Asphalt, sulfonated, sodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation assay (OECD 471), SAS was considered to be non-mutagenic under the conditions of this test. In a chromosomal aberration test (OECD 473) the test substance was considered to be non-clastogenic to human lymphocytes in vitro. Finally, in an in vitro mammalian cell gene mutation test (OECD 476), the test substance was non-mutagenic in the L5178Y mouse lymphoma cell line. Therefore, we consider that this endpoint has been adequately tested and no additional studies are required.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

N/A

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Materials and methods were limited.

Principles of method if other than guideline:

N/A

GLP compliance:

not specified

Remarks:

HPV document indicated that the test was GLP. However, information was limited on the testing laboratory.

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine (S. typhimurium strains) and tryptophan locus (E. coli strain)

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

N/A

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

N/A

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

N/A

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

N/A

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

N/A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and B-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: N/A

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (test substance vehicle)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracine (2AA): 1 ug/plate for TA100; 2 ug/plate for TA1535 and TA1537; 10 ug/plate for WP2uvrA-

Remarks:

With metabolic activation

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (test substance vehicle)

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With metabolic activation 5 ug/plate for TA98

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (test substance vehicle)

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without metabolic activation 2 ug/plate for WP2uvrA-; 3 ug/plate for TA100; 5 ug/plate for TA1535

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (test substance vehicle)

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation 80 ug/plate for TA1537

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (test substance vehicle)

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation 0.2 ug/plate for TA98

Details on test system and experimental conditions:

METHOD OF APPLICATION: N/A


DURATION
- Preincubation period: N/A
- Exposure duration: N/A
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A


SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A


NUMBER OF REPLICATIONS: 3


NUMBER OF CELLS EVALUATED: N/A


DETERMINATION OF CYTOTOXICITY
- Method: reduction in growth of bacterial background lawn


OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Other: N/A


OTHER: N/A

Evaluation criteria:

N/A

Statistics:

N/A

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: N/A
- Effects of osmolality: N/A
- Evaporation from medium: N/A
- Water solubility: N/A
- Precipitation: No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. A brown colour was observed from 1500 ug/plate, but this did not prevent the scoring of revertant colonies.
- Other confounding effects: N/A


RANGE-FINDING/SCREENING STUDIES: N/A


COMPARISON WITH HISTORICAL CONTROL DATA: N/A


ADDITIONAL INFORMATION ON CYTOTOXICITY: The test substance caused no visible reduction in the growth of the bacterial background lawn at any dose level, although small decreases in revertant colony frequency were noted to several of the tester strains at 5000 ug/plate, predominantly in the presence of S9. The test substance was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 -mix were validated. No significant increases in the frequency of revertant colonies were recorded for any of the strains with any dose of the test substance, either with or without metabolic activation.

Conclusions:

Interpretation of results:
negative No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

The test substance was evaluated for mutagenic potential using the Ames test. Under the conditions of the test, the test substance was determined to be non-mutagenic with or without metabolic activation.

Executive summary:

N/A

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

N/A

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

Information on materials and methods was lacking.

Principles of method if other than guideline:

N/A

GLP compliance:

not specified

Remarks:

HPV document indicated that the test was GLP. However information was limited on the testing laboratory.

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

N/A

Species / strain / cell type:

other: Human lymphocytes

Details on mammalian cell type (if applicable):

N/A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and B-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1 with and without S9: 0, 39.06, 78.13, 156.25, 312.5, 625, 1250 ug/mL
Experiment 2 with S9: 0, 39.06, 78.13, 156.25, 312.5, 625, 1250 ug/mL
Experiment 2 without S9: 0, 312.5, 625, 1250, 2500, 3750, 5000 ug/mL
4(20-h) (+S9): 0, 39.06, 78.13, 156.25, 312.5, 625, 1250 ug/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: No data was provided on the identity of the vehicle used.
- Justification for choice of solvent/vehicle: N/A

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

no data on the identity

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 mix 0.4 (Exp. 1) and 0.2 (Exp. 2) ug/mL

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

no data on the identity

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix 5.0 (Exp. 1) and 7.5 (Exp. 2) ug/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: N/A
- Exposure duration: 4 hours (Exp. 1 with and without S9; Exp. 2 with S9); 24 hours (Exp. 2 without S9)
- Expression time (cells in growth medium): An expression time could not be determined from the available data, based on addition time of a spindle inhibitor was not provided.
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours


SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A


NUMBER OF REPLICATIONS: N/A


NUMBER OF CELLS EVALUATED: N/A


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: N/A
- Other: N/A


OTHER: N/A

Evaluation criteria:

N/A

Statistics:

N/A

Key result

Species / strain:

other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9 (see below for details)

