Reaction mass of Benzene sulfonic acid, hexadecyl(sulfophenoxy)-,disodium salt and Benzene sulfonic acid, - oxibis[hexadecyl]-, disodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Dowfax 8390 powder was negative in three key studies, an in vitro CHO/HGPRT forward mutation assay, an in vitro chromosomal aberration assay with rat lymphocytes and an in vivo rat bone marrow assay. In addition, two supporting studies of a liquid formulation were also negative, an in vitro Ames and an in vitro chromosomal aberration study with human lymphocytes. Studies with other category members were also negative; refer to category document for more details.

Link to relevant study records

Reference

Reference 1

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:

1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, 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:

no

Remarks:

Not specified in report

Qualifier:

according to guideline

Guideline:

other: U.K. EMS (1990), EEC (1992) and U.S. EPA (1990).

Deviations:

no

Remarks:

Not specified in report.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

rat lymphocytes

Species / strain / cell type:

lymphocytes: rat/Sprague-Dawley

Details on mammalian cell type (if applicable):

Spraguw-Dawley rat, outbred Crl:CD BR strain purchased from Charles River, Kingston, NY

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 liver homogenate prepared from Aroclor 1254 treated (500 mg/kg) male Sprague-Dawley rats

Test concentrations with justification for top dose:

In Assay 1, cultures were treated with the test material in the absence and presence of S-9 activation at concentrations of 1.7, 5, 16.7, 50, 100, 167, 500, and 1000 μg/ml.

In the confirmatory assay, rat lymphocyte cultures were treated continuously for 24 h with 16.7, 25, 50, 75, 100, and 150 μg/ml of the test material in the absence of S-9 activation. In the presence of S9, cultures were exposed for 4 h to concentrations of 5, 16.7, 50, 75, 100, 150, and 500 μg/ml.

Vehicle / solvent:

culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Mitomycin C- non-activation assay (without S-9), cyclophosphamide- activation assay (with S-9)

Details on test system and experimental conditions:

Lymphocyte Cultures
Blood samples were collected by cardiac puncture, following euthanasia with carbon dioxide, from 2 rats and pooled. Whole blood cultures were set up in RPMI 1640 medium (with 25 mM HEPES, GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO), antibiotics and antimycotics (Fungizone 0.25 μg/ml; penicillin G, 100 u/ml; and streptomycin sulfate, 0.1 mg/ml; GIBCO), 20 μg/ml PHA (HA16-17, Murex Diagnostics Ltd., Dartford, England), and an additional 2 mM L-glutamine (GIBCO). Cultures were initiated by inoculating approximately 0.5 ml of whole blood/5 ml of culture medium. Cultures were set up in duplicate at each dose level in T25 plastic tissue culture flasks and incubated at 37°C.

Controls
The solvent selected for dissolving the test material was used as the negative control treatment. Mitomycin C (MMC, Sigma, St. Louis, MO, CAS No. 50-07-7) was used as the positive control chemical for the non-activation assay at a concentration of 0.05 μg/ml in Assay 1 and 0.05 and 0.075 μg/ml in Assay 2, while cyclophosphamide (CP, Sigma, CAS No. 6055-19-2) was the positive control for the activation assay at a final concentration of 4 μg/ml in Assay 1 and 4 and 6 mg/ml in Assay 2.

In vitro Metabolic Activation System
S-9 liver homogenate prepared from Aroclor 1254 treated (500 mg/kg) male Sprague-Dawley rats was purchased from Molecular Toxicology, Inc., Annapolis, MD and stored at -100°C or below. Thawed S-9 was reconstituted at a final concentration of 10% (V/V) in a "mix" (O'Neill et al., 1982). The mix consists of 10 mM MgCl2·6H2O (Sigma), 5 mM glucose-6-phosphate (Sigma), 4mM nicotinamide adenine dinucleotide phosphate (Sigma), 10mM CaCl2 (Fisher, Fair Lawn, NJ), 30 mM KCl (Sigma), and 50 mM sodium phosphate (pH 8.0, Sigma and Fisher). The reconstituted mix was added to the culture medium to obtain the desired final concentration of S- 9 in the culture, i.e., 2% V/V.

Preparation of the Treatment Solution
Stock solutions of the test material were prepared fresh just prior to treatment. The test material was dissolved in culture medium and further diluted (1:10) with the treatment medium to obtain the desired concentrations. MMC and CP were dissolved directly in treatment medium. The treatment medium was RPMI 1640 without the serum and the PHA. The pH and the osmolality of treatment medium containing approximately 36655 μg/ml of the test material were determined using a Corning M140 pH meter (Corning Medical and Scientific, Medfield, MA) and an Osmette A freezing point osmometer (Precision Systems, Inc., Natick, MA). There was no appreciable change in the pH of the culture medium following the addition of the test material as compared to the culture medium alone and the slight increase in osmolality was interpreted to be inconsequential to the conduct of the assay (culture medium with the test material, pH = 7.48, osmolality = 299 mOsm/kg H2O; culture medium, pH = 7.46, osmolality = 261 mOsm/kg H2O).

Identification of the Test System
All test cultures were identified using self adhesive labels containing a code system which identified the test material, experiment number, treatment, and replicate.

Treatment Procedure Without Metabolic Activation
Approximately forty-eight hours after initiation of the cultures (Assay 1), the cell suspension was dispensed into 15 ml sterile disposable centrifuge tubes (approximately 5.5 ml/tube, 2 cultures per dose level). The cells were sedimented by centrifugation and the culture medium removed and saved. The cells were exposed to medium (RPMI 1640, HEPES, and antibiotics) containing the test or positive or negative control treatments for approximately 4 h at 37°C and the exposure was terminated by washing the cells with culture medium. The cells were then placed in individual sterile disposable tissue culture flasks (T-25) along with approximately 4.5 ml of the original culture medium until the time of harvest. The cultures were harvested at approximately 24 h after treatment initiation (i.e., approximately 20 h after treatment termination). An additional experiment (Assay 2) was conducted with continuous treatment with the test material for approximately 1.5 cell cycle length. In this assay, the negative control, positive control, and test material were added directly to the culture flasks approximately 48 h after initiation of the cultures and the cultures were harvested approximately 24 h later (1.5X normal cell cycle length). A second harvest time of 48 h after treatment initiation was also included for negative control and test material-treated cultures with a rinse and culture medium change (to complete culture medium without the solvent or the test material) after 24 h of treatment.

Treatment Procedure with Metabolic Activation
Approximately 48 hours after initiation of the cultures, the cell suspension was dispensed into sterile disposable centrifuge tubes. The cells were sedimented by centrifugation and the culture medium removed and saved. The cells were exposed to medium (RPMI 1640, HEPES, antibiotics, and S-9) containing the test or positive or negative control treatments for approximately 4 h at 37°C and the exposure was terminated by washing the cells with culture medium (without serum and PHA). The cells were then placed in individual sterile disposable tissue culture flasks (T-25) along with approximately 4.5 ml of the original culture media until the time of harvest. In Assay 1, the cultures were harvested approximately 24 h after treatment initiation (i.e., approximately 20 h after treatment termination), and in Assay 2, negative control and test material-treated cultures were also harvested at an additional time of 48 h post-treatment initiation (i.e., 44 h after the end of the treatment termination).

Harvesting of Cultures
Colcemid was added approximately 3 h prior to harvest at a final concentration of 0.2 μg/ml. The cells were swollen by hypotonic treatment (0.075 M KCl), fixed with methanol:acetic acid (3:1), dropped on microscope slides, and stained in Giemsa. All slides were coded prior to evaluation.

Evaluation criteria:

Mitotic indices were determined as the number of cells in metaphase among 1000 cells/replicate and expressed as percentages. One hundred metaphases/replicate were examined from coded slides at each selected concentration of the test chemical and the negative controls (a total of 200 cells/treatment) for structural abnormalities (Buckton and Evans, 1973; Sinha et al., 1984; Gollapudi et al., 1986) where possible. In the positive control cultures, 50 metaphases/replicate (a total of 100 cells/treatment) were examined for abnormalities. The microscopic coordinates of each metaphase were recorded. Only those metaphases that contained 42 ± 2 centromeres were scored with the exception of cells with multiple aberrations, in which case accurate counts of the chromosomes were not possible. Those cells having 5 or more aberrations/cell were classified as cells with multiple aberrations. Gaps were not included in calculations of total cytogenetic aberrations.

