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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are no in vitro genotoxicity studies on the reaction mass of 4-sulphophthalic acid and 3-sulphophthalic acid (read across target substance). However, genotoxicity studies are available for the reaction mass of trisodium 4-sulphonophthalate, trisodium 3-sulphonatophthalate, disodium phthalate, and sulphate (read across source substance) and it was used for read-across purposes. Below the results of these studies are briefly described.

Bacterial mutagenicity: In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535 and TA 1537 of S. typhimurium were exposed to the reaction mass of sulphophthalic acid sodium salt (read across source substance), at concentrations of 1000, 2000, 3000, 4000 and 5000 µg/plate in the presence and absence of mammalian metabolic activation. According to the results, the read across source substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay).

Mammalian Mutagenicity: The sulphophthalic acid sodium salt reaction mass (read across source substance) was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). Following attachment of the cells for 20-24 hours, cells were treated with the test substance for 4 and 24 hours in the absence of metabolic activation and for 4 hours in the presence of metabolic activation. Subsequently, cells were cultured for 6-8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in three independent experiments. Thus, under the experimental conditions of this study, the read across source substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Chromosomal Aberration: The read across source substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). Based on the results of the present study, the read across source substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other. Thus, under the experimental conditions described, the read across source substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read across follows Scenario 5 - Qualitatively and quantitatively similar effects are caused by a common compound, which is formed from all category members (as described in the 2017 Read-Across Assessment Framework document).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
TARGET: Reaction Mass of 4-sulphophthalic acid and 3-sulphophthalic acid (ratio 3:1)
SOURCE: Sulfophthalic acid sodium salts

3. CATEGORY APPROACH JUSTIFICATION
Sulphophthalic acid sodium and ammonium salts are different organic salts of a common acid, sulphophthalic acid. The salts dissociate rapidly in the medium (water) to the common sulphophthalic acid anion and to their different counter ions. The counter ions ammonium and sodium do not influence the solubility and the toxicity of the category members.

4. DATA MATRIX
See Read Across document attached to CSR

Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
N/A
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source
supplemented with
− 10% (v/v) fetal calf serum (FCS)
− 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL)
− 1% (v/v) amphotericine B (250 μg/mL)
During exposure to the test substance for 4 hours only, MEM medium was used without FCS supplementation.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
1st Experiment
4 hours exposure; 24 hours harvest time; without S9 mix:
0; 165.6; 331.3; 662.5; 1325.0; 2650.0; 5300.0 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix:
0; 165.6; 331.3; 662.5; 1325.0; 2650.0; 5300.0 μg/mL
2nd Experiment
24 hours exposure, 24 hours harvest time, without S9 mix
0; 165.6; 331.3; 662.5; 1325.0; 2650.0; 5300.0 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix
0; 1325.0; 2650.0; 3975.0; 5300.0 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [Minimal Essential Medium: MEM]
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, culture medium (Minimal Essential Medium: MEM) was selected as vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 6 hours
- Exposure duration: 4 or 24 hours
- Recovery time: 0 or 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours after addition of the test substance (4 h exposure + 20 h recovery or 24 h of exposure and no recovery)

SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): After drying, the slides were stained in Wrights solution (modified May-Grünwald solution) for about 3 minutes. After being rinsed once in Titrisol solution pH 7.2, the slides were counterstained with 2.6% (v/v) Giemsa/Titrisol solution pH 7.2 for about 20 minutes. After being rinsed twice in Titrisol solution pH 7.2 and clarified in xylene, the slides were mounted using Corbit-Balsam.

NUMBER OF REPLICATIONS:
The cell cycle of the untreated V79 cells lasts for about 12 - 14 hours under the selected culture conditions. Thus, the selected harvest time of 24 hours is about 2 times the normal cell cycle length.

NUMBER OF CELLS EVALUATED: A sample of at least 1 000 cells for each culture were analyzed for micronuclei, i.e. at least 2 000 cells for each test group. Due to inhomogeneous data the sample of evaluation was increased up to 4 000 cells per test group in both experimental parts of the 1st Experiment.

DETERMINATION OF CYTOTOXICITY
For additional information about the cytotoxic potential of the test substance cells were seeded in flasks (2.5x10^5 cells per 25 cm^2 flask) about 24 – 30 hours prior to exposure. The cells were treated similar to the slides using the same media and test substance concentrations. At the end of recovery period single cell suspensions were prepared from each test group (all test groups, except the positive controls) and the cells were counted
using a cell counter. Based on these data the relative increase in cell count (RICC) was calculated.
Evaluation criteria:
Acceptance criteria
The in vitro micronucleus assay is considered valid if the following criteria are met:
• The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells.
• The number of cells containing micronuclei in the negative control was within the range of the historical negative control data.
• The positive control substances both with and without S9 mix induced a significant increase in the number of micronucleated cells.