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: N/A
- Effects of osmolality: N/A
- Evaporation from medium: N/A
- Water solubility: N/A
- Precipitation: Yes. See range-finding study below. Precipitate observations in the Preliminary Toxicity Test were considered to be representative for the study.
- Other confounding effects: N/A


RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 19.53 to 5000 ug/mL. The maximum dose was based on the maximum recommended dose level. A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 19.53 ug/mL, in all exposure groups. The precipitate was considered to be too fine to be observed by the naked eye at dose levels below 1250 ug/mL, although the precipitate became obvious after centrifugation. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 5000 ug/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation. The maximum dose with metaphases present in the 24-hour continuous exposure was 2500 ug/mL. There was a steep toxicity curve in the 24-hour exposure group as there were no metaphases observed at 5000 ug/mL. The selection of the maximum dose level was based on the lowest precipitating dose level for the short-term exposure groups and on toxicity for the continuous exposure group used in Experiment 2.

COMPARISON WITH HISTORICAL CONTROL DATA: N/A


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1: The qualitative assessment of the slides determined that there was no toxicity, as had been observed in the Preliminary Toxicity Test, and that there were scorable metaphases present at all dose levels in both exposure groups. The mitotic index data confirm the qualitative observations in that no inhibition of mitotic index was observed in either exposure group. The maximum dose level selected for metaphase analysis was the maximum dose level used in the experiment and was based on the lowest observable precipitating dose level.
Experiment 2: The qualitative assessment of the slides determined that there were scorable metaphases present at the maximum test substance dose level of 1250 ug/mL in the presence of S9. In the continuous exposure in the absence of S9, the maximum test substance dose level with scorable metaphases was 2500 ug/mL and was expected from the toxicity seen in the Preliminary Toxicity Test. The mitotic index data confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed in the absence of S9 and that 56% mitotic inhibition was achieved at 2500 ug/mL. In the presence of S9, there was no evidence of toxicity. The maximum dose level selected for metaphase analysis was based on toxicity for the 24-hour exposure group in the absence of S9. In the presence of S9, the maximum dose level tested was selected for analysis and was based on the lowest observable precipitating dose level.

Experiment 1: All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control substances induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test substance did not induce any statistically significant increases in the frequency of cells with aberrations, either in the absence or presence of metabolic activation. The test substance did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Experiment 2: All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control substances induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test substance did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, either in the absence or presence of metabolic activation. The test substance did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Conclusions:

Interpretation of results: negative with and without S9

The test substance was evaluated for the induction of chromosome aberrations in human lymphocytes. The test substance did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of metabolic activation, and was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

N/A

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-01-14 to 2010-06-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate from The Department of Health of the Government of the United Kingdom

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase locues

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: The cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 ug/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 ug/ml) and 10 % donor horse serum (giving R10 media) at 37 degrees C with 5 % CO2 in air. RPMI 1640 with 20 % donor horse serum (R20) and without serum (R0) were used during the course of the study.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: N/A
- Periodically checked for karyotype stability: N/A
- Periodically "cleansed" against high spontaneous background: yes; Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 ug/ml), Hypoxanthine (15 ug/ml), Methotrexate (0.3 ug/ml) and Glycine (22.5 ug/ml). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Additional strain / cell type characteristics:

other: The cell line used was heterozygous at the thymidine kinase locus (TK +/-) with a generation time of approximately 12 hours.

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/B-naphthoflavone induced rat liver S9 (final concentration of 2% and 1 % for Experiment 1 and 2, respectively)

Test concentrations with justification for top dose:

Experiments 1 and 2: 156.25, 312.5, 625, 1250, 1875 and 2500 ug/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 medium without serum (R0)
- Justification for choice of solvent/vehicle: N/A

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

R0 medium (test substance vehicle)

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation 400 ug/ml and 150 ug/ml for Experiment 1 and Experiment 2, respectively

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

R0 medium (test substance vehicle)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation 2 ug/ml for Experiments 1 and 2

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: N/A
- Exposure duration: Experiment 1: 4 hours in the presence and absence of metabolic activation; Experiment 2: 4 hours in the presence of metabolic activation and 24 hours in the absence of metabolic activation
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-14 days
- Fixation time (start of exposure up to fixation or harvest of cells):Cell cultures were scored for mutation frequency at the end of the selection time. To assist in the scoring of mutant colonies, MTT solution stain was added for two hours.

SELECTION AGENT (mutation assays): 5-trifluorothymidine
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: N/A

DETERMINATION OF CYTOTOXICITY
- Method: Relative Suspension Growth and Relative Total Growth; See "any other information on materials and method" section for calculations

OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Other: Large and small mutant colony determinations were made. Large colonies grow at a normal rate and represent events within the gene (base-pair substitutions or deletions) while small colonies represent large genetic changes involving chromosome 11b (indicative of clastogenic activity). Large colonies are defined as those that covered approximately 1/4 to 3/4 of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25 % of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick.

