Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three GLP in vitro guideline studies are available for the registered substance: bacterial Ames assay, mammalian CHO/HGPRT mutation assay and mammalian RLCAT chromosome aberration assay. All three studies were negative (non-mutagenic). The dataset for the registered substance is supported by its category members (refer to full ADPODS category justification document).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD 471 (July 1997), EC B.13/14 and OPPTS
870.5100 (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 fraction
Test concentrations with justification for top dose:
159.375, 318.75, 637.5, 1275, 2550 and 5100 μg (in the presence and absence of metabolic activation).
Test material concentrations based on active ingredient and the top dose set based on initial cytotoxicity screen.
Vehicle / solvent:
distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
Evaluation criteria:
Once criteria for a valid assay have been met, responses observed in the assay were evaluated. The conditions necessary for determining a positive result were that there should be a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test article either in the absence or presence of the metabolic activation system.

Strains TA98, TA1535 and TA1537
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean negative control value.

Strains TA100 and Escherichia coli WP2 uvrA (pKM101)
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean negative control value.

A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness,
reproducibility) was determined to be non-mutagenic.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5100 micrgram per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5100 micrgram per plate
Vehicle controls validity:
valid
Remarks:
water
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks:
without S9: 9-Aminoacridine hydrochloride hydrate with S9: 2-Aminoanthracene
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5100 micrgram per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks:
with S9: 2-Aminoanthracene w/o S9: 2-Nitrofluorene
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5100 micrgram per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks:
with S9: 2-Aminoanthracene w/o S9: Sodium azide
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5100 micrgram per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks:
w/o S9: 4-Nitroquinoline N-oxide with S9: 2-Aminoanthracene
Conclusions:
The test material was non-mutagenic under the conditions of this assay
Executive summary:

The potential of DOWFAX™™ 2A1 Solution Surfactant to induce reverse mutations in Salmonella typhimurium strains TA1537, TA1535, TA98 and TA100 and a tryptophan deficient strain, Escherichia coli WP2uvrA (pKM101) was evaluated in the bacterial reverse mutation test using the pre-incubation method. DOWFAX™ 2A1 Solution Surfactant was tested in the absence and presence of metabolic activation using sterile distilled water (DW) as the solvent. In the Initial Toxicity-mutation Assay, bacterial cultures were exposed to DOWFAX™ 2A1 Solution Surfactant at concentrations of 1.53, 5.1, 15.3, 51, 153, 510, 1530 and 5100 μg/plate (two plates/concentration) based on the active ingredient (a.i.) concentration. Precipitation was not observed up to the concentration of 5100 μg/plate. Partial inhibition of background lawn was observed at tested concentration of 5100 μg/plate in all the tester strains in absence of metabolic activation system. No inhibition of background lawn was observed up to the tested concentration of 5100 μg/plate in all the tester strains in presence of metabolic activation system. No increase in the number of revertant colonies over the background was observed with the test item in any of the tester strains at any of the tested concentrations in the absence or presence of metabolic activation (5% v/v S9 mix) when compared with the concurrent negative control. The recommended top dose indicated in the guidelines for testing in the Confirmatory Mutation Assay is 5000 μg/plate. Dose calculations for the confirmatory assay were re-calculated using the active ingredient concentration (46.1%) from the concurrently conducted GLP characterization once the results became available. In the Confirmatory Mutation Assay, bacterial cultures were exposed to DOWFAX™ 2A1 Solution Surfactant at concentrations of 159.375, 318.75, 637.5, 1275, 2550 and 5100 μg/plate (three plates/concentration) both in the absence and presence of metabolic activation (10% v/v S9 mix). Precipitation was not observed up to the concentration of 5100 μg/plate. Partial inhibition of background lawn was observed at tested concentration of 5100 μg/plate in all the tester strains in absence of metabolic activation system. No inhibition of background lawn was observed up to the tested concentration of 5100 μg/plate in all the tester strains in presence of metabolic activation system. No increase in the number of revertant colonies over the background was observed in any of the tester strains with the test item at any of the tested concentrations in the absence or presence of metabolic activation when compared with the concurrent negative control. All the values for the negative controls were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system. The DOWFAX™ 2A1 Solution Surfactant stock concentrations 1593.75, 3187.5, 6375, 12750, 25500 and 51000 μg/mL were found to be within acceptable range of ± 15% of nominal concentrations during the Confirmatory Mutation Assay. The 0 hour concentrations of DOWFAX™ 2A1 Solution Surfactant in the dose formulation were found to be 98.0 %, 95.3 %, 92.5 %, 93.1 %, 96.5 %, 97.6% of the nominal concentrations of dose level T1 (1593.75 μg/mL), T2 (3187.5 μg/mL), T3 (6375 μg/mL), T4 (12750 μg/mL), T5 (25500 μg/mL) and T6 (51000 μg/mL), respectively. The concentrations of DOWFAX™ 2A1 Solution Surfactant in the dose formulation after 4 hours were found to be 100 % and 97.5 % of the 0 hour concentrations of dose level T1 (1593.75 μg/mL) and T6 (51000 μg/mL), respectively. Therefore, the doses complied with the presence of test item for claimed concentration (± 15 %) of active ingredient. All criteria for a valid study were met as described in the protocol. From the results of this study, under the specified experimental conditions, DOWFAX™ 2A1 Solution Surfactant is concluded to be nonmutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium and Escherichia coli WP2uvrA (pKM101).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: Chromosomal aberration assay
Target gene:
N/a
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
10 week old Male Crl:CD (SD) rat lymphocytes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate prepared from Aroclor 1254 treated (500 mg/kg body weight) male Sprague Dawley rats
Test concentrations with justification for top dose:
4-hour treatment (-S9): 54.7, 164.2, 1478.2 ug/ml
4-hour treatment (+S9): 6.1, 164.2, 492.7 ug/ml
24-hour treatment (-S9): 54.7, 164.2, 492.7 ug/ml
Dose levels selected based oncytotoxicity
Vehicle / solvent:
water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Key result
Species / strain:
lymphocytes: rat
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