References:
Buckton, K.E., and H.J. Evans, eds. (1973): Methods for the analysis of human chromosome aberrations, World Health Organization, Geneva.

Gollapudi, B.B., D.J. Sutcliffe, and A.K. Sinha (1986): Assessment of cytogenetic response to folic acid deprivation in rat lymphocytes, In vitro, 22, 681-684.

Sinha, A.K., V.A. Linscombe, B.B. Gollapudi, M.L. McClintock, R.E. Flake, and K.M. Bodner (1984). The incidence of spontaneous cytogenetic aberrations in lymphocytes cultured from normal humans for 48 and 72 h, Can. J. Genet. Cytol., 26, 528-531.

Statistics:

The frequencies of cells with aberrations (excluding gaps) were compared by the following statistical methods. At each dose level, data from the replicates were pooled. A two way contingency table was constructed to analyze the frequencies of cytogenetic abnormalities. An overall Chi-square statistic, based on the table, was partitioned into components of interest. Specifically, statistics were generated to test the two global hypotheses of (1) no differences in average number of cells with aberrations among the dose groups, and (2) no linear trend of increasing number of cells with aberrations with increasing dose (Armitage, 1971). An ordinal metric (0, 1, 2, etc.) was used for the doses in the statistical evaluation. If either statistic was found to be significant at alpha=0.01 versus a one-sided increasing alternative, pairwise tests (i.e., control vs treatment) were performed at each dose level and evaluated at alpha=0.01 again versus a one-sided alternative.

For a test to be acceptable, the chromosomal aberration frequency in the positive control cultures should be significantly higher than the negative controls. The aberration frequency in the negative control should be within reasonable limits of the laboratory historical values. A test chemical is considered positive in this assay if it induces a significant dose-related, and reproducible increase in the frequency of cells with aberrations.

Reference:
Armitage, P. (1971): Statistical Methods in Medical Research, John Wiley & Sons, Inc., New York, NY.

Species / strain:

lymphocytes: Sprague-Dawley rat

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In an initial assay, cultures were treated with the test material in the absence or presence of S-9 activation at concentrations of 50, 167, 500, 1000, 1667, 2500, and the limit dose of 5000 μg/ml. Dose levels of 1000 μg/ml and greater in the absence of S-9 activation, showed excessive toxicity (mitotic indices of <0.1%). The reductions in mitotic index at 500 and 167 μg/ml were 72 and 56%, respectively. In cultures treated with the test material in the presence of S-9 activation, there was excessive toxicity at concentrations > or = 500 μg/ml. The only dose with a moderate reduction in mitotic index was 50 μg/ml (i.e., 21%,). Because of the excessive toxicity observed at several of the concentrations assayed and the resulting unavailability of the desired number of treatment levels for aberration scoring, cultures from this assay were not selected for scoring and another assay (designated as Assay 1) was conducted using a lower concentration range.

In Assay 1, cultures were treated with the test material in the absence and presence of S-9 activation at concentrations of 1.7, 5, 16.7, 50, 100, 167, 500, and 1000 μg/ml. Without metabolic activation, cultures treated with 500 and 1000 μg/ml had > or = 60% reduction in mitotic activity while the this value at the 167 μg/ml was 45% reduction. In the presence of S-9 activation, dose levels of 167, 500 and 1000 μg/ml were excessively toxic (> or = 70% reduction in mitotic activity). Based upon these results, cultures treated with 50, 100, and 167 μg/ml in the absence of S-9 activation and cultures treated with 16.7, 50, and 100 μg/ml in the presence of S-9 were selected for determining the chromosomal aberration frequencies.

In the non-activation assay, the frequency of cells with aberrations in the negative control was 1.5% and the corresponding values at treatment levels 50, 100, and 167 μg/ml were 3.0, 2.5, and 2.5%, respectively. In the activation assay, cultures treated with the test material at concentrations of 16.7, 50, and 100 μg/ml had aberrant cell frequencies of 2.5, 3.0, and 2.5%, respectively. Statistical analyses of these data did not identify significant differences between the negative control and any of the treated cultures either with or without S-9 activation. The frequencies of aberrant cells observed in the test material treated cultures were within the laboratory historical background range.

Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemicals, MMC (without S-9) and CP (with S-9).

In the confirmatory assay, rat lymphocyte cultures were treated continuously for 24 h with 16.7, 25, 50, 75, 100, and 150 μg/ml of the test material in the absence of S-9 activation. In the presence of S9, cultures were exposed for 4 h to concentrations of 5, 16.7, 50, 75, 100, 150, and 500 μg/ml. Cultures were harvested either at 24 h or 48 h after treatment initiation. The additional 48 h harvest time is expected to provide adequate time for the cells to recover from any cell cycle delay induced by the treatments. The highest concentration tested in the absence of S-9, i.e. 150 μg/ml, induced a 79% reduction in the mitotic index at the 24 h harvest and a 39% reduction at the 48 h harvest. The reduction in mitotic activity observed at the 100 μg/ml dose level was 50% at the 24 h harvest with a complete recovery by the 48 h harvest. In the presence of S-9 activation, the highest concentration tested, 500 μg/ml, showed excessive toxicity at both harvest times while dose levels of 75, 100 and 150 μg/ml had mitotic index reductions of 57, 64, and 80%, respectively at the 24 h harvest and 44, 57 and 52 at the 48 h harvest. Based upon these data, slides prepared from the following treatments of DOWFAX 8390D Surfactant were selected for aberration scoring:

Without Activation, 24 h harvest:
0, 25, 50, and 100 μg/ml

Without Activation, 48 h harvest:
0, 150 μg/ml

With Activation, 24 h harvest:
0, 16.7, 50, and 75 μg/ml

With Activation, 48 h harvest:
0, 150 μg/ml

Among the cultures treated with the positive control chemicals, 0.075 μg/ml of MMC and 6 μg/ml of CP were selected for scoring aberrations.

The frequency of cells with aberrations among the treated cells were not significantly different from the corresponding negative control values under any activation conditions and these values were within the historical background aberration rates. The frequency of cells with aberrations among the treated cultures ranged from 0.5 to 4.5% while negative control values ranged from 2.5 to 4.0% .

Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemicals, MMC (without S-9) and CP (with S-9).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

TABLE 1A

RESULTS OF THE CHROMOSOMAL ABERRATION ASSAY 24 HOURS AFTER TREATMENT IN THE ABSENCE OF S-9

(Results of replicates designated A & B are combined)

ASSAY 1

Test Chemical: DOWFAX 8390 D Surfactant Negative Control: 10% Medium Positive Control: MMC (0.5 μg/ml)

	Negative Control	50 ug/ml	100 ug/ml	167 ug/ml	Pos. Control
Number of cells scored	200	200	200	200	100
Chromatid Gaps	0	5	4	0	3
Chromosome Gaps	0	0	0	0	0
Chromatid Breaks	3	4	3	5	24
Chromatid Exchanges	0	0	0	0	36
Chromosome Breaks	0	2	2	0	11
Chromosome Exchanges	0	0	0	0	2
Total Aberrations (excluding gaps)^a	3 (1.5)	6 (3.0)	5 (2.5)	5 (2.5)	73 (73.0)
No. of cells with Aberr.	3 (1.5)	6 (3.0)	6 (3.0)	5 (2.5)	58^b (58.0)
Miscellaneous Aberr.	0	0	1	0	0
Cells with Multiple Aberr. (5 or more aberr.)	0	0	0	0	5

^aValues in parentheses are percentages.

^bSignificantly (alpha<0.01) different from the negative control.

TABLE 1B

RESULTS OF THE CHROMOSOMAL ABERRATION ASSAY 24 HOURS AFTER TREATMENT IN THE PRESENCE OF S-9

(Results of replicates designated A & B are combined)

ASSAY 1

Test Chemical: DOWFAX 8390 D Surfactant Negative Control: 10% Medium Positive Control: CP (4.0 mg/ml)

	Negative Control	16.7 ug/ml	50 ug/ml	100 ug/ml	Pos. Control
Number of cells scored	200	200	200	200	100
Chromatid Gaps	2	1	1	4	2
Chromosome Gaps	0	0	0	0	0
Chromatid Breaks	0	3	4	2	18
Chromatid Exchanges	0	0	0	1	12
Chromosome Breaks	0	2	1	2	7
Chromosome Exchanges	0	0	1	0	1
Total Aberrations (excluding gaps)^a	0 (0.0)	5 (2.5)	6 (3.0)	5 (2.5)	38 (38.0)
No. of cells with Aberr.	0 (0.0)	5 (2.5)	6 (3.0)	5 (2.5)	29^b (29.0)
Miscellaneous Aberr.	0	0	0	0	0
Cells with Multiple Aberr. (5 or more aberr.)	0	0	0	0	0

^aValues in parentheses are percentages.