Assessment criteria
A test substance is considered "positive" if the following criteria are met:
• A significant, dose-related and reproducible increase in the number of cells containing micronuclei.
• The number of micronucleated cells exceeds both the value of the concurrent negative control and the range of the historical negative control data.
A test substance generally is considered "negative" if the following criteria are met:
• The number of micronucleated cells in the dose groups is not significant increased above the concurrent negative control value and is within the range of the historical negative control data.
Statistics:
The statistical evaluation of the data was carried out using the MUVIKE program system (BASF SE). The proportion of cells containing micronuclei was calculated for each group. A comparison of each dose group with the concurrent negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.
If the results of this test were statistically significant compared with the respective negative control, labels (* p ≤ 0.05, ** p ≤ 0.01) have been be printed in the tables.
Key result
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

MICRONUCLEUS ANALYSIS

In this study, no biologically relevant increase in the number of micronucleated cells was observed either without S9 mix or after the addition of a metabolizing system.

In both experiments in the absence and presence of metabolic activation after 4 and 24 hours treatment with the test substance the values (0.5 – 1.7% micronucleated cells) were close to the concurrent negative control values (0.5 – 1.5% micronucleated cells) and clearly within our historical negative control data range (0.1 - 1.8% micronucleated cells). In the 1st Experiment in the absence and presence of metabolic activation due to inhomogeneous data the sample of evaluation of all test groups was 4 000 cells.

The positive control substances EMS (without S9 mix; 500 μg/mL) and CPP (with S9 mix; 2.5 μg/mL) induced statistically significant increased micronucleus frequencies in both independently performed experiments. In this study, in the absence and presence of metabolic activation the frequency of micronucleated cells (3.7 – 15.2% micronucleated cells) was clearly above the range of our historical negative control data range (0.1 - 1.8% micronucleated cells) and within our historical positive control data range (2.3 – 26.6% micronucleated cells).

PROLIFERATION INDEX

The proliferation index (PI) is based on the scoring of at least 1 000 cells per culture (at least 2 000 cells per test group) for the different test groups without and with metabolic activation and includes the measurement of colony size.

In this study, in the absence and the presence of S9 mix no cytotoxicity indicated by reduced PI values was observed.

RELATIVE INCREASE IN CELL COUNT

In addition, in both main experiments in the absence and the presence of S9 mix no growth inhibition indicated by clearly reduced cell counts was observed.

CELL MORPHOLOGY

In this study cell attachment was not relevant influenced at any dose evaluated for micronuclei.

TREATMENT CONDITIONS

A slight increase of the osmolarity values was measured at all experimental conditions of the study. At the highest applied concentration of 5 300 μg/mL an increase of 37 to 87 mOsm compared to the respective vehicle control value was obtained. The pH values were not influenced by test substance treatment. No precipitation of the test substance in culture medium was observed.

DISCUSSION

According to the results of the present in vitro micronucleus assay, the test substance Produkt SPS did not lead to a biologically relevant increase in the number of micronucleated cells either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. The frequencies of micronuclei after test substance treatment were close to the range of the concurrent negative control values at both exposure times and clearly within the range of our historical negative control data.

The number of micronucleated cells in the vehicle control groups were within our historical negative control data range and, thus, fulfilled the acceptance criteria of this study.

The increase in the frequencies of micronuclei induced by the positive control substances EMS and CPP clearly demonstrated the sensitivity of the test system and 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.

CONCLUSION

Thus, under the experimental conditions chosen here, the conclusion is drawn that Produkt SPS has not the potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic

activation.

Conclusions:
Under the experimental conditions described, the read across source substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.
Executive summary:

There are no in vitro cytogenicity studies on the reaction mass of 4-sulphophthalic acid and 3-sulphophthalic acid (read target substance). However, data is available for the reaction mass of trisodium 4-sulphonophthalate, trisodium 3-sulphonatophthalate, disodium phthalate, and sulphate (read across source substance) and it was used for read-across purposes