pH and Osmolality

The pH and osmolality of treatment medium containing approximately 5120 μg/ml of the test material and medium containing 1% (distilled water) were determined using a Denver Basic pH meter (Denver Instrument Co., Arvada, Colorado) and an OSMETTE A freezing point osmometer (Precision Systems, Inc., Natick, Massachusetts), respectively. There was no appreciable change in either the pH or osmolality at this concentration as compared to the treatment medium with solvent alone (treatment medium with the test material, pH = 7.59, osmolality = 274 mOsm/kg H2O; treatment medium with 1% distilled water , pH = 7.61, osmolality = 265 mOsm/kg H2O).

Assay A1

Cultures were treated with the test material for 4 hours in the absence and presence of S9 activation at concentrations of 0 (vehicle control), 6.1, 18.2, 54.7, 164.2, 492.7, 1478.2, and 4434.6 μg/ml (Tables 1 and 2). Cultures were also treated continuously for 24 hours in the absence of S9 with the above concentrations plus an additional lower concentration of 2.0 μg/ml . Analytically detected concentrations of the test material in the stock solutions (Assay A1) varied from 99.2 to 110.0% of the target and verified that concentrations used for treatment were within the acceptable range.

Short Treatment

In the absence of S9, the cultures displayed excessive toxicity at the highest concentration (i.e., 4434.6 μg/ml) as indicated by the lack of observable mitotic figures. The remaining cultures had relative mitotic indices ranging from 44.2 to 106.3% compared to the vehicle control values (Table 1). In the presence of S9, excessive toxicity was exhibited in the two highest concentrations (i.e., 1478.2 and 4434.6 μg/ml) as indicated by the lack of observable mitotic figures. The remaining cultures had relative mitotic indices ranging from 47.5 to 107.5% as compared to the vehicle control values (Table 2). Based upon these results, cultures treated with targeted concentrations of 0 (vehicle control), 54.7, 164.2, and 1478.2 μg/ml were chosen for the determination of chromosomal aberration frequency and incidence of polyploidy in the absence of S9 and targeted concentrations of 0 (vehicle control), 6.1, 164.2, and 492.7 μg/ml in the presence of S9 activation. Among the cultures treated with the positive control chemicals for 4 hours, 0.5 μg/ml of MMC and 2 μg/ml of CP were selected for evaluation of aberrations in the absence and presence of S9, respectively. There were no significant increases in the incidence of polyploid cells in any of the test material treated cultures as compared to the vehicle control values. In the 4-hour non-activation assay (Table 5), the frequency of cells with aberrations in the vehicle control was 1.0% and the corresponding values at treatment levels of 54.7, 164.2, and 1478.2 μg/ml were 1.3, 0.0, and 0.7%, respectively. In the activation assay, cultures treated with the test material at concentrations of 6.1, 164.2, and 492.7 μg/ml had aberrant cell frequencies of 0.7, 0.7 and 0.0%, respectively, as compared to the vehicle control value of 1.0%. Statistical analyses of these data did not identify significant differences between the vehicle control and any of the treated cultures without or with S9 activation. The frequencies of aberrant cells observed in the test material treated cultures were within the control limits of the laboratory historical vehicle control range (Table 8). Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemicals. Aberrant cell frequencies in MMC (-S9, 4-hour treatment), and CP (+S9, 4-hour treatment) cultures were 28.0% and 26.7%, respectively. All values were within the control limits of the laboratory historical positive control range.

Continuous Treatment