OTHER:
N/A

Evaluation criteria:

For the test substance to have demonstrated a mutagenic response it had to produce a statistically significant increase in the induced mutation frequency over the concurrent vehicle mutant frequency value. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the induced mutation frequency must exceed some value based on the global background mutant frequency for each method (agar or microwell). This Global Evaluation factor value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 E-6 for the microwell method. Therefore, any test substance dose level that had a mutation frequency value that was greater than the corresponding vehicle control by the Global Evaluation Factor of 126 E-6 was considered positive. However, if the test substance produced a modest increase in mutation frequency, which only marginally exceeded the Global Evaluation Factor value and was not reproducible or part of a dose-related response, it was considered to have no toxicological significance. Conversely, when the test substance induced modest reproducible increases in the mutation frequencies that did not exceed the Global Evaluation factor value then scientific judgment was applied. If the reproducible responses were significantly dose-related and included increases in the absolute numbers of mutant colonies then they may have been considered to be toxicologically significant.

Statistics:

The experimental data was analyzed using a dedicated computer program which followed the statistical guidelines recommended by the UKEMS. Significant results were reviewed by the above evaluation criteria.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no marked change in pH when the test substance was dosed into media.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm when the test substance was dosed into media.
- Evaporation from medium: N/A
- Water solubility:N/A
- Precipitation: In the preliminary toxicity test, a precipitate of the test substance was observed at and above 19.53 ug/ml and increased with increase in dose concentration in all three of the exposure groups. In Experiment 1 and 2, a precipitate of test substance was observed at and above 156.25 ug/ml in both the absence and presence of metabolic activation.
- Other confounding effects: N/A

RANGE-FINDING/SCREENING STUDIES: A range-finding study was performed to determine the correct dose levels to be used in the main gene mutation assay. The test was performed on cell cultures at 5 E+5 cells/ml, using a 4-hour exposure time both with and without metabolic activation (S9), and at 1.5 E+5 cells/ml using a 24-hour exposure without S9. The dose range used was 19.53-5000 ug/ml for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 E5 cells/ml. The cultures were incubated and sub-cultured after 24 hours by counting and dilution to 2 E+5 cells/ml. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth values. The Suspension Growth values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment Suspension Growth value to the concurrent vehicle control performed to give a % Relative Suspension Growth Value. Maximum dose levels for the main gene mutation assay were selected using the following criteria:
1. Maximum recommended dose level, 5000 ug/ml or 10 mM.
2. The presence of excessive precipitate where no test substance-induced toxicity was observed.
3. Test substance-induced toxicity, where the maximum dose level used should produce 10-20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002).
In all three of the exposure groups there were marked reduction in the relative suspension growth of cells treated with the test substance when compared to the concurrent vehicle controls.
In the main gene mutation experiment the maximum dose was limited to 2500 ug/ml primarily by toxicity, and due to the nature of the precipitate observed at 5000 ug/ml that resulted in handling problems during the washing stage of the test.

COMPARISON WITH HISTORICAL CONTROL DATA: All of the vehicle and positive control mutation frequency values were within historical data control range, with the exception of the vehicle control of both experiments in the presence of S9 which were slightly below.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
1. Experiment 1: There was evidence of toxicity following exposure to the test substance in both the absence and presence of metabolic activation, as indication by the % Relative Suspension Growth (27-120 % and 48-112 % in the absence and presence of metabolic activation, respectively) and Relative Total Growth (0.36-1.44 and 0.65-1.20 in the absence and presence of metabolic activation, respectively) values. The levels of toxicity were very similar to those observed in the preliminary toxicity test with near optimum levels of toxicity achieved in the absence of metabolic activation. While optimum levels of toxicity were not achieved in the presence of metabolic activation, the test substance was considered to have been adequately tested as dose levels above 2500 ug/ml were impractical due to the nature of the test substance precipitate observed. There was no evidence of any reduction in viabilities, therefore indicating the residual toxicity had not occurred in either the absence or presence of metabolic activation. Acceptable levels of toxicity were seen with both positive control substances.
2. As was seen previously there was evidence of toxicity following exposure to the test substance in both the absence and presence of metabolic activation, as indicated by the % Relative Suspension Growth (43-136 % and 27-113 % in the absence and presence of metabolic activation, respectively) and Relative Total Growth (0.90-1.76 and 0.37-1.25 in the absence and presence of metabolic activation, respectively) values. Once again the levels of toxicity observed in the 24-h exposure group in the absence of metabolic activation were very similar to that of the preliminary toxicity test. However, the lowering of the S9 concentration to 1% in this second experiment resulted in greater levels of toxicity than those observed in the presence of 2% S9 in the first experiment and near optimum levels of toxicity were achieved on this occasion. While optimum levels of toxicity were not achieved in the absence of metabolic activation, the test substance was considered to have been adequately tested as dose levels above 2500 ug/ml were impractical due to the nature of the test substance precipitate observed. There was no evidence of any reductions in viability, indicating residual toxicity had not occurred in either the absence or presence of metabolic activation. Both positive controls induced acceptable levels of toxicity. Based on these observations and those from Experiment 1 it was considered that the test substance had been adequately tested during the performance of this study. The 24-h exposure without metabolic activation demonstrated that the extended time point had no effect on the toxicity of the test substance.