The read across source substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). A sample of at least 1 000 cells for each culture were analyzed for micronuclei, i.e. at least 2000 cells for each test group. Due to inhomogeneous data, the sample of evaluation was increased up to 4000 cells per test group in both experimental parts of the 1st Experiment. Based on the results of the present study, the read across source substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other. Thus, under the experimental conditions described, the read across source substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read across follows Scenario 5 - Qualitatively and quantitatively similar effects are caused by a common compound, which is formed from all category members (as described in the 2017 Read-Across Assessment Framework document).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
TARGET: Reaction Mass of 4-sulphophthalic acid and 3-sulphophthalic acid (ratio 3:1)
SOURCE: Sulfophthalic acid sodium salts
3. CATEGORY APPROACH JUSTIFICATION
Sulphophthalic acid sodium and ammonium salts are different organic salts of a common acid, sulphophthalic acid. The salts dissociate rapidly in the medium (water) to the common sulphophthalic acid anion and to their different counter ions. The counter ions ammonium and sodium do not influence the solubility and the toxicity of the category members.
4. DATA MATRIX
See Read Across document attached to CSR
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
HIS/TRP
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
N/A
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
N/A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
33 μg - 5 300 μg/plate (SPT and PIT)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [water]
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in ultrapure water, ultrapure water was used as vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine
Remarks:
+ S9: 2-aminoanthracene; - S9: N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine, 9-aminoacridine, 4-nitroquinoline-N-oxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Incubation period: 20 min in medium (PIT) and incubation at 37°C for 48 – 72 hours (PIT and SPT)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.
Statistics:
not applicable
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A weak bacteriotoxic effect was occasionally observed depending on the strain only in the Standard plate test from about 2 650 μg/plate onward
Vehicle controls validity:
valid
Untreated negative 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:
cytotoxicity
Remarks:
A weak bacteriotoxic effect was occasionally observed depending on the strain only in the Standard plate test from about 2 650 μg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the standard plate test with the strain TA 98 from about 2 650 μg/plate onward.
In the preincubation assay no bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed up to the highest required concentration.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay).

Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.

In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

Conclusions:
In the absence and the presence of metabolic activation, the sulphophthalic acid sodium salt reaction mass (read across source substance) is not a mutagenic substance in the Salmonella assay (Ames test) under the experimental conditions chosen.
Executive summary:

There are no in vitro gene mutation studies in bacteria on the reaction mass of 4-sulphophthalic acid and 3-sulphophthalic acid (read across target substance). However, a bacterial mutagenicity study is available for the reaction mass of trisodium 4-sulphonophthalate, trisodium 3-sulphonatophthalate, disodium phthalate, and sulphate (read across source substance) and it was used for read-across purposes.

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535 and TA 1537 of S. typhimurium were exposed to the reaction mass of sulphophthalic acid sodium salt (read across source substance), at concentrations of 1000, 2000, 3000, 4000 and 5000 µg/plate in the presence and absence of mammalian metabolic activation.  The positive controls induced the appropriate responses in the corresponding strains.   There was no evidence of induced mutant colonies over background with the test substance. This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data. According to the results of the present study, the read across source substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay).

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read across follows Scenario 5 - Qualitatively and quantitatively similar effects are caused by a common compound, which is formed from all category members (as described in the 2017 Read-Across Assessment Framework document).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
TARGET: Reaction Mass of 4-sulphophthalic acid and 3-sulphophthalic acid (ratio 3:1)
SOURCE: Sulfophthalic acid sodium salts
3. CATEGORY APPROACH JUSTIFICATION
Sulphophthalic acid sodium and ammonium salts are different organic salts of a common acid, sulphophthalic acid. The salts dissociate rapidly in the medium (water) to the common sulphophthalic acid anion and to their different counter ions. The counter ions ammonium and sodium do not influence the solubility and the toxicity of the category members.
4. DATA MATRIX
See Read Across document attached to CSR
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media:
Culture media
All media were supplemented with:
- 1% (v/v) penicillin/streptomycin (stock solution: 10 000 IU / 10 000 μg/mL)
- 1% (v/v) amphotericine B (stock solution: 250 μg/mL)

Treatment medium (4-hour exposure period)
Ham's F12 medium containing stable glutamine and hypoxanthine (Biochrom; Cat. No. FG 0815).

Culture medium and Treatment medium (24-hour exposure)
Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) fetal calf serum (FCS).