Based upon the negative findings in the 4-hour treatment, slides from the continuous 24-hour treatment were evaluated. Cultures treated continuously for 24 hours in the absence of S9 activation had excessive toxicity at the two highest concentrations (i.e., 1478.2 and 4434.6 μg/ml) as indicated by the lack of observable mitotic figures. The remaining concentrations had relative mitotic indices ranging from 27.5 to 103.3% relative to the vehicle control value. Based upon these results, cultures treated with targeted concentrations of 0 (vehicle control), 54.7, 164.2, and 492.7 μg/ml were chosen for the determination of chromosomal aberration frequencies and incidence of polyploidy. Cultures treated with 0.075 μg/ml MMC were selected to serve as the positive control. There were no significant increases in the incidence of polyploid cells in any of the test material treated cultures as compared to the vehicle control values. The frequency of aberrant cells in the vehicle control was 0.0% and the corresponding values at concentration levels of 54.7, 164.2, and 492.7 μg/ml were 0.3, 0.3, and 0.0%, respectively. There were no statistically significant differences between the test material treated cultures and the vehicle control values and all values were within the control limits of the laboratory historical vehicle control range. A significant increase in the frequency of cells with aberrations was observed in cultures treated with the positive control chemical. Aberrant cell frequency in MMC treated cultures was 5.7%. All values were within the control limits of the laboratory historical positive control range.

Conclusions:
It was concluded that under the experimental conditions used, DOWFAX™ 2A1 Solution Surfactant was negative in this in vitro chromosomal aberration test.
Executive summary:

DOWFAX™ 2A1 Solution Surfactant (A product containing 1,1’-oxybisbenzene tetrapropylene derivatives, sulfonated, sodium salts) was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes. Approximately 48 hours after the initiation of whole blood cultures, cells were treated either in the absence or presence of S9 activation with concentrations ranging from 0 (vehicle control) to 4434.6 μg DOWFAX™ 2A1 solution surfactant per ml of culture medium. The duration of treatment was 4 hours without and with S9 and 24 hours without S9, and the highest concentration tested was based on the 2000 μg/ml assay limit adjusted for purity of the active ingredient. The analytically determined concentrations of DOWFAX™ 2A1 Solution Surfactant in the dose preparations ranged from 99.2 to 110% of the targeted values. Selection of concentrations for the determination of the incidence of chromosomal aberrations was based upon the acceptable limit of cytotoxicity as defined in the guideline. In this study cultures treated for 4 hours with targeted concentrations of 0 (vehicle control), 54.7, 164.2, and 1478.2 μg/ml in the absence of S9 and 0 (vehicle control), 6.1, 164.2, and 492.7 μg/ml in the presence of S9 and cultures treated for 24 hours with targeted concentrations of 0 (vehicle control), 54.7, 164.2, and 492.7 μg/ml were analyzed. There were no significant increases in the frequency of cells with aberrations administered DOWFAX™ 2A1 solution surfactant in either the absence or presence of S9 activation. Cultures treated with the positive control chemicals (i.e., mitomycin C without S9 and cyclophosphamide with S9) had significantly higher incidences of aberrant cells. Based upon these results, DOWFAX™ 2A1 solution surfactant was considered to be negative in this in vitro chromosomal aberration assay utilizing rat lymphocytes.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: CHO/HGPRT forward mutation assay
Target gene:
Hypoxanthine-guanine-phosphoribosyl transferase (Hgprt)
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, Tennessee, was used in this study. The CHO-K1-BH4
cell line was selected as the test system for this study because it is sensitive to mutagens,
has a low background mutant frequency, and is readily available.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
without S9: 75, 150, 200, 225, 250, 300 ug/ml
with S9: 400, 500, 600, 700, 800, 900 ug/ml
Top dose levls with and without metabolic activation determined by cytotoxicity
Vehicle / solvent:
distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: 20-methylcholanthrene (20-MCA, CAS No. 56-49-5)
Key result
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:
not applicable
Positive controls validity:
valid