^bSignificantly (alpha<0.01) different from the negative control.

TABLE 2A

RESULTS OF THE CHROMOSOMAL ABERRATION ASSAY 24 HOURS AFTER TREATMENT IN THE ABSENCE OF S-9

(Results of replicates designated A & B are combined)

ASSAY 2

Test Chemical: DOWFAX 8390 D Surfactant Negative Control: 10% Medium Positive Control: MMC (0.075 μg/ml)

	Negative Control	25 ug/ml	50 ug/ml	100 ug/ml	Pos. Control
Number of cells scored	200	200	200	200	125
Chromatid Gaps	2	3	1	4	3
Chromosome Gaps	0	0	0	0	1
Chromatid Breaks	5	1	2	5	19
Chromatid Exchanges	0	0	0	0	19
Chromosome Breaks	0	0	2	1	8
Chromosome Exchanges	0	0	0	0	1
Total Aberrations (excluding gaps)^a	5 (2.5)	1 (0.5)	4 (2.0)	6 (3.0)	47 (37.6)^b
No. of cells with Aberr.	5 (2.5)	1 (0.5)	4 (2.0)	5 (2.5)	39^b (31.0)
Miscellaneous Aberr.	0	0	0	0	0
Cells with Multiple Aberr. (5 or more aberr.)	0	0	0	0	3

^aValues in parentheses are percentages.

^b Table 5A of the report erroneously states the percentage as 47%. Following reanalysis of the data, the percentage was changed to 37.6%.

^cSignificantly (alpha<0.01) different from the negative control.

TABLE 2B

RESULTS OF THE CHROMOSOMAL ABERRATION ASSAY 48 HOURS AFTER TREATMENT IN THE ABSENCE OF S-9

(Results of replicates designated A & B are combined)

ASSAY 2

Test Chemical: DOWFAX 8390 D Surfactant Negative Control: 10% Medium

	Negative Control	150 ug/ml
Number of cells scored	200	200
Chromatid Gaps	1	1
Chromosome Gaps	0	0
Chromatid Breaks	4	6
Chromatid Exchanges	0	0
Chromosome Breaks	2	0
Chromosome Exchanges	1	1
Total Aberrations (excluding gaps)^a	7 (3.5)	7 (3.5)
No. of cells with Aberr.	7 (3.5)	7 (3.5)
Miscellaneous Aberr.	0	0
Cells with Multiple Aberr. (5 or more aberr.)	0	0

^aValues in parentheses are percentages.

^bSignificantly (alpha<0.01) different from the negative control.

TABLE 2C

RESULTS OF THE CHROMOSOMAL ABERRATION ASSAY 24 HOURS AFTER TREATMENT IN THE PRESENCE OF S-9

(Results of replicates designated A & B are combined)

ASSAY 2

Test Chemical: DOWFAX 8390 D Surfactant Negative Control: 10% Medium Positive Control: CP (6 μg/ml)

	Negative Control	16.7 ug/ml	50.0 ug/ml	75.0 ug/ml	Pos. Control
Number of cells scored	200	200	200	200	100
Chromatid Gaps	0	2	0	1	3
Chromosome Gaps	0	0	0	0	0
Chromatid Breaks	5	4	4	3	20
Chromatid Exchanges	0	0	0	0	11
Chromosome Breaks	3	1	0	0	5
Chromosome Exchanges	0	0	0	0	0
Total Aberrations (excluding gaps)^a	8 (4.0)	5 (2.5)	4 (2.0)	3 (1.5)	36 (36.0)
No. of cells with Aberr.	8 (4.0)	3 (3.0)	3 (1.5)	3 (1.5)	29^b (29.0)
Miscellaneous Aberr.	0	1	0	0	0
Cells with Multiple Aberr. (5 or more aberr.)	0	0	0	0	1

^aValues in parentheses are percentages.

^bSignificantly (alpha<0.01) different from the negative control.

TABLE 2D

RESULTS OF THE CHROMOSOMAL ABERRATION ASSAY 48 HOURS AFTER TREATMENT IN THE PRESENCE OF S-9

(Results of replicates designated A & B are combined)

ASSAY 2

Test Chemical: DOWFAX 8390 D Surfactant Negative Control: 10% Medium

	Negative Control	150 ug/ml
Number of cells scored	200	200
Chromatid Gaps	0	2
Chromosome Gaps	0	0
Chromatid Breaks	3	8
Chromatid Exchanges	0	0
Chromosome Breaks	1	1
Chromosome Exchanges	0	0
Total Aberrations (excluding gaps)^a	4 (2.0)	9 (4.5)
No. of cells with Aberr.	4 (2.0)	9 (4.5)
Miscellaneous Aberr.	0	0
Cells with Multiple Aberr. (5 or more aberr.)	0	0

^aValues in parentheses are percentages.

Conclusions:

Interpretation of results (migrated information):
negative

The test material, DOWFAX 8390D Surfactant, did not induce an increase in the frequency of cells with chromosomal abnormalities. Hence, it was concluded that under the experimental conditions used, DOWFAX 8390D Surfactant was negative in this in vitro chromosomal aberration test.

Executive summary:

DOWFAX 8390D Surfactant was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes. Approximately 48 h after the initiation of whole blood cultures, cells in the absence and presence of rat liver S-9 activation were treated for 4 h with concentrations ranging from 0 (negative control), 50, 166.7, 500, 1000, 1667, 2500, and 5000 μg DOWFAX 8390D Surfactant per ml of culture medium. The treated cultures were harvested approximately 20 h after the termination of the treatments. Mitotic indices data from this experiment indicated excessive toxicity at dose levels of 500 μg/ml. Hence, the experiment was repeated using dose levels of 1.7, 5, 16.7, 50, 100, 166.7, 500, and 1000 μg/ml. Based upon the mitotic indices, cultures treated with concentrations of 0, 50, 100, and 166.7 μg/ml in the absence of S-9 activation and cultures treated with targeted doses of 0, 16.7, 50, and 100 μg/ml in the presence of S-9 activation were selected for determining the incidence of chromosomal aberrations. In this assay, there were no significant increases in the incidence of aberrant cells in DOWFAX 8390D Surfactant treated cultures.

In a confirmatory assay with S9, rat lymphocytes were treated as described above and harvested at 20 h and 44 h after treatment termination. In the absence of S9, the treatment time was increased to 24 h and the treated cultures were harvest either at the end of the treatments or 24 h later. The incidence of chromosomal abnormalities was determined from cultures treated with 0, 25, 50, and 100 μg/ml in the absence of S-9 at the first harvest time, and from cultures treated with 0 and 150 μg/ml at the second harvest. In the presence of S9, cultures treated with 0, 16.7, 50, and 75 μg/ml were used for evaluation at the first harvest and 0 and 150 μg/ml at the second harvest. No significant increase in the incidence of aberrant cells was noticed at any of the treatment levels when compared to the corresponding negative control values. Hence, DOWFAX 8390D Surfactant was considered to be negative in the in vitro chromosomal aberration assay utilizing rat lymphocytes.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, 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

Remarks:

Not specified in report.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Remarks:

Not specified in report.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

Not specified in report.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT locus of Chinese hamster ovary (CHO) cells in culture

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The cell line CHO-K1-BH4, originally obtained from Dr. Abraham Hsie, Oak Ridge National Laboratory, Oak Ridge, TN, was used in this study. Stock cultures were stored at about –100 °C or below. The cultures were periodically checked for mycoplasma contamination (American Type Culture Collection, Manassas, VA). The cells were grown as monolayer cultures in plastic disposable tissue culture labware under standard conditions of approximately 5% CO2 in air at 37 °C in a humidified incubator.

Metabolic activation:

with and without

Metabolic activation system:

S-9 liver homogenate prepared from Aroclor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

Assay B1: 10, 20, 40, 60, 80, 100, 120, 150, 200, and 300 ug/ml with and without S-9
Assay C1: 2.5, 5, 10, 20, 40, 50, 60, 70, 80 and 100 ug/ml without S-9, and 10, 20, 40, 80, 120, 140, 160, 180 and 200 ug/ml with S-9

Vehicle / solvent:

culture medium (Ham’s F-12 nutrient mix)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethyl methanesulfonate (EMS) was used as the positive control for the non-activation system (without S-9 factor) at 621 ug/ml. The positive control for assays performed with S-9 (activation system) was 20- methylcholanthrene (20-MCA) at 4 ug/ml.

