) covering the intervals at which maximum aberration frequencies were expected.

TREATMENT OF TEST CULTURES
About 24 - 30 hours after seeding and incubating the cells, the medium was replaced by fresh medium. The test article, dissolved in 1 mL serum-free medium (4-hour treatment) or in medium with FCS (18-hour treatment), was added to the culture medium with or without 1 mL S-9 mix. Concurrent negative and positive controls were tested in parallel. After incubation (5 % CO2, 37 °C and >= 90 % humidity) for 4 hours with S-9 mix the serum-free medium was replaced by MEM supplemented with 10 % FCS after being rinsed twice with Hanks' balanced salt solution (HBSS). Subsequently, the Quadriperm dishes were incubated again for another 14 hours or 24 hours until the cells were harvested. In the experiments without S-9 mix, cells were treated for 18 hours in culture medium supplemented with 10 % FCS.

CELL HARVEST AND PREPARATION OF METAPHASE SPREADS
The cells were prepared based on the methods described by SCHMID, W. and SPEIT, G. and S. HAUPTER.
- 2 - 3 hours prior to harvesting the cells, 0.2 µg colcemid/mL culture medium (= 1 µg colcemid dissolved in 0.1 mL PBS/culture) was added to each
chamber in order to arrest mitosis in the metaphase.
- After incubation at 37 °C, the culture medium was completely removed.
- For hypotonic treatment, 5 mL of a 0.4 % KCl solution which was at 37 °C was added for about 20 minutes.
- Subsequently, 5 mL of fixative (methanol : glacial acetic acid/3 : 1) which was at 4 °C was added and kept for at least 15 minutes and then replaced.
After about another 10 minutes, the fixative was replaced again and kept for at least 5 minutes at room temperature for complete fixation.
- The slides were taken out of the Quadriperm chambers, briefly dripped off and then rapidly passed through a Bunsen burner flame.
- The preparations were dried in the air and subsequently stained in a solution of Giemsa and Titrisol (15 mL Giemsa, 185 mL Titrisol pH 7.2) for 10 minutes.
- After being rinsed twice in purified water and clarified in xylene, the preparations were mounted in Corbit-Balsam.

CHROMOSOME ANALYSIS
As a rule, the first 100 consecutive well-spread metaphases of each culture were counted for all test groups, and if cells had 20 - 22 chromosomes, they were analyzed for structural chromosome aberrations. Numerical chromosome aberrations were also recorded. lf there is a clear increase in chromosomally damaged cells the number of metaphases to be analyzed is reduced from the planned 200 mitoses/test group.

MITOTIC INDEX
A mitotic index based on 1000 cells/culture was determined for all test groups.

CELL COUNTS
For the determination of cytotoxicity, additional cell cultures (using 25 cm2 plastic flasks) were treated in the same way as in the main experiment. Growth inhibition was estimated by counting the number of cells in the dose groups in comparison with the concurrent vehicle control at the end of the
culture period using a counting chamber.

CELL MORPHOLOGY
About 3 hours after test substance treatment cultures of all test groups were checked for cell morphology, which is an indication of attachment of the cells to the slides.

TREATMENT CONDITIONS
pH values and osmolality were measured. The solubility of the test substance in the vehicle used and in the aqueous culture medium about 3 hours after treatment was checked to ensure proper culturing and to avoid extreme treatment conditions.
Evaluation criteria:
The test chemical is to be considered positive in this assay if the following criteria are met:
- A dose-related and reproducible significant increase in the number of structural chromosomal aberrations.
- The proportion of aberrations exceeded both the concurrent negative control range and the negative historical control range.
A test substance is generally considered nonclastogenic in this test system if:
- There was no significant increase in the number of chromosomally damaged cells at any dose above concurrent control frequencies.
- The aberration frequencies were within the historical control range.
Statistics:
The statistical evaluation of the data was carried out using the MUCHIAN program System (BASF AG). The proportion of metaphases with aberrations was calculated for each group. A comparison of each dose group with the vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
other: slight but statistically significant and dose-dependent increase in the number of structurally aberrant metaphases incl. and excl. gaps after a sampling time of 28 hours in two experiments independent of each other
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Both of the positive control chemicals, i.e. EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. According to the results of the present study, the test substance caused a slight but statistically significant and dose-dependent increase in the number of structurally aberrant metaphases incl. and excl. gaps after a sampling time of 28 hours without S-9 mix in two experiments independent of each other.