pH and Osmolality

The pH and osmolality of treatment medium containing approximately 5120.0 μg/ml of the test material and medium containing 1% distilled water were determined using a Denver Basic pH meter (Denver Instrument Co., Arvada, Colorado) and an OSMETTE A freezing point osmometer (Precision Systems, Inc., Natick, Massachusetts). Alterations in the pH and osmolality of the culture medium have been shown to induce false positive responses in in vitro genotoxicity assays (Thilagar et al., 1984; Galloway et al., 1985; Cifone, 1985). There was no appreciable change in either the pH or osmolality at this concentration as compared to the treatment medium with solvent alone (treatment medium with the test material, pH = 7.49, osmolality = 322 mOsm/kgH2O; treatment medium with 1% distilled water, pH = 7.45, osmolality = 314 mOsm/kgH2O).

Assay A1 – Preliminary Toxicity Assay

In the preliminary toxicity assay, the test material was tested at concentrations of 0 (vehicle control), 17.3, 34.6, 69.3, 138.6, 277.2, 554.3, 1108.7, 2217.3, and 4434.6 μg/ml in the absence and presence of an externally supplied metabolic activation system (S9). The highest concentration tested was based upon 2000 μg/ml assay limit, adjusted for purity of the active ingredient in the test material. The cultures treated at the four highest target concentrations (i.e., 554.3, 1108.7, 2217.3, and 4434.6 μg/ml) without S9 activation had no cells surviving after treatment. The surviving cells in the remaining cultures had relative cell survival (RCS) values ranging from 88.7 to 110.7%. In the presence of S9 activation, no cells were detected at target concentrations of 1108.7, 2217.3, and 4434.6 μg/ml following treatment. The surviving cells in the remaining cultures displayed RCS values ranging from 53.2 to 108.9%.

Based upon the unique toxicity profile of the test material, the adjusted RCS (i.e., RCS adjusted by loss of cells during treatment) was examined in cultures treated in the absence and presence of S9. In the absence of S9, cultures exhibited excessively low adjusted RCS (i.e., < 10%) at the five highest target concentrations (i.e., 277.2, 554.3, 1108.7, 2217.3, and 4434.6 μg/ml). The remaining cultures had adjusted RCS values ranging from 55.6 to 112.3%. In the presence of S9, excessive toxicity was observed at the three highest target concentrations (i.e., 1108.7, 2217.3, and 4434.6 μg/ml). The remaining cultures displayed adjusted RCS values ranging from 43.0 to 92.2%. Based upon the results of this assay, target concentrations of 0 (vehicle control), 75, 150, 300, 400, 450, 500, and 600 μg/ml of the test material were selected for the initial gene mutation assay in the absence of S9 and 0 (vehicle control), 150, 400, 500, 600, 700, 800, and 1100 μg/ml in the presence of S9.

Assay B1 – Initial Mutagenicity Assay

In the initial mutagenicity assay (Assay B1), in the absence of S9, no cells were detected in cultures at the three highest target concentrations (i.e., 450, 500, and 600 μg/ml) following treatment. The surviving cells in the cultures at the remaining target concentrations had RCS values ranging from 55.5 to 107.5%. Excessive cell loss was observed at target concentrations of 300 and 400 μg/ml. In the presence of S9, cultures treated with the highest target concentration (i.e., 1100 μg/ml) exhibited no cells following treatment. The surviving cells in the remaining cultures had RCS values ranging from 62.1 to 114.6%.