Details on test system and experimental conditions:

Indicator Cells
The cell line CHO-K1-BH4, originally obtained from Dr. Abraham Hsie, Oak Ridge National Laboratory, Oak Ridge, TN, was used in this study. Stock cultures were stored at about –100 °C or below. The cultures were periodically checked for mycoplasma contamination (American Type Culture Collection, Manassas, VA). The cells were grown as monolayer cultures in plastic disposable tissue culture labware under standard conditions of approximately 5% CO2 in air at 37 °C in a humidified incubator.

Media
The cells were routinely maintained in Ham's F-12 nutrient mix (GIBCO, Grand Island, NY) supplemented with 5% (v/v) heat-inactivated (56 °C, 30 min), dialyzed fetal bovine serum (GIBCO), antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone, 25 mg/ml; GIBCO) and an additional 2 mM L-glutamine (GIBCO). The selection medium used for the detection of HGPRT- mutants was Ham's F-12 nutrient mix without hypoxanthine, supplemented with 10 μM 6-thioguanine (GIBCO) and 5% serum and the above-mentioned antibiotics.

Controls
The solvent used to dissolve the test material was used as the negative control treatment. Ethyl methanesulfonate (EMS, CAS No. 62-50-0) was used as the positive control for the non-activation system (without S-9 factor) at a final concentration of 621 mg/ml. The positive control for assays performed with S-9 (activation system) was 20- methylcholanthrene (20-MCA, CAS No. 56-49-5) at a concentration of 4 mg/ml. The dose levels of EMS and 20-MCA were based upon our unpublished findings.

Treatment Procedure
Cells in logarithmic growth phase were trypsinized and placed in medium containing 5% serum at a standard density of 3.0 X 10E6 cells/T-75 flask approximately 24 hr prior to treatment. At the time of treatment, the culture medium was replaced with serum-free medium, S-9 mix (when applicable) and the test chemical, the negative control solvent or the positive control chemical. The cells were treated for approximately 4 hr at 37 °C and the exposure was terminated by washing the cells with phosphate buffered saline.

Identification of the Test System
All test cultures were identified using self adhesive labels containing a code system which identified the test material, experiment number, treatment and replicate.

Preparation of the Treatment Solution
The test material was first dissolved in culture medium (Ham’s F-12 nutrient mix) and further diluted (1:10) with the medium to obtain the desired concentrations. EMS was dissolved in treatment medium. 20-MCA was dissolved first in dimethyl sulfoxide and further diluted in the culture medium. All dosing units were expressed in μg/ml. During initial solubility testing, the pH and osmolality of the medium and the treatment medium containing approximately 5000 μg/ml of the test material (limit dose) were determined using a Denver Basic pH meter (Denver Instrument Co., Arvada, CO) and an Osmette A freezing point osmometer (Precision Systems, Inc., Natick, MA). There was no appreciable change in either the pH or the osmolality at this concentration as compared to the culture medium alone (culture medium with the test material, pH = 8.08, osmolality = 320 mOsm/kgH2O; culture medium alone, pH = 7.76, osmolality = 314 mOsm/kgH2O.

In Vitro Metabolic Activation
S-9 liver homogenate prepared from Aroclor 1254-induced male Sprague-Dawley rats was purchased from Molecular Toxicology, Inc., Boone, North Carolina, and stored at approximately –100 °C or below. Thawed S-9 was reconstituted at a final concentration of 10% (V/V) in a "mix" (O'Neill et al., 1982). The mix consisted of the following cofactors: 10 mM MgCl2·6H2O, 5 mM glucose-6-phosphate, 4 mM nicotinamide adenine dinucleotide phosphate, 10 mM CaCl2, 30 mM KCl, and 50 mM sodium phosphate (pH 8.0). The reconstituted mix was added to the culture medium to obtain the desired final concentration of S-9 in the culture (i.e., 2% V/V). Hence, the final concentration of the co-factors in the culture medium is 1/5 of the concentrations stated above.

Toxicity Assay
The cytotoxicity of the test material was assessed by determining the ability of the treated cells to form colonies. This assay was conducted for selecting concentrations of the test material to be used in the gene mutation assay. Cells were seeded into T-25 flasks (1.0 X 10E6 cells/flask) approximately 24 hr prior to treatment. Treatment was for approximately 4 hr with various concentrations of the test material in the presence and absence of S-9
factor. After termination of treatment, the cells were trypsinized and replated at a density of 200 cells/dish into 60 mm dishes (3/dose) and the dishes incubated for 6-7 days to allow colony formation. The colonies were then fixed/stained with methanol/crystal violet. The numbers of colonies/dish were counted and the mean colonies/treatment were expressed relative to the negative control value.

Gene Mutation Assay
Each dose level was set up in duplicate from the time of treatment until the completion of the assay. The number of cells treated at each dose level was adjusted to yield at least 1 X 10E6 surviving cells. The cultures were trypsinized at the end of the treatment and replated at a density of 1 X 10E6 cells/100 mm dish (at least 2 dishes/replicate) for phenotypic expression (O'Neill et al., 1977a, O'Neill and Hsie, 1979). In addition, 200 cells/60 mm dish (3 dishes/replicate) were also plated to determine the toxicity and incubated for approximately 6-8 days to permit colony formation. During the phenotypic expression period (7-9 days), cells in the larger petri dishes were subcultured every 2-3 days and plated (at least 2 dishes/replicate) at a density of about 1 X 10E6 cells /100 mm petri dish. At each subculture, cells from various dishes within each replicate were pooled prior to replating. At the end of the expression period, the cultures were trypsinized and plated at a density of 2 X 10E5 cells/100 mm dish (a total of 10 dishes/treatment) in the selection media (Ham's F-12 without hypoxanthine and with 6- thioguanine) for the determination of HGPRT- mutants and 200 cells/60 mm dish (3 dishes/treatment) in Ham's F-12 medium without hypoxanthine for determination of cloning efficiency. Treatments resulting in less than approximately 10% relative cell survival (based upon the concurrent toxicity assay results) were not used for determining either the cloning efficiency or mutation frequencies. The dishes were incubated for about 6-10 days and the colonies were fixed/stained with methanol/crystal violet. The mutation frequency (expressed as mutants per 10E6 clonable cells) for each replicate were calculated by the following formula (Kirkland, 1989):

MF = K x (m/c)

Where:
K = (Pc x 10E6)/Pm

Pc = the number of cells plated for the survival plates
Pm = the number of cells plated for the mutation plates
c = the mean colonies per plate for survival plates for each replicate
m = the mean colonies per plate for mutation plates for each replicate

Evaluation criteria:

For an assay to be acceptable, the mutation frequency in positive controls should have been significantly higher than the negative controls. An additional criteria was that the mutation frequency in the negative controls should have been within reasonable limits of the laboratory historical control values and literature values. The test chemical was considered positive if it induced a statistically significant, dose related, reproducible increase in mutation frequency. The final interpretation of the data took into consideration such factors as the mutation frequency and cloning efficiencies in the negative controls.

Statistics:

The frequencies of mutants per 10E6 clonable cells were statistically evaluated using a weighted analysis of variance (Hsie et al., 1980); weights were derived from the inverse of the mutation frequency variance. The actual plate counts were assumed to follow a Poisson distribution, therefore the mean plate count was used as an estimate of variance (Kirkland, 1989).

A linear trend test and lack of fit test were employed (alpha = 0.05) as omnibus tests to compare treated groups to the negative control. If there was a significant increasing trend or a significant lack of fit, a Dunnett's t-test was conducted (Winer, 1971), comparing each treated group and the positive control to the negative control (alpha = 0.05, one-sided). The lack of fit test was just an indicator that further analysis needed to be done (i.e., the Dunnett’s test). An additional comparison of the positive control to the negative control (alpha = 0.05) was conducted using a linear contrast statement.


References:
Hsie, A.W., Brimer, P.A., O’Neill J.P., Epler, J.L., Guerin, M.R. and Hsie, M.H. (1980). Mutagenicity of alkaline constituents of a coal-liquefied crude oil in mammalian cells. Mutation Research 78:79-84.

Kirkland, D. J. (editor) (1989). Statistical Evaluation of Mutagenicity Test Data, Cambridge University Press, New York, NY, Pgs. 78-87.