Thus, under the experimental conditions of this assay, the test substance is considered to have a weakly chromosome-damaging (clastogenic) effect under in vitro conditions in V79 cells.

1st Experiment

Without S-9 Mix, 18 hours harvest time

Dose (active compound) [µg/mL] H. Metaphases Incl. Gaps Excl. Gaps Exchanges Mul. Aber. Chr. Dis. Aneupl. Polypl.
N % N % N % N % N % N % N %
Vehicle MEM 18 200 7 3.5 4 2 3 1.5 0 0 0 0 0 0 1 0.5
500 (198) 18 200 19 9.5* 3 1.5 2 1 0 0 0 0 0 0 3 1.5
1000 (395) 18 200 19 9.5* 4 2 0 0 1 0.5 0 0 0 0 0 0
2000 (790) 18 200 34 17.0** 9 4.5 2 1 0 0 0 0 0 0 1 0.5
EMS 350 18 100 15 15.0** 13 13.0** 8 8.0* 0 0 0 0 0 0 0 0

With S-9 Mix, 18 hours harvest time

Dose (active compound) [µg/mL] H.  Metaphases Incl. Gaps Excl. Gaps Exchanges Mul. Aber. Chr. Dis. Aneupl. Polypl.
N % N % N % N % N % N % N %
Vehicle MEM 18 200 13 6.5 4 2 3 1.5 0 0 0 0 1 0.5 4 2
3500 (1383) 18 200 15 7.5 10 5 6 3 0 0 0 0 0 0 2 1
5000 (1975) 18 200 11 5.5 4 2 1 0.5 1 0.5 0 0 0 0 1 0.5
6500 (2568) 18 200 9 4.5 5 2.5 3 1.5 0 0 0 0 3 1.5 1 0.5
CPP 0.5 18 100 15 15.0* 13 13.0** 9 9.0** 0 0 0 0 0 0 0 0

2nd Experiment

Without S-9 mix, 18 hours harvest time

Dose (active compound) [µg/mL] H. Metaphases Incl. Gaps Excl. Gaps Exchanges Mul. Aber. Chr. Dis. Aneupl. Polypl.
N % N % N % N % N % N % N %
Vehicle MEM 18 200 8 4 1 0.5 1 0,5 0 0 0 0 0 0 0 0
500 (198) 18 200 9 4.5 3 1.5 2 1 1 0.5 0 0 0 0 0 0
1000 (395) 18 200 4 2 3 1.5 3 1.5 0 0 0 0 0 0 0 0
2000 (790) 18 200 18 9 6 3 4 2 0 0 0 0 0 0 0 0
EMS 350 18 100 13 13.0* 13 13.0** 10 10.0** 0 0 0 0 0 0 0 0

With S-9 Mix, 18 hours harvest time

Dose (active compound) [µg/mL] H. Meta-phases Incl. Gaps Excl. Gaps Exchanges Mul. Aber. Chr. Dis. Aneupl. Polypl.
N % N % N % N % N % N % N %
Vehicle MEM 18 200 8 4 2 1 0 0 0 0 0 0 1 0.5 0 0
3500 (1383) 18 200 18 9 8 4 3 1.5 0 0 0 0 0 0 0 0
5000 (1975) 18 200 13 6.5 2 1 1 0.5 0 0 0 0 0 0 0 0
6500 (2568) 18 200 11 5.5 5 2.5 1 0.5 0 0 0 0 1 0.5 2 1
CPP 0.5 18 100 17 17.0** 15 15** 9 9.0** 0 0 0 0 0 0 0 0

Without S-9 Mix, 28 hours harvest time

Dose (active compound) [µg/mL] H. Meta-phases Incl. Gaps Excl. Gaps Exchanges Mul. Aber. Chr. Dis. Aneupl. Polypl.
N % N % N % N % N % N % N %
Vehicle MEM 28 200 10 5 3 1.5 0 0 0 0 0 0 1 0.5 0 0
2000 (790) 28 200 24 12.0* 2 1 0 0 0 0 0 0 1 0.5 1 0.5
3000 (1185) 28 200 21 10.5* 13 6.5* 8 4.0** 0 0 0 0 0 0 1 0.5

With S-9 Mix, 28 hours harvest time

Dose (active compound) [µg/mL] H. cultures Meta-phases Incl. Gaps Excl. Gaps Exchanges Mul. Aber. Chr. Dis. Aneupl. Polypl.
N % N % N % N % N % N % N %
Vehicle MEM 28 200 6 3 3 1.5 0 0 0 0 0 0 0 0 0 0
5000 (1975) 28 200 11 5.5 5 2.5 2 1 0 0 0 0 0 0 0 0
6500 (2568) 28 200 21 10.5** 0 0 0 0 0 0 0 0 0 0 1 0.5

3rd Experiment

Without S-9 Mix, 28 hours harvest time

Dose (active compound) [µg/mL] H. Meta-phases Incl. Gaps Excl. Gaps Exchanges Mul. Aber. Chr. Dis. Aneupl. Polypl.
N % N % N % N % N % N % N %
Vehicle MEM 28 200 11 5.5 5 2.5 2 1 0 0 1 0.5 3 1.5 0 0
1000 (395) 28 200 25 12.5* 8 4 4 2 0 0 0 0 0 0 2 1
2000 (790) 28 200 26 13.0* 16 8.0* 9 4.5 0 0 0 0 1 0.5 0 0
3000 (1185) 28 200 39 19.5** 16 8.0* 10 5 0 0 0 0 1 0.5 0 0
EMS 350 28 100 19 19.0** 16 16.00** 6 6 1 1 0 0 0 0 0 0

With S-9 Mix, 28 hours harvest time

Dose (active compound) [µg/mL] H. Meta-phases Incl. Gaps Excl. Gaps Exchanges Mul. Aber. Chr. Dis. Aneupl. Polypl.
N % N % N % N % N % N % N %
Vehicle MEM 28 200 11 5.5 6 3 0 0 0 0 0 0 0 0 0 0
6000 (2370) 28 200 19 9.5 8 4 3 1.5 0 0 0 0 2 1 1 0.5
7000 (2765) 28 200 16 8 9 4.5 4 2 0 0 0 0 0 0 1 0.5
CPP 0.5 28 100 18 18.0** 16 16.0** 6 6.0** 0 0 0 0 0 0 0 0

Fisher's Exact Test (one-sided) with Bonferroni-Holm correction: *: p <= 0.05, **: p <= 0.01

A pairwise comparison of each dose group with the solvent control group, Bonferroni-holm corrected for each time.

MEM: Minimal essential medium incl. glutamine

CPP: Cyclophosphate

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-10-17 to 1995-03-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted at a concentration of ca. 39.5 % test substance in an aqueous solution.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
testing lab.
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- ot/batch No.of test material: 80-9159

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix
Test concentrations with justification for top dose:
1st experiment: with and without S-9 mix 0, 40, 200, 1000, 5000, 10000 µg/plate i.e. 16, 79, 395, 1975, 3950 µg/plate of active compound (TA1535, TA100, TA1537, TA98)
2nd experiment: with and without S-9 mix 0, 40, 200, 1000, 5000, 10000 µg/plate i.e. 16, 79, 395, 1975, 3950 µg/plate of active compound(TA1535, TA100, TA1537, TA98)
3rd experiment: with and without S-9 mix 0, 40, 200, 1000, 5000, 10000 µg/plate i.e. 16, 79, 395, 1975, 3950 µg/plate of active compound (E. coli WP2 uvrA)
4th experiment: with and without S-9 mix 0, 40, 200, 1000, 5000, 10000 µg/plate i.e. 16, 79, 395, 1975, 3950 µg/plate of active compound (E. coli WP2 uvrA)
Vehicle / solvent:
- Vehicle used: purified water
- Justification for choice of vehicle: Good water solubility of the test substance.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 100, TA 98, TA 1537,TA 1535, WP2 uvrA (with S-9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitroso-guanidine
Remarks:
TA100, TA1535 (without S-9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine
Remarks:
TA 98 (without S-9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 (without S-9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
E. coli WP2 uvrA (without S-9 mix)
Details on test system and experimental conditions:
Bacteria
For testing, deep-frozen (-70 °C to -80 °C) bacterial cultures are thawed at room temperature, 0.1 mL of this bacterial suspension is inoculated in nutrient broth solution (8 g Difco nutrient broth + 5 g NaCl/liter) and incubated in the shaking water bath at 37 °C for about 10 - 12 hours. As a rule, a germ density of >= 10E08 bacteria/mL is reached. These cultures grown overnight are kept in iced water from the beginning of the experiment until the end in order to prevent further growth.

MUTAGENITY TEST (Standard plate test)

Salmonella typhimurium:
The experimental procedure is based on the method of Ames et al.. Test tubes containing 2 mL portions of soft agar which consists of 100 mL agar (0.6 % agar + 0.6 % NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45 °C, and the remaining components are added in the following order:
- 0.1 mL test solution or vehicle
- 0.1 mL bacterial suspension
- 0.5 mL S-9 mix (in tests with metabolic activation)
or
- 0.5 mL phosphate buffer (in tests without metabolic activation)

After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar:
- 980 mL aqua dest.
- 20 mL Vogel-Bonner E medium
- 15 g Difco bacto agar
- 20 g D-glucose, monohydrate
After incubation at 37 °C for 48 hours in the dark, the bacterial colonies are counted.

Escherichia coli:
The experimental procedure is based on the method of Ames et al.. Test tubes containing 2 mL portions of soft agar which consists of 100 mL agar (0.6 % agar + 0.6 % NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at 45 °C and the remaining components are added in the following order:
- 0.1 mL test solution or vehicle
- 0.1 mL bacterial suspension
- 0.5 mL S-9 mix (in tests with metabolic activation)
or
-0.5 mL phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds.

Composition of the minimal agar:
The composition of the minimal agar (SA1 selective agar) is based on the description of Green, M.H.L. and Muriel, W.J., with the exception of solution E (tryptophan solution), which has been added to the soft agar before:
- 300 mL solution B (agar)
- 100 mL solution A (saline solution)
- 8 mL solution C (glucose solution)
- 10 mL solution D (casein solution)

After incubation at 37 °C for 48 hours in the dark, the bacterial colonies are counted.


PREINCUBATION TEST:

The experimental procedure is based on the method described by Yahagi et al. and Matsushima et al. 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S-9 mix are incubated at 37 °C for the duration of 20 minutes. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37 °C for 48 hours in the dark, the bacterial colonies are counted.

The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character; UV sensitivity; ampicillin resistance.
Histidine and tryptophan auxotrophy is automatically checked in each experiment via the spontaneous rate.
Evaluation criteria:
In general, a substance to be characterized as positive in the bacterial tests has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: A weakly bacteriotoxic effect was occasionally observed depending on the strain and test conditions at doses > 5000 µg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
An increase in the number of his+ or trp+ revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Standard Plate Test

Dose (active compound) [µg/plate]

 Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and E.coli

TA 1535

TA 100

TA 1537

TA 98

E. coli WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Vehicle

19

18

107

117

12

11

28

46

37

43

40 (16)

18

17

112

114

11

10

27

31

47

37

200 (79)

19

15

95

102

9

10

27

26

41

51

1000 (395)

16

15

99

121

10

11

28

28

40

49

5000 (1975)

11

15

108

110

8

8

28

26

48

51

10000 (3950)

10

12

107

96

8

7

20

23

40

53

Positive control

853

110

851

782

651

105

779

1124

793

200

Preincubation Test

Dose [µg/plate]

 Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and E.coli

TA 1535

TA 100

TA 1537

TA 98

E. coli WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Vehicle

19

18

134

140

8

9

28

37

35

46

40 (16)

16

17

132

145

9

11

26

32

39

43

200 (79)

15

14

131

117

8

10

24

34

38

46

1000 (395)

14

14

118

129

5

8

19

32

36

45

5000 (1975)

13

13

100

117

8

6

19

25

30

42

10000 (3950)

15

13

111

126

8

4

16

25

37

39

Positive control

1146

112

969

888

757

112

1071

747

591

200

M= mean

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-23 - 2018-02-20
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)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: Gene mutation test in mammalian cells (HPRT locus)
Specific details on test material used for the study:
Name of test substance: Disodium (sulphonatothio)acetate; dried
Test substance No.: 14/0318-2
Batch identification: Sample 14/078 aus Mollescal HW, charge 10924344R0
Identity: Confirmed
Purity: approx. 60.9 g/100 g (see analytical report, Study code: 15L00094)
Content: 60.9 g/100 g (1H-NMR)
79.0 g/100 g (titration) (see analytical report, Study code: 15L00094)
Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance preparations.
Storage stability: The stability of the test substance under storage conditions was guaranteed until 28 Apr 2019 as indicated by the sponsor, and the sponsor holds this responsibility.
The test facility is organizationally independent from the BASF SE sponsor division.
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes.
Stocks of the CHO cell line (1-mL portions) are maintained at -196°C in liquid nitrogen using 7% (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma contamination.
Test concentrations with justification for top dose:
Based on the data and the observations from the pretest and taking into account the current guidelines, the following doses were selected in this study.
1st Experiment:
With and without S9 mix (4-hour exposure)
206.25 μg/mL
412.50 μg/mL
825.00 μg/mL
1650.00 μg/mL
3300.00 μg/mL

2nd Experiment:
With and Without S9 mix (4-hour exposure)
375.0 μg/mL
750.0 μg/mL
1500.0 μg/mL
3000.0 μg/mL
3300.0 μg/mL
At least four concentrations were evaluated to describe a possible dose-response relationship. In general in this study, the concentrations are given as rounded values by using a dilution factor of 2
Vehicle / solvent:
Due to the good solubility of the test substance in water, the aqueous culture medium (Ham's F12) was selected as vehicle.
Untreated negative controls:
yes
Positive controls:
yes
Details on test system and experimental conditions:
All media were supplemented with:
- 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL)
- 1% (v/v) amphotericine B (stock solution: 250 μg/mL)
Culture medium: Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) supplemented with 10% (v/v) fetal calf serum (FCS).
Treatment medium (without S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) FCS.
Treatment medium (with S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine.
Pretreatment medium ("HAT" medium): Ham's F12 medium supplemented with:
- hypoxanthine (13.6 x 10-3 mg/mL)
- aminopterin (0.18 x 10-3 mg/mL)
- thymidine (3.88 x 10-3 mg/mL)
- 10% (v/v) FCS
Selection medium ("TG" medium): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with:
- 6-thioguanine (10 μg/mL)
- 10% (v/v) FCS

For cell cultivation, deep-frozen cell suspensions were thawed at 37°C in a water bath, and volumes of 0.5 mL were transferred into 25 cm2 plastic flasks containing about 5 mL Ham's F12 medium including 10% (v/v) FCS. Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity up to approximate confluence and sub-cultured twice weekly (routine passage in 75 cm2 plastic flasks). Routine passage (preparation of a single cell suspension)
- Cell medium was removed and cells were washed with 5 mL PBS or HBSS (both Ca-Mgfree).
- Cells were trypsinized with 2 mL HBSS (Hanks balanced salt solution; Ca-Mg-free) and 2 mL trypsin (0.25% [w/v]) to remove the cells from the bottom of the plastic flasks.
- This reaction was stopped by adding 6 mL culture medium incl. 10% (v/v) FCS.
- Cells were pipetted up and down to separate them and to prepare a homogeneous single cell suspension.
- Cells were counted in a counting chamber or using a cell counter.
- Cell suspensions were diluted with complete culture medium to the desired cell count.
Evaluation criteria:
Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative controls should be within our historical negative control data range (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• The positive controls both with and without S9 mix should induce a distinct, statistically significant increase in mutant frequencies in the expected range (see Appendix 7).
Assessment criteria
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).
Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).
Statistics:
An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report (6). The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction (7, 8). The calculation was performed using R (9). If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
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:
TREATMENT CONDITIONS
In the first experiment, the pH values were not influenced by test substance treatment. Osmolality was slightly increased by test substance treatment in the highest applied concentrations with and without S9 mix. In detail, in the absence of S9 mix the osmolality was increased from 291 mOsm (negative control) to 344 mOsm (test group 3300.00 μg/mL) and in the presence of S9 mix from 269 mOsm (vehicle control) to 314 mOsm (test group influenced by test substance treatment. However, the increased values obtained for osmolality did not influence the results on cytotoxicity and genotoxicity of this study. In this study, in the absence and the presence of S9 mix, no precipitation in culture medium was observed macroscopically up to the highest applied test substance concentration.
CELL MORPHOLOGY
After 4 hours treatment either in the absence or presence of metabolic activation, the cell morphology and attachment of the cells was not adversely influenced (grade > 2) in any test group tested for gene mutations.
CYTOTOXICITY
No cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective negative control values were observed in both experiments either in the absence or presence of S9 mix.
MUTANT FREQUENCY
In this study, no relevant increase in the number of mutant colonies was observed with or without S9 mix. In the absence of S9 mix the corrected mutation frequencies ranged between 0.58 – 2.35 per 106 cells in the first experiment and 3.57-6.51 per 106 cells in the second experiment. The negative control values were 4.39 and 2.40 in the first and second experiments, respectively. The obtained values were within the range of the 95% control limit of our historical negative control data (without S9 mix: MFcorr.: 0.00 – 5.97 per 106 cells; see Appendix 6), except the value obtained at 3000 μg/mL in the second experiment (6.51 per 106 cells). However, this increased value was not statistically significant and not dose related. Thus, the increase is not considered as biologically relevant. In the presence of S9 mix the corrected mutation frequencies ranged between 0.37 – 5.88 per 106 cells in the first experiment and 1.79-8.37 per 106 cells in the second experiment. The negative control values were 2.53 and 4.95 in the first and second experiments, respectively. The obtained values were within the range of the 95% control limit of our historical negative control data (without S9 mix: MFcorr.: 0.00 – 7.91 per 106 cells; see Appendix 6), the value obtained at 1500 μg/mL in the second experiment (8.37 per 106 cells). However, this increased value was not statistically significant and not dose related. Thus, the increase is also not considered as biologically relevant. In all experiments, no statistically significant and dose-related increase (trend analysis) in the mutant frequency was found in cells after 4 hours of treatment either in the absence or presence of S9 mix. The positive control substances EMS (without S9 mix; 400 μg/mL) and DMBA (with S9 mix; 1.25 μg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 101.15 – 202.44 per 106 cells; with S9 mix: MFcorr.: 159.49 – 160.82 per 106 cells) were clearly within our historical positive control data range (without S9 mix: MFcorr.: 42.47 – 419.90 per 106 cells; with S9 mix: MFcorr.: 21.52 – 270.48 per 106 cells).

Mutant frequency and viability - 1st Experiment without S9 mix; 4-hour exposure period

 

 

Test groups [µg/mL]

Cloning efficiency 2 (viability)

Mutant frequency

Number of colonies

CE2

Number of coloniesa

MF (per 106cells)

Dish 1

Dish 2

Abs. [%]

Rel. [%]

Flask 1

Flask 2

Uncorrected

Correctedb

Negative control

180

162

85.5

100.0

8

7

3.75

4.39

206.25

n.c.

412.50

177

140

79.3

92.7

5

2

1.75

2.21

825.00

149

149

74.5

87.1

5

2

1.75

2.35

1650.00

148

165

78.3

91.5

1

4

1.25

1.60

3300.00

172

173

86.3

100.9

1

1

0.50

0.58

EMS 400.0

127

134

65.3

76.3

134

130

66.00

101.15S

S       Mutant frequency statistically significant higher than corresponding control values (p ≤0.05)


 

Mutant frequency and viability - 1st Experiment with S9 mix; 4-hour exposure period

 

 

Test groups [µg/mL]

Cloning efficiency 2 (viability)

Mutant frequency

Number of colonies

CE2

Number of coloniesa

MF (per 106cells)

Dish 1

Dish 2

Abs. [%]

Rel. [%]

Flask 1

Flask 2

Uncorrected

Correctedb

Negative control

151

165

79.0

100.0

3

5

2.00

2.53

206.25

n.c.

412.50

152

143

73.8

93.4

7

4

2.75

3.73

825.00

131

139

67.5

85.4

1

0

0.25

0.37

1650.00

144

145

72.3

91.5

9

8

4.25

5.88

3300.00

166

153

79.8

100.9

0

3

0.75

0.94

DMBA 1.25

120

125

61.3

77.5

205

189

98.50

160.82S

S       Mutant frequency statistically significant higher than corresponding control values (p ≤0.05)


 

Mutant frequency and viability – 2nd Experiment without S9 mix; 4-hour exposure period

 

 

Test groups [µg/mL]

 

Cloning efficiency 2 (viability)

 

Mutant frequency

Number of colonies

CE2

Number of coloniesa

MF (per 106cells)

Dish 1

Dish 2

Abs. [%]

Rel. [%]

Flask 1

Flask 2

Uncorrected

Correctedb

Negative control

151

141

73.0

100.0

4

3

1.75

2.40

375.0

n.c.

750.0

150

152

75.5

103.4

6

8

3.50

4.64

1500.0

127

129

64.0

87.7

5

5

2.50

3.91

3000.0

142

150

73.0

100.0

7

12

4.75

6.51

3300.0

114

110

56.0

76.7

4

4

2.00

3.57

EMS 400.0

104

101

51.3

70.2

210

205

103.75

202.44S

S       Mutant frequency statistically significant higher than corresponding control values (p ≤0.05)


 

Mutant frequency and viability – 2nd Experiment with S9 mix; 4-hour exposure period

 

 

Test groups [µg/mL]

 

Cloning efficiency 2 (viability)

 

Mutant frequency

Number of colonies

CE2

Number of coloniesa

MF (per 106cells)

Dish 1

Dish 2

Abs. [%]

Rel. [%]

Flask 1

Flask 2

Uncorrected

Correctedb

Negative control

149

134

70.8

100.0

7

7

3.50

4.95

375.0

n.c.

750.0

117

116

58.3

82.3

5

4

2.25

3.86

1500.0

119

120

59.8

84.5

9

11

5.00

8.37

3000.0

118

127

61.3

86.6

2

7

2.25

3.67

3300.0

116

107

55.8

78.8

2

2

1.00

1.79

DMBA 1.25

96

99

48.8

68.9

161

150

77.75

159.49S

S       Mutant frequency statistically significant higher than corresponding control values (p ≤0.05)

Conclusions:
Thus, in the absence and the presence of metabolic activation, Disodium (sulphonatothio)acetate; dried is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.
Executive summary:

According to the results of the present in vitro study, in two experiments performed independently of each other the test substance Disodium (sulphonatothio)acetate; dried did not lead to a biologically relevant or dose-dependent increase the number of mutant colonies, either without S9 mix or after the addition of a metabolizing system. The mutant frequencies at any concentration were close to or within the range of the concurrent negative control values and close to or within the 95% control limit of our historical negative control data. Additionally, no statistically significant dose-dependent increase in mutant colonies was observed in any experimental part of this study after 4 hours treatment either in the absence or presence of metabolic activation. The mutation frequencies of the vehicle control groups were within our historical negative control data range (95% control limit) and, thus, fulfilled the acceptance criteria of this study. The proficiency of the laboratory to perform the HPRT assay in CHO cells was demonstrated by the laboratory’s historical control database on vehicle and positive controls and by X-bar chart to identify the variability of the vehicle control data. The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method and/or of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

Genetic toxicity in vitro

Bacterial Reverse Mutation Assay (Ames Test)