Based upon the unique toxicity profile of the test material, the adjusted RCS values were examined in cultures treated in the absence and presence of S9. In the absence of S9, cultures at concentrations ranging from 300 to 600 μg/ml exhibited excessively low adjusted RCS values and the remaining cultures displayed adjusted RCS values ranging from 55.9 to 91.3%. In the presence of S9, cultures at the highest concentration (i.e., 1100 μg/ml) exhibited an excessively low adjusted RCS and the remaining cultures at concentrations ranging from 150 to 800 μg/ml exhibited adjusted RCS values ranging from 11.2 to 113.7%. The top five concentrations (i.e., 300, 400, 450, 500, and 600 μg/ml) in the absence of S9 and the highest concentration (i.e., 1100 μg/ml) in the presence of S9 were unable to be evaluated for phenotypic expression and mutation frequency due to excessive toxicity following treatment. The mutant frequencies observed in cultures treated with the test material in the absence and presence of S9 at the remaining concentration levels were not significantly different from the concurrent vehicle control values. Mutant frequencies of the vehicle controls were within the control limits of the laboratory historical vehicle control range.

Based upon the results of the initial mutation assay (Assay B1), the guideline required level of cytotoxicity (i.e., 80 – 90% cytotoxicity) was not achieved in the absence of S9; therefore, this portion of the assay was repeated in a separate assay. In the presence of S9, the required level of cytotoxicity was achieved at the target concentration of 800 μg/ml, therefore a confirmatory assay was conducted in a separate assay to verify these results.

Assay C1 – Repeat/Confirmatory Mutagenicity Assay

In the repeat/confirmatory assay (Assay C1), the concentrations ranged from 75 to 300 μg/ml in the absence of S9 and 400 to 1100 μg/ml in the presence of S9. In the absence of S9, excessive cell loss was observed at 300 μg/ml and the surviving cells in cultures treated with target concentrations ranging from 75 to 300 μg/ml had RCS values ranging from 64.2 to 106.0%. In the presence of S9, no cells were observed at the highest concentration (i.e., 1100 μg/ml) and excessive cell loss was observed at 800 μg/ml. The surviving cells in cultures treated at target concentrations ranging from 400 to 800 μg/ml had RCS values ranging from 83.1 to 131.8%. Based upon the unique toxicity profile of the test material, the adjusted RCS values were examined in cultures treated in the absence and presence of S9 In the absence of S9, cultures exhibited excessively low adjusted RCS at the target concentration of 300 μg/ml and the remaining cultures displayed adjusted RCS values ranging from 17.6 to 81.7%. In the presence of S9, cultures treated with the two highest target concentrations (i.e., 800 and 1100 μg/ml) exhibited excessively low adjusted RCS values and the remaining cultures displayed adjusted RCS values ranging from 17.5 to 117.6%. The highest target concentration (i.e., 300 μg/ml) in the absence of S9 and the highest two target concentrations (i.e., 800 and 1100 μg/ml) in the presence of S9 were not evaluated for phenotypic expression and mutation frequency as a result of the excessive toxicity of the test material following treatment. The mutant frequencies observed in cultures treated with the test material in the absence of S9 and presence of S9 were not significantly different from the concurrent vehicle control values. Mutant frequencies of the vehicle controls were within the control limits of the laboratory historical vehicle control range. Based upon the results of the repeat/confirmatory mutation assay (Assay C1), a separate assay was conducted to confirm the results in the absence and presence of S9.