Winer, B.J. (1971). Statistical Principles in Experimental Design (2nd Edition). McGraw-Hill, New York.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In a preliminary toxicity assay (A1), the test material was assayed at concentrations ranging from 1.2 to 5000 μg/ml in the absence and presence of an externally supplied metabolic activation system (S-9). The highest concentration tested represented the limit dose for this assay system. Cultures treated in the absence of S-9 showed excessive toxicity at concentrations of 156.3 μg/ml and higher, and this was evident from the absence of cells in culture dishes at the end of treatments. At the two lower concentrations of 39.1 and 78.1 mg/ml, the relative cell survival (RCS) was 32 and 40%,
respectively. The absence of colonies in cultures treated with 19.5 mg/ml was attributed to a likely technical error rather than toxicity. There was no evidence of toxicity at the remaining lower treatment levels.

Cultures treated in the presence of S-9 activation showed excessive toxicity at concentrations of 312.5 through 5000 ug/ml as reflected by the absence of cells in culture dishes at the end of the treatment. The remaining concentrations induced moderate to no toxicity (42 to 111% RCS).

Based upon the results of this assay, concentration levels in the range of 10 to 300 ug/ml of the test material were selected for the gene mutation assay in both the presence and absence of metabolic activation.

Assay 1
In Assay 1 (B1), cultures treated with concentrations of 60 μg/ml and higher without S-9 displayed excessive toxicity. At these concentrations, there were either no cells in the culture dishes at the end of the treatments or insufficient number of cells to permit their propagation through the expression period. Cultures treated with the next lower concentration of 40 mg/ml displayed little toxicity when assessed by the relative cell survival. These observations suggest a steep dose-response relationship for DOWFAX 8390-D induced cytotoxicity, i.e., a very narrow concentration threshold for
compatibility with cell survival and cell death. Such a mode of cytotoxicity is typically observed with detergents and surfactants in these cell systems. There were no increases in the mutant frequencies at the concentration levels of DOWFAX 8390-D that were compatible to cell survival.

In the presence of S-9, cultures treated with the two highest concentrations of 200 and 300 μg/ml were lost to toxicity. Again, the mode of cytotoxicity with S-9 was similar to that observed without S-9. The mutant frequencies observed in the cultures treated with the test material at concentrations that were compatible with cell survival were not significantly different from the negative control values. The mutant frequencies in the test material treated cultures were within a reasonable range of historical background values. The positive control chemicals, EMS and 20-MCA, induced significant increases in mutant frequencies. All other criteria for a valid assay were also satisfied.

Assay 2
The concentrations tested in this assay were adjusted based upon Assay 1 results and included dose(s) in the steep portion of the dose-response curve. This was done in an attempt to secure doses that would result in varying levels of cytotoxicity. However, similar to Assay 1, the test material displayed a threshold concentration for compatibility with cell survival and an “all or none” type of cytotoxic effect despite the use of a very narrow dose range.

In the absence of S-9, cultures treated with > 50 ug/ml were lost due to excessive cytotoxicity at the end of the treatments. There were no significant treatment related increases in mutant frequencies at the lower concentrations evaluated. In the presence of S9, no significant increases in mutant frequencies were observed at any of the concentrations tested. The mutant frequencies of the test material treated cultures were in the range of the laboratory historical background values. The positive control chemicals induced significant increases in mutant frequencies and this data confirmed the adequacy of the experimental conditions for detecting induced mutations.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1A. Results of the Gene Mutation Assay in CHO Cells Treated with DOWFAX 8390-D Surfactant in the Absence of S-9

Assay B1 Cell Line: CHO-K1-BH4 Positive Control: 621 μg/ml EMS

Passage: 37 Negative Control: Culture medium

Treatment	Toxicity Assay	Mutation Assay	Cloning Efficiency (CE)	TG^r Mutants/10^6
ug/ml	RCS (%)^a	Total	CE (%)^b	Clonable Cells^c
Neg. Control	100.5	14	68.0	10.3
Neg. Control	99.5	5	54.3	4.6
10	178.1	6	77.3	3.9
10	204.1	4	73.3	2.7
20	94.6	5	74.0	3.4
20	128.3	8	79.3	5.0
40	126.0	20	97.5	10.3
40	125.0	11	91.8	6.0
60	-^e	-^e	-^e	-^e
60	-^e	-^e	-^e	-^e
80	-^e	-^e	-^e	-^e
80	-^e	-^e	-^e	-^e
100	-^e	-^e	-^e	-^e
100	-^e	-^e	-^e	-^e
120	-^e	-^e	-^e	-^e
120	-^e	-^e	-^e	-^e
150	-^e	-^e	-^e	-^e
150	-^e	-^e	-^e	-^e
200	-^e	-^e	-^e	-^e
200	-^e	-^e	-^e	-^e
300	-^e	-^e	-^e	-^e
300	-^e	-^e	-^e	-^e
Pos. Control	49.4	501	48.0	521.9^d
Pos. Control	39.2	396	45.7	433.6^d

^a Relative cell survival (%) = (Mean number of colonies/dish in the treated x 100)/Mean number of colonies/dish in the negative control (avg. of replicates)

^b CE (%) = (Mean number of colonies/dish x 100)/No. of cells seeded/dish

^c TG^r= 6-Thioguanine resistant

^d The frequency of TG^r mutants is significantly higher than the concurrent negative control value (alpha=0.05)

^e Lost to toxicity due to either no cells in the culture dishes at the end of the treatments or insufficient number of cells to permit their propagation through the expression period

Table 1B. Results of the Gene Mutation Assay in CHO Cells Treated with DOWFAX 8390-D Surfactant in the Absence of S-9

Assay C1 Cell Line: CHO-K1-BH 4 Positive Control: 621 μg/ml EMS

Passage: 34 Negative Control: Culture medium

Treatment	Toxicity Assay	Mutation Assay	Cloning Efficiency (CE)	TG^rMutants/10^6
ug/ml	RCS (%)^a	Total	CE (%)^b	Clonable Cells^c
Neg. Control	105.0	+	-^e	+
Neg. Control	95.0	20	65.0	17.1
2.5	106.4	34	72.2	23.6
2.5	117.3	15	62.0	12.1
5	137.9	17	66.5	12.8
5	144.2	3	61.8	2.4
10	126.3	+	59.0	+
10	117.1	14	77.8	10.0
20	91.6	18	77.2	11.7
20	108.4	19	53.0	17.9
40	96.7	!	!	!
40	92.6	11	64.5	8.5
50	-^e	-^e	-^e	-^e
50	-^e	-^e	-^e	-^e
60	-^e	-^e	-^e	-^e
60	-^e	-^e	-^e	-^e
70	-^e	-^e	-^e	-^e
70	-^e	-^e	-^e	-^e
80	-^e	-^e	-^e	-^e
80	-^e	-^e	-^e	-^e
100	-^e	-^e	-^e	-^e
100	-^e	-^e	-^e	-^e
Pos. Control	28.6	269	29.7	503.7^d
Pos. Control	33.7	266	29.7	448.3^d

^aRelative cell survival (%) = (Mean number of colonies/dish in the treated x 100)/Mean number of colonies/dish in the negative control (avg. of replicates)

^bCE (%) = (Mean number of colonies/dish x 100)/No. of cells seeded/dish

^cTG^r= 6-Thioguanine resistant

^dThe frequency of TG^rmutants is significantly higher than the concurrent negative control value (alpha=0.05)

^eLost to toxicity due to either no cells in the culture dishes at the end of the treatments or insufficient number of cells to permit their propagation through the expression period

+ Lost to contamination

! Cells not plated due to technical error.