In both experiments, none of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 E-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional. The test substance did not induce any statistically significant or dose related (linear trend) increase in the mutant frequency x E-6 per viable cell in either the absence or presence of metabolic activation.

Conclusions:

Interpretation of results: negative

The test substance was evaluated for the potential to induce mutations at the thymidine kinase locus of the L5178Y mouse lymphoma cell line. Based on the results of the study, it was concluded that the test substance was negative for mutagenic potential in the absence and presence of metabolic activation up to dose levels that produced toxicity.

Executive summary:

N/A

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

SAS was tested in the reverse bacterial mutation assay. The test substance caused no visible reduction in the growth of the bacterial background lawn at any dose level, although small decreases in revertant colony frequency were noted to several of the tester strains at 5000 µg/plate, predominantly in the presence of metabolic activation (S-9, from rat liver, induced with phenobarbital and B-naphthoflavone). The test substance was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A brown color was observed from 1500 µg/plate, but this did not prevent the scoring of revertant colonies. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the five bacterial strains, with any dose of the test substance, either with or without metabolic activation. In a separate study, sodium sulfonated asphalt was assayed in a chromosomal aberration test using human lymphocytes. The selection of the maximum dose level was based on the lowest precipitating dose level for the short-term exposure studies and on toxicity for the continuous exposure studies. The test substance did not induce any statistically significant increases in the frequency of cells with aberrations, either in the absence or presence of metabolic activation in short-term exposure experiments. In the continuous exposure studies in the absence of S9, the maximum test substance dose level with scorable metaphases was 2500 µg/mL. The mitotic index data confirmed the qualitative observations in that a dose-related inhibition of mitotic index was observed in the absence of S9, and that 56% mitotic inhibition was achieved at 2500 µg/mL. In the presence of S9, there was no evidence of toxicity. The maximum dose level selected for metaphase analysis was based on toxicity for the 24-hour exposure group in the absence of S9. In the presence of S9, the maximum dose level tested was selected for analysis and was based on the lowest observable precipitating dose level. Based on the continuous exposure experiments, the test substance did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, either in the absence or presence of metabolic activation. Finally, sodium sulfonated asphalt was evaluated in a mammalian cell gene mutation test using the L5178Y mouse lymphoma cell line. Two independent experiments were performed with dose levels ranging from 156.25 to 2500 µg /mL. The maximum dose level used was limited to 2500 µg/ml by toxicity, and the nature of the precipitate observed at 5000 µg/ml in a preliminary experiment. In experiment 1, cells were treated for 4 hours in the presence and absence of metabolic activation (S9) and in experiment 2, the cells were treated for 4 hours in the presence of S9 and for a continuous 24 hour exposure in the absence of S9. Precipitate of the test substance was observed at all dose levels in both experiments. The test substance failed to induce any toxicologically significant dose-related increases in the mutant frequency at the thymidine kinase locus at any dose level, in the presence or absence of S9, in either experiment. Therefore, the test substance was considered to be non-mutagenic to L5178Y cells.

 

Justification for classification or non-classification

SAS was tested in the reverse bacterial mutation assay and in a chromosomal aberration test using human lymphocytes. In the assay, at 5000µg/plate, no significant increases in the frequency of revertant colonies were recorded for any of the five bacterial strains, with any dose of the test substance, either with or without metabolic activation.Based on the short or continuous exposure experiments, the test substance did not induce any statistically significant increases in the frequency of human lymphocytes with chromosome aberrations, either in the absence or presence of metabolic activation. In addition, SAS was evaluated in the mammalian cell gene mutation test using L5178Y mouse lymphoma cells. The test substance failed to induce any toxicologically significant dose-related increases in the mutant frequency at the thymidine kinase locus at any dose level, in the presence or absence of S9.

Therefore, the available data do not support a classification under DSD or CLP/GHS based on the weight of evidence.