A GLP compliant bacterial reverse mutation test was performed according to OECD 471, OECD 472 and EU method B.13/14 (BASF, 1995). The test substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9 mix (induced by 1 volume S-9 rat liver fraction and 9 volumes S-9 supplement). Parallel with each experiment with and without S-9 mix, a negative control (vehicle control, sterility control) was carried out for each tester strain in order to determine the spontaneous mutation rate. In the Ames test (standard plate test) and in preincubation test, the test substance was tested at the concentrations 40, 200, 10000, 5000 and 10000 µg/plate (i.e. 16, 79, 395, 1975, 3950 µg/plate of active compound) in the absence and presence of 10% (v/v) S9 mix in tester strains TA1535, TA1537, TA 100, TA98 and Escherichia coli WP2 uvrA (4 experiments). Regarding mutagenicity no increase the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA was observed both in the absence and presence of S9 metabolic activation. However a weakly bacteriotoxic effect (slight decrease in the number of his revertants) was occasionally observed in the Ames test depending in the strain and test conditions at doses >= 5,000 µg/plate.

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

Chromosome Aberration Assay

A GLP compliant chromosome aberration assay was performed according to OECD 473 and EU method B.10 and EPA OTS 798.5375 (BASF, 1995). The test substance was assessed for its potential to induce structural chromosomal aberrations in V79 cells in vitro both in the presence and in the absence of a metabolizing system (S-9 mix). In a pretest the doses for the cytotoxicity tests were determined (with and without S-9 mix). On the basis of these findings without S-9 mix, 2,000 µg/mL (i.e. 790 µg/mL of active compound) aqueous test substance were selected as the top dose. For safety reasons, a dose of 5,000 µg/mL (i.e. 1,975 µg/mL of active compound)

was also included, which should only be evaluated if the obtained results regarding cytotoxicity deviated from those in the pretest. With metabolic activation 6,500 µg/mL (i.e. 2,568 µg/mL of active compound) was chosen as top dose. This selection was based on respective  recommendations (Higher doses were not tested to avoid concentrations > 10 mM which may lead to artefactual chromosome breakage.)

Chromosomes were prepared 18 hours and 28 hours after test substance treatment, which lasted for about 4 hours in the experiments with S-9 mix and for about 18 hours without metabolic activation. Duplicate cultures were used for all experimental groups. About 2 - 3 hours prior to harvesting the cells, colcemid was added to arrest cells in a metaphase like stage of mitosis (c-metaphases). After preparation of the chromosomes and staining with Giemsa, 100 metaphases of each culture in the case of the test substance, negative controls and vehicle controls, or 50 cells of each culture in the case of the concurrent positive controls, were analyzed for chromosomal aberrations.

The negative controls (untreated and vehicle controls) gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive control chemicals, i.e. EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations.

According to the results of the present study, the test substance caused a slight but statistically significant and dose-dependent increase in the number of structurally aberrant metaphases incl. and excl. gaps after a sampling time of 28 hours without S-9 mix in two experiments independent of each other.

Thus, under the experimental conditions of this assay, the test substance is considered to have a weakly chromosome-damaging (clastogenic) effect under in vitro conditions in V79 cells.

HPRT gene mutation assay

According to the results of the present in vitro study, in two experiments performed independently of each other the test substance Disodium (sulphonatothio)acetate; dried did not lead to a biologically relevant or dose-dependent increase the number of mutant colonies, either without S9 mix or after the addition of a metabolizing system. The mutant frequencies at any concentration were close to or within the range of the concurrent negative control values and close to or within the 95% control limit of our historical negative control data. Additionally, no statistically significant dose-dependent increase in mutant colonies was observed in any experimental part of this study after 4 hours treatment either in the absence or presence of metabolic activation. The mutation frequencies of the vehicle control groups were within our historical negative control data range (95% control limit) and, thus, fulfilled the acceptance criteria of this study. The proficiency of the laboratory to perform the HPRT assay in CHO cells was demonstrated by the laboratory’s historical control database on vehicle and positive controls and by X-bar chart to identify the variability of the vehicle control data. The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method and/or of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

Thus, in the absence and the presence of metabolic activation, Disodium (sulphonatothio)acetate;dried is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

Justification for classification or non-classification

The available experimental test data are reliable but not suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776 based on the currently available data.