Table 2A. Results of the Gene Mutation Assay in CHO Cells Treated with DOWFAX 8390-D Surfactant in the Presence of S-9

Assay B1 Cell Line: CHO-K 1-BH4 Positive Control: 4.0 μg/ml MCA

Passage: 37 Negative Control: Culture Medium

Treatment	Toxicity Assay	Mutation Assay	Cloning Efficiency (CE)	TG^rMutants/10^6
ug/ml	RCS (%)^a	Total	CE (%)^b	Clonable Cells^c
Neg. Control	96.7	23	83.3	13.8
Neg. Control	103.3	28	66.5	21.1
10	151.1	7	75.2	4.7
10	107.1	15	65.0	11.5
20	104.4	6	61.3	4.9
20	120.5	6	66.3	4.5
40	171.5	22	106.2	10.4
40	206.6	11	87.3	6.3
60	103.5	29	79.8	18.2
60	119.7	2	72.7	1.4
80	140.3	9	54.0	8.3
80	162.6	9	65.2	6.9
100	91.0	23	73.0	15.8
100	85.4	39	66.2	29.5
120	76.9	32	97.7	16.4
120	78.6	40	81.7	24.5
150	68.9	5	84.2	3.0
150	78.2	5	63.3	3.9
200	-^e	-^e	-^e	-^e
200	-^e	-^e	-^e	-^e
300	-^e	-^e	-^e	-^e
300	-^e	-^e	-^e	-^e
Pos. Control	73.3	328	86.5	189.6^d
Pos. Control	98.8	270	61.5	219.5^d

^aRelative cell survival (%) = (Mean number of colonies/dish in the treated x 100)/Mean number of colonies/dish in the negative control (avg. of replicates)

^bCE (%) = (Mean number of colonies/dish x 100)/No. of cells seeded/dish

^cTG^r= 6-Thioguanine resistant

^dThe frequency of TG^rmutants is significantly higher than the concurrent negative control value (alpha=0.05)

^eLost to toxicity due to either no cells in the culture dishes at the end of the treatments or insufficient number of cells to permit their propagation through the expression period

Table 2B. Results of the Gene Mutation Assay in CHO Cells Treated with DOWFAX 8390-D Surfactant in the Presence of S-9

Assay C1 Cell Line: CHO-K1-BH4 Positive Control: 4.0 μg/ml MCA

Passage: 34 Negative Control: Culture Medium

Treatment	Toxicity Assay	Mutation Assay	Cloning Efficiency (CE)	TG^rMutants/10^6
ug/ml	RCS (%)^a	Total	CE (%)^b	Clonable Cells^c
Neg. Control	100.1	10	84.5	5.9
Neg. Control	99.9	10	79.7	6.3
10	86.5	7	85.7	4.1
10	101.0	11	75.5	7.3
20	65.4	13	66.8	9.7
20	83.7	+	+	+
40	68.4	12	73.3	8.2
40	73.4	19	66.3	14.3
80	77.4	+	+	+
80	77.9	18	69.5	13.0
120	59.6	12	68.0	8.8
120	73.3	6	71.0	4.2
140	72.7	+	+	+
140	92.8	15	78.5	9.6
160	80.5	8	72.0	5.6
160	66.0	13	63.8	10.2
180	80.0	11	58.5	9.4
180	80.0	21	71.7	14.7
200	57.8	5	59.2	4.2
200	67.3	12	77.8	7.7
Pos. Control	39.5	+	+	+
Pos. Control	42.5	301	81.3	185.0^d

^aRelative cell survival (%) = (Mean number of colonies/dish in the treated x 100)/Mean number of colonies/dish in the negative control (avg. of replicates)

^bCE (%) = (Mean number of colonies/dish x 100)/No. of cells seeded/dish

^cTG^r= 6-Thioguanine resistant

^dThe frequency of TG^rmutants is significantly higher than the concurrent negative control value (alpha=0.05)

+Lost to contamination

Conclusions:

Interpretation of results (migrated information):
negative

DOWFAX 8390-D exhibited a steep dose response for cytotoxicity with an apparent threshold separating total cytotoxicity and compatibility with cell survival – a phenomenon typical to a number of detergents and surfactants. Because of this, the test material could be evaluated for mutagenicity only at non-cytotoxic concentrations. Based upon the frequency of TGr mutants recovered in cultures treated with the test material, it was concluded that DOWFAX 8390-D Surfactant did not induce a mutagenic response in the CHO/HGPRT gene mutation assay.

Executive summary:

DOWFAX 8390-D Surfactant was evaluated in the in vitro Chinese hamster ovary cell/hypoxanthineguanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation (S-9) system with concentrations ranging from 2.5 to 300 μg/ml. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assays without S-9 and 20- methylcholanthrene for assays with S-9. Negative control cultures were treated with the solvent used to dissolve the test material. Based upon the frequency of TGr mutants recovered in cultures treated with the test material, it was concluded that DOWFAX 8390-D Surfactant did not induce a mutagenic response in the assay system employed.

Reference 3

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:

1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, 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:

no

Remarks:

Not specified in report.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

TA1535- hisG46, TA1537- hisC3076, TA1538-hisD3052 , TA98- hisD3052 and TA100- hisG46

Species / strain / cell type:

other: TA1535, TA1537, TA1538, TA98 and TA100

Details on mammalian cell type (if applicable):

The Salmonella tester strains have been provided by Dr. Bruce N. Ames, University of California at Berkeley, USA.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-1254 induced rat liver S9-mix

Test concentrations with justification for top dose:

10, 33, 100, 333 and 500 ug/plate with and without S-9

Vehicle / solvent:

dimethylsulphoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S-9: sodium azide (SA)-TA1535, 9-aminoanthracene (9AC)-TA1537, 4-nitro-o-phenylenediamine (4NPD)-TA1538, daunomycine (DM)-TA98, methylmethanesulfonate (MMS)-TA100; with S-9: 2-aminoanthracene (2AA)-all strains

Details on test system and experimental conditions:

PRINCIPLE OF THE TEST METHOD
Bacteria; plated onto minimal medium agar plates; are exposed to the test chemical with and without metabolic activation ( S9-mix) . After 48h of
incubation, revertant colonies showing histidine-independent growth are counted and compared to the number of spontaneous revertants in
solvent-treated control cultures.

MATERIALS AND METHODS
Salmonella tester strains
The Salmonella tester strains have been provided by Dr. Bruce N. Ames, University of California at Berkeley, USA.

Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA1538 hisD3052 Frameshift
TA98 his D 3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TAl00 his G46/R-factor* Base-pair substitutions

*R-factor = plasmid pKMlOl (increases error-prone DNArepair) .

Each tester strain contains the following additional mutations :
r f a : deep rough (defective lipopolysaccharide cell coat).
gal : mutation in the galactose metabolism.
chl : mutation in nitrate reductase.
bio : defective biotin synthesis.
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene).
Stock cultures of the five strains are stored in liquid nitrogen (-196°C). Strains are regularly checked for their histidine requirement,
crystal violet sensitivity, ampicillin resistance (TA98 and TA100) and the frequency of spontaneous revertants.

Preparations
Bacterial cultures
Samples of frozen stock cultures of bacteria are transferred into enriched nutrient broth (Oxoid No.2) and incubated in a shaking water bath (37"C, 150 spm) until the cultures reach an O.D. of 0.4 at 700 nm ( 10E9 cells/ml ) . Freshly grown cultures of each strain are used for a test.

Selective agar plates
Selective plates (9 cm) contain 25 ml of glucose agar medium; Glucose agar medium contains per liter : 18 g purified agar (Oxoid, code L28) in
Vogel-Bonner Medium E (reference 5), 10 g glucose, 0.5 mg biotin and 0.6 mg histidine.

Top agar
Vogel -Bonner Medium E containing 0.6% (W/V) purified agar is heated to dissolve the agar. Samples of 3 ml of top agar are transferred into 10
ml glass tubes with metal caps. Top agar tubes are autoclaved for 20 min at 120°C.

Metabolic activation system
Adult male Wistar rats(3 months old) are injected intraperitoneally with a solution. (20% W/V) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days after injection, rats are killed by decapitation and their livers are removed aseptically. Livers are washed in cold (0°C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA, minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate is centrifuged for 15 min at 9000 g. The supernatant (S9) is transferred into sterile ampulles, which are stored in liquid nitrogen (-196°C). S9-mix contains per 10 ml : 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 ml water of bidistilled quality ; 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgC12 solution; 1 ml 0.33 M KCl solution, and 0.5 ml S9. S9-mix is prepared immediately before use *and kept on ice during the test.

Test procedure
Standard plate test
Top agar in top agar tubes is melted and heated to 45°C. The following solutions are successively added to 3 ml of top agar: 0.1 ml of a fresh
bacterial culture ( 10 E9 cells/ml ) of one of the tester strains, 0.1 ml of a dilution of the test substance in dimethylsulphoxide (DMSO) of
spectroscopic quality (Merck), and in the case of activation assays 0.5 ml of S9-mix. The ingredients are mixed on a Vortex and the contents of
the top agar tube are poured onto a selective agar plate. After solidification of the top agar, the plates are turned and incubated in the dark at 37°C for 48h. After this period revertant colonies ( histidine independent) are counted automatically with an Artek model 880 colony counter or manually.

Evaluation criteria:

ACCEPTABILITY OF ASSAY AND CRITERIA FOR RESPONSE
An Ames test is considered acceptable if it meets the following criteria : a) The negative control data (number of spontaneous revertants per
plate) should reasonably fall within the laboratory background historical range for each tester strain. b) The positive control chemicals should produce responses in all tester strains which also reasonably fall within the laboratory historical range documented for each positive control substance.
c) The selected dose range should include a clearly toxic concentration as demonstrated by a preliminary toxicity range-fi nding test with strain TA100.

A test substance is considered negative (not mutagenic) in the Ames test if :
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control
value, with o r without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the Ames test if:
a) It induces at least a 2-fold and statistically significant ( p< 0.05; determined by appropriate statistical analysis) increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. Moreover, the positive response should be dose-related. If the test substance shows in the first test only a positive response at one or two concentrations, the assay i s repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria are not absolute and other extenuating factors may enter into the final evaluation decision.

Statistics:

Yes , student's t-test (p < 0.05)

Species / strain:

other: TA1535, TA1537, TA1538, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY DETERMINATION OF THE TEST SUBSTANCE

Eleven serial dilutions of the test substance, in approximately ha1f-log steps, were plated with an appropriately diluted TA100 culture (equal numbers of bacterial cells/plate) onto non-selective agar ( viability determination). The percentage survival of the TA100 culture is determined by comparing the number of colonies on the solvent control plate with those on the plates containing the test substance. The survival of strain TA100 is reduced a test substance concentrations from 100 ug/plate upwards and is eliminated at test substance concentrations from 3330 ug/plate upwards. Based on these data, the test substance was tested up to a concentration of 500 ug/plate.

The. Ames Salmonellal microsome plate test
In the first two experiments with some strains several plates were infected with other bacteria. Therefore, these parts of the test were repeated. All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. ~ no statistically significant dose-related increase in the number of revertants in two independently repeated experiments. The negative and strain specific positive control values fell within our laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly.
Based on these results, the test substance can be considered as nonmutagenic in the Ames Salmonella/microsome assay.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1 Mutagenic Response of DOWFAX 8390 in the Ames Salmonella/Microsome Plate Test

Experiment 1

Dose	Mean number of revertants (His^+) colonies/3 replicate plates (+S.D.) with different strains of S. typhimurium
ug/plate	TA1535	TA1537	TA1538	TA98	TA100
	Without S9 -mix
10	6 +1	^e)	^e)	24 +5	^e)
33	5;5^b)	^e)	^e)	23 +5	^e)
100	6 +2	^e)	^e)	32 +4	^e)
333	4 +3	^e)	^e)	34 +2	^e)
500	5 +2	^e)	^e)	29 +6	^e)
Solvent control^a)	6 +2	10 +4	^e)	20 +7	^e)
Positive control	177^d)	869 +163	^e)	475; 461^c)	^e)
	With S9 -mix
10	9 +2	10 +2	17 +2	37 +3	88 +13
33	10 +2	10 +3	15 +5	38 +5	81;80^b)
100	10 +3	10 +4	14 +3	39 +8	69 +7
333	9 +3	11 +4	11 +6	33 +9	66 +6
500	9 +2	11 +3	16 +2	43 +4	83 +7
Solvent control^a)	9 +2	11 +1	19 +3	44 +9	70 +7
Positive control	287 +60	154 +6	1225 +75	2006 +81	1103 +263

^a) 0.1 ml DMSO

^b) one plate infected with other bacteria

^c) one plate lost due to technical error

^d) two plates infected with other bacteria

 ^e) all three plates infected with other bacteria

Table 2 Mutagenic Response of DOWFAX XD 8390 in the Ames Salmonella/Microsome Plate Test

Experiment 2

Dose	Mean number of revertants (His^+) colonies/3 replicate plates (+S.D.) with different strains of S. typhimurium
ug/plate	TA1535	TA1537	TA1538	TA98	TA100
	Without S9 -mix
10	18 +2	11 +4	^e)	34 +7	90; 61^b)
33	20 +5	13 +2	^e)	28 +5	51 +7
100	10; 15^b)	12 +2	^e)	27 +8	51 +5
333	^e)	13d)	^e)	27 +3	44^d)
500	18 +3	^e)	^e)	19 +3	25 +9
Solvent control^a)	18 +2	14 +2	^e)	33 +2	110 +30
Positive control	225; 205^b)	823 +58	^e)	1400; 1150^c)	770 +50
	With S9 -mix
10	13 +8	12 +3	^e)	24 +7	118 +10
33	8 +3	9 +2	^e)	25 +3	87 +5
100	18 +5	7 +2	^e)	21 +5	95 +22
333	12 +2	8 +2	^e)	22 +5	87 +8
500	7 +1	9 +1	^e)	25 +5	85 +13
Solvent control^a)	13 +5	8 +2	^e)	26 +2	123 +9
Positive control	237 +9	261 +48	^e)	1596 +200	2987 +163

^a) 0.1 ml DMSO

^b) one plate infected with other bacteria

^c) one plate lost due to technical error

^d) two plates infected with other bacteria

^e) all three plates infected with other bacteria

Table 3 Mutagenic Response of DOWFAX XD 8390 in the Ames Salmonella/Microsome Plate Test

Experiment 3

Dose	Mean number of revertants (His^+) colonies/3 replicate plates (+S.D.) with different strains of S. typhimurium
ug/plate	TA1537	TA1538	TA100
	Without S9 -mix
10	11 +1	23 +1	119 +6
33	9 +2	13 +2	120 +12
100	8 +1	14 +4	118 +9
333	9 +1	13 +3	108 +6
500	9 +2	8 +1	77 +8
Solvent control^a)	12 +2	15 +3	108 +11
Positive control	825 +51	1241 +244	1020 +44
	With S9 -mix
10	ND	17 +3	ND
33	ND	18 +2	ND
100	ND	22 +4	ND
333	ND	17 +2	ND
500	ND	11 +4	ND
Solvent control^a)	ND	27 +2	ND
Positive control	ND	1127 +66	ND

^a) 0.1 ml DMSO              

ND - No Data

Table 4 Mutagenic Response of DOWFAX XD 8390 in the Ames Salmonella/Microsome Plate Test

Experiment 4

Dose	Mean number of revertants (His^+) colonies/3 replicate plates (+S.D.) with different strains of S. typhimurium
ug/plate	TA1535	TA1537	TA1538
	Without S9 -mix
10	7 +1	6 +1	10 +2
33	7 +1	6 +1	12 +2
100	10 +2	6 +1	10 +1
333	10 +2	9 +2	11 +2
555	9 +1	5 +1	11 +3
Solvent control^a)	8 +3	7 +4	9 +1
Positive control	214 +22	692 +98	910 +39
	With S9 -mix
10	ND	ND	13 +2
33	ND	ND	19 +5
100	ND	ND	16 +3
333	ND	ND	13 +1
500	ND	ND	10 +2
Solvent control^a)	ND	ND	17 +1
Positive control	ND	ND	1779 +103

^a) 0.1 ml DMSO

ND - No Data

Conclusions:

Interpretation of results (migrated information):
negative

The test substance was considered as nonmutagenic under conditions of the assay.

Executive summary:

DOWFAX XD 8390 was tested in the Ames Salmonella typhimurium reverse mutation assay at concentrations up to the limit of toxicity (500 ug per plate). The test substance induced no statistically significant dose-related increases in the numbers of revertant (His+ ) colonies in each of the five tester strains (TA1535, TA1537, TA1538, TA98 and TA100). The results were confirmed in an independently repeated experiment. The test substance was considered as nonmutagenic under conditions of the assay.

Genetic toxicity in vivo

Description of key information

The cytogenetic effects of Dowfax Surfactant XD-8390 were determined on bone marrow cells of rats fed diets containing sufficient quantity of the test material to provide doses of 0, 50, 100, 200 or 600 mg/kg body weight/day for 90 days. No significant difference was found in the aberration rate of metaphase chromosomes of the treated and control animals.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was not conducted according to guideline/s and GLP but the report contains sufficient data for interpretation of study results

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Similar to: Buckton, K.E. and B. J. Evans. Methods for the Analysis of Human Chromosome Aberrations. World Health Organization, Geneva (1973).

GLP compliance:

no

Type of assay:

other: cytogenetic effects on rat bone marrow cells

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Groups of 5 Sprague-Dawley (Spartan substrain) rats/sex

Route of administration:

oral: feed

Vehicle:

None- Undiluted, dried material

Details on exposure:

The Dowfax surfactant identified as XD-8390 is a 36% aqueous solution of disodium hexadecyldiphenyl oxide disulfonate.

For incorporation in the diet of rats, the solution was dried to a fine hydroscopic powder which contained 92.5% active ingredient, 5.4% NaCl, 2.1% Na2S04 and 7.5 ppm iron. The test diets were prepared weekly with the concentration of powdered XD-8390 adjusted according to the rate of food consumption and body weight change to maintain the designated dose levels on a mg/kg body weight/day basis.

For this cytogenetic study, five groups of 5 male Sprague-Dawley (Spartan substrain) rats and five groups of 5 female rats were fed diets ( Purina Laboratory Chow) containing amounts of XD-8390 calculated to provide dosages of 0, 50, 100, 200 or 600 mg/kg body weight per day for 90 days.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Post exposure period:

None

Remarks:

Doses / Concentrations:
0, 50, 100, 200 or 300 mg/kg/day
Basis:
nominal in diet

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Positive control(s):

No

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

After the 90-day treatment, bone marrow cells fromall the rats were processed. The methods used for the chromosome analysis are similar to those described in ''Methods for the Analysis of Human Chromosomal Aberrations" published by the World Health Organization .

The slides were coded so that the identity of the animal was unknown to the scorer. The plan was to score 50 metaphase spreads per animal, and only diploid (2n=42) or 2n-1 cells were scored.

Cell Collection and Preparation
1. The rats were injected intraperitoneally with colchicine (0.4 mg/kg) four hours before being decapitated. The head of the femur was removed and the bone marrow was aspirated using a 10 cc syringe and a 20 gauge needle.
2. The bone marrow cells were mixed in 10 ml Hank's balanced salt solution using a disposable pipette.
3. The cells were centrifuged at 2000 RPM for 10 minutes.
4. The Hank's balanced salt solution was removed and 10 ml of 0.4% KC1 was added to the tubes. They were then incubated for 20 minutes in a 37C water bath. ( Critical )
5. The cells were then centrifuged for 10 minutes at 2000 RPM .
6. The supernatant was removed and a 3:l methanol/acetic acid fixative was used to resuspend the cells.
7 . The cells were then stored at 4C for later preparation of slides.

B. Slide Preparation
1. Centrifuge as in A-3 above, decant the fixative and add fresh 3:1 fixtive.
2. Remove the slides and place approximately 5 drops of a cell suspension on a slide; pass the slide through a flame. The slides were then dried on a warming plate at 60°.
3. The slides were rinsed gentley in distilled H20 and were placed in Giemsa stain for 10 minutes. Dehydration was through 2 acetone washes, 2 acetone-xylol changes. The slides were coverslipped from xylol using Harleco Synthetic Resin (HSR) as a mounting medium and were dried in an oven overnight.

Evaluation criteria:

The plan was to score 50 metaphase spreads per animal, and only diploid (2n=42) or 2n-1 cells were scored.

Statistics:

None

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

A careful analysis of 50 metaphase spreads from each of the 25 male rats revealed no chromosomal aberrations among the 1250 cells examined. Analysis of 1244 spreads from the 25 female rats revealed only one aberration (a chromatid break) in one animal at the 100 mg/kg dose level. Although this abnormality was found in atreated animal, it is probably inconsequential because in similar studies with this strain of rats , 27 of 3750 cells (0.72%) from 100 untreated control rats contained one or more abnormalities . When this strain of rats was treated with a potent mutagen ( triethylene-melamine), 55% of the bone marrow cells contained abnormal chromosomes.

TABLE 1

THE MJMBER OF CELLS CONTAINING CHROMATID OR CHROMOSOME

ABERRATIONS IN METAPHASE SPREADS OF BONE MARROW CELLS

OF MALE RATS FED DOWFAXB SURFACTANT XD-8390

Animal Number	Dose mg/kg	Number of cells scored	Chromatid Aberrations	Chromosome Aberrations	Abnormal Cells
75 -2602	0	50	0	0	0
75 -2603	0	50	0	0	0
75 -2604	0	50	0	0	0
75 -2605	0	50	0	0	0
75 -2924	0	50	0	0	0
75 -2621	50	50	0	0	0
75 -2622	50	50	0	0	0
75 -2623	50	50	0	0	0
75 -2624	50	50	0	0	0
75 -2925	50	50	0	0	0
75 -2640	100	50	0	0	0
75 -2641	100	50	0	0	0
75 -2642	100	50	0	0	0
75 -2643	100	50	0	0	0
75 -2926	100	50	0	0	0
75 -2659	200	50	0	0	0
75 -2660	200	50	0	0	0
75 -2661	200	50	0	0	0
75 -2662	200	50	0	0	0
75 -2927	200	50	0	0	0
75 -2678	600	50	0	0	0
75 -2679	600	50	0	0	0
75 -2680	600	50	0	0	0
75 -2681	600	60	0	0	0
75 -2928	600	60	0	0	0

TABLE 3

THE NUMBER OF CELLS CONTAINING CHROMATID OR CHROMOSOME ABERRATIONS

IN METAPKASE SPREADS OF BONE MARROW CELLS OF FEMALE RATS FED

DOWFAXB SURFACTANT XD-8390

Animal Number	Dose mg/kg	Number of cells scored	Chromatid Aberrations	Chromosome Aberrations	Abnormal Cells
75 -2697	0	50	0	0	0
75 -2698	0	50	0	0	0
75 -2699	0	50	0	0	0
75 -2700	0	50	0	0	0
75 -2929	0	50	0	0	0
75 -2716	50	50	0	0	0
75 -2717	50	50	0	0	0
75 -2718	50	50	0	0	0
75 -2719	50	50	0	0	0
75 -2930	50	50	0	0	0
75 -2735	100	50	0	0	0
75 -2736	100	50	0	0	0
75 -2737	100	50	0	0	0
75 -2738	100	50	0	0	0
75 -2931	100	50	0	0	0
75 -2754	200	50	0	0	0
75 -2755	200	50	0	0	0
75 -2756	200	50	0	0	0
75 -2757	200	50	0	0	0
75 -2932	200	50	0	0	0
75 -2773	600	50	0	0	0
75 -2774	600	50	0	0	0
75 -2775	600	50	0	0	0
75 -2776	600	60	0	0	0
75 -2933	600	60	0	0	0

Conclusions:

Interpretation of results (migrated information): negative
No significant difference was found in the aberration rate of metaphase chromosomes of the treated and control animals.

Executive summary:

INTRODUCTION: This cytogenetic study was conducted as a supplement to a subchronic toxicity study to determine if chromosomal aberrations are induced in rats ingesting XD-8390 at various concentrations in the diet for 90 days.

MATERIALS AND METHODS: Five groups of 5 male Sprague-Dawley rats and five groups of 5 female rats were fed diets containing amounts of XD-8390 calculated to provide dosages of 0, 50, 100, 200 or 600 meg/kg body weight per day for 90 days. After the 90-day treatment, bone marrow cells from all rats were processed. The slides were coded. The plan was to score 50 metaphase spreads per animal, and only diploid (2n=42) or 2n-1 cells were scored.

RESULTS AND DISCUSSION: A careful analysis of the 50 metaphase spreads from each of the 25 male rats revealed no chromosomal aberrations among the 1250 cells examined. Analysis of the 1244 spreads from the 25 female rats revealed only one aberration (a chromatid break) in one animal at the 100 mg/kg dose level. Although this abnormality was found in a treated animal, it is probably inconsequential because in similar studies with this strain of rat, 27 of 3750 cells (0.72%) from 100 untreated control rats contained one or more abnormalities. When this strain of rat was treated with a potent mutagen (triethylenemelamine), 55% of the bone marrow cells contained normal chromosomes.

SUMMARY: The cytogenetic effects of Dowfax® Surfactant XD-8390 were determined on bone marrow cells of rats fed diets containing sufficient quantity of the test material to provide doses of 0, 50, 100, 200 or 600 mg/kg body weight/day for 90 days. No significant difference was found in the aberration rate of metaphase

chromosomes of the treated and control animals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Dowfax 8390 was negative in all in vitro and in vivo assays that have been conducted.

Justification for classification or non-classification

EU classification regarding genotoxicity is not warranted, as Dowfax 8390 was negative in all in vitro and in vivo assays conducted.