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) fetal calf serum (FCS)

Selection medium ("TG" medium)
Hypoxanthine-free Ham's F12 medium supplemented with:
- 6-thioguanine (10 μg/mL)
- 1% (v/v) stable glutamine (200 mM)
- 10% (v/v) fetal calf serum (FCS)

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: N/A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9 Mix
Test concentrations with justification for top dose:
1st Experiment
without S9 mix (4-hour exposure period) (top dose too low)
0; 362.5; 725.0; 1450.0; 2900.0 μg/mL
with S9 mix (4-hour exposure period) (top dose too low)
0; 362.5; 725.0; 1450.0; 2900.0 μg/mL
2nd Experiment
without S9 mix (4-hour exposure period)
0; 331.3; 662.5; 1325.0; 2650.0; 5300.0 μg/mL
with S9 mix (4-hour exposure period)
0; 331.3; 662.5; 1325.0; 2650.0; 5300.0 μg/mL
3rd Experiment
without S9 mix (24-hour exposure period)
0; 331.3; 662.5; 1325.0; 2650.0; 5300.0 μg/mL
with S9 mix (4-hour exposure period)
0; 750.0; 1500.0; 3000.0; 5300.0 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [aqueous culture medium]
- Justification for choice of solvent/vehicle: 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
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20 – 24 hours
- Exposure duration: 4-hour and 24-hour
- Expression time (cells in growth medium): 3 days (4-hour treatment) or 2 days (24-hour treatment). This was followed by the 1st passage. After an entire expression period of 7 – 9 days the cells were transferred into selection medium (2nd passage).
- Selection time (if incubation with a selection agent): about 6 - 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): from day 16

SELECTION AGENT (mutation assays): "TG" medium (6-thioguanine)

NUMBER OF CELLS EVALUATED: all cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth
Evaluation criteria:
Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
- The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
- The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range of 0.00 – 16.43 mutants per 10^6 clonable cells.
- The positive controls both with and without S9 mix have to induce distinctly increased mutant frequencies.
- At least 4 dose levels should be tested ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

Assessment criteria
A finding is assessed as positive if the following criteria are met:
- Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
- Evidence of the reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10^6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
- The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.
Statistics:
An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative control groups. A trend is judged
as statistically significant whenever the p-value (probability value) is below 0.10 and the slope is greater than 0. However, both, biological and statistical significance will be 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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

MUTANT FREQUENCY

In this study, no increase in the number of mutant colonies was observed with or without S9 mix. In all experiments after 4 and 24 hours treatment with the test substance the values for the corrected mutation frequencies (MFcorr.: 0.00 – 5.00 per 10^6 cells) were close to the respective vehicle control values (MFcorr.: 0.37 – 4.66 per 10^6 cells) and clearly within the range of our historical negative control data (without S9 mix: MFcorr.: 0.00 – 16.43 per 10^6 cells; with S9 mix: MFcorr.: 0.00 – 15.83 per 10^6 cells)

The positive control substances EMS (without S9 mix; 300 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.: 95.80 – 506.18 per 10^6 cells; with S9 mix: MFcorr.: 255.88 – 320.75 per 10^6 cells) were clearly within our historical positive control data range (without S9 mix: MFcorr.: 47.35 – 1338.10 per 10^6 cells; with S9 mix: MFcorr.: 131.35 – 1250.00 per 10^6 cells).

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 all experiments under all test conditions up to the highest applied concentrations.

The cell densities were not distinctly reduced.

CELL MORPHOLOGY

There were no adverse observations on cell morphology (cell attachment).

TREATMENT CONDITIONS

Osmolarity and pH values were not relevantly influenced by test substance treatment.

In this study, in the absence and the presence of S9 mix, no precipitation in culture medium was observed up to the highest applied test substance concentration.

DISCUSSION

According to the results of the present in vitro study, in two experiments performed independently of each other the test substance Produkt SPS did not 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 the range of the concurrent negative control values and within the range of our historical negative control data.

The mutation frequencies of the vehicle control groups were within our historical negative control data range including all vehicles used in our laboratory and, thus, fulfilled the acceptance criteria of this study.

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 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.

CONCLUSION

Thus, in the absence and the presence of metabolic activation, Produkt SPS is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

Conclusions:
In the absence and the presence of metabolic activation, the sulphophthalic acid sodium salt reaction mass (read across source substance) is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.
Executive summary:

There are no in vitro gene mutation studies in mammalian cells on the reaction mass of 4-sulphophthalic acid and 3-sulphophthalic acid (read across target substance). However, a mammalian mutagenicity study is available for the reaction mass of trisodium 4-sulphonophthalate, trisodium 3-sulphonatophthalate, disodium phthalate, and sulphate (read across source substance) and it was used for read-across purposes.

The sulphophthalic acid sodium salt reaction mass (read across source substance) was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). Following attachment of the cells for 20-24 hours, cells were treated with the test substance for 4 and 24 hours in the absence of metabolic activation and for 4 hours in the presence of metabolic activation. Subsequently, cells were cultured for 6-8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in three independent experiments. Thus, under the experimental conditions of this study, the read across source substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification