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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Produkt SPS
- Test-substance No.: 11/0629-1
- Lot/batch No.: 78522224U0
- Purity: Trisodium 4-sulphonophthalate: 25.5 g/100 g; Trisodium 3-sulphonatophthalate: 7.6 g/100 g; Disodium phthalate: 1 g/100 g; Sulphate: 60.6 g/100 g; Water: 4.8 g/100 g; Sum: 99.5 g/100 g; Determined by 1H-NMR-analysis
- Homogeneity: The test substance was homogeneous by visual inspection
- Storage stability: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility
- Storage conditions: Room temperature
- Physical state/ colour: Solid/ white
- Expiry date: December 10, 2012

Method

Target gene:
N/A
Species / strain
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.
Controls
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.

Results and discussion

Test results
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'.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.