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

Genetic toxicity: in vitro

Administrative data

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Remarks:

Not specified in report

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

405-430-6

EC Name:

-

Cas Number:

65143-89-7

Molecular formula:

UVCB

IUPAC Name:

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

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

DOWFAX® 8390 D Surfactant
Appearance: powder
Reference: Lot # 9401205-50A
Source: Dow Europe S. A.
Purity: 97.5% active ingredient

Method

Target gene:

rat lymphocytes

Species / strain

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

Controls

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.