Assay D1 – Confirmatory Mutagenicity Assay

In the confirmatory mutagenicity assay, concentrations ranged from 75 to 300 μg/ml in the absence of S9 and 400 to 900 μg/ml in the presence of S9. In the absence of S9, excessive cell loss was observed at a target concentration of 300 μg/ml and the surviving cells treated at target concentrations ranging from 75 to 300 μg/ml had RCS values ranging from 58.3 to 112.9%. In the presence of S9, excessive cell loss was observed at target concentrations of 800 and 900 μg/ml and the surviving cells in cultures treated at target concentrations ranging from 400 to 900 μg/ml had RCS values ranging from 44.1 to 113.2%. Based upon the unique toxicity profile of the test material, the adjusted RCS values were examined in cultures treated in the absence and presence of S9. In the absence of S9, cultures treated with the highest target concentration 300 μg/ml exhibited excessively low adjusted RCS and the remaining cultures displayed adjusted RCS values ranging from 19.3 to 90.0%. In the presence of S9, the cultures treated with the two highest target concentrations (i.e., 800 and 900 μg/ml) exhibited excessively low adjusted RCS values and the remaining cultures displayed adjusted RCS values ranging from 14.3 to 98.2%. The highest target concentration (i.e., 300 μg/ml) in the absence of S9 and the two highest target concentrations (i.e., 800 and 900 μg/ml) in the presence of S9 were not evaluated for phenotypic expression and mutation frequency as a result of the excessive toxicity of the test material following treatment. The mutant frequencies observed in cultures treated with the test material in the absence of S9 and presence of S9 were not significantly different from the concurrent vehicle control values. Mutant frequencies of the vehicle controls were within the control limits of the laboratory historical vehicle control range. In both the initial and confirmatory mutagenicity assays, the positive control chemicals induced significant increases in mutation frequencies and this data confirmed the adequacy of the experimental conditions for detecting induced mutations. The mutant frequencies exhibited by the positive control chemicals in each individual assay were either within or exceeded control limits of the laboratory historical positive control range, which had no impact on the integrity of the study. The analytically observed concentrations of the test material in the stock dosing solutions in Assay B1 ranged from 95.5 to 105.0% of target and verified that concentrations used for treatment were within acceptable range.

Conclusions:
It was concluded that under the experimental conditions used, DOWFAX™ 2A1 Solution Surfactant was negative in this in vitro CHO/HGPRT forward gene mutation assay.
Executive summary:

DOWFAX™ 2A1 Solution Surfactant (A product containing 1,1’-oxybisbenzene tetrapropylene derivatives, sulfonated, sodium salts) was evaluated in the in vitro Chinese Hamster Ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in three independent assays in the absence and presence of an externally supplied metabolic activation system (S9). The concentrations ranged from 75 to 1100 μg/ml in the absence and presence of S9. The highest concentration was based on the cytotoxicity of the test material. The analytically determined concentrations of DOWFAX™ 2A1 Solution Surfactant in the dose preparations ranged from 95.5 to 103.0%. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assays in the absence of S9 and 20-methylcholanthrene for assays in the presence of S9. Vehicle control cultures were treated with the solvent used to dissolve the test material (i.e. distilled water). There were no statistically significant treatment-related increases in the mutant frequency in the test material-treated cultures compared to the vehicle control cultures in either the absence or presence of S9. Cultures treated with the positive control chemicals had significantly higher mutant frequencies. Based upon these results, DOWFAX™ 2A1 Solution Surfactant was considered to be negative in this in vitro CHO/HGPRT forward gene mutation assay.

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

Genetic toxicity in vivo

Description of key information

No in vivo data are available for the REACH substance itself; however, DOWFAX 2A1 was negative in three in vitro genetic toxicity assays. Moreover, 4 in vitro and 1 in vivo studies are available for a structural analogue of the registered substance from the ADPODS category (DOWFAX 8390) that clearly demonstrate negative genotoxic potential of this type of chemistry. These two category members differ in structure and length of their alkyl side chains. This structural difference would not affect/change the overall genotoxic potential. See justification document for further justification. Thus, DOWFAX 2A1 is not considered to be a genotoxicant.

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:
read-across based on grouping of substances (category approach)
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
Justification for type of information:
Please see read across justification document.
Reason / purpose:
reference to same study
Reason / purpose:
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
Remarks:
pre-GLP
Type of assay:
other: cytogenetic effects on rat bone marrow cells
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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 8390 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.

None

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 DOWFAX 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 DOWFAX XD-8390 calculated to provide dosages of 0, 50, 100, 200 or 600 mg/kg bw 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 inconsequential because of historical control data in similar studies with this strain of rat, where 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 bw/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:

All 4 in vitro studies (bacterial mutation, mammalian mutation and 2 chromosome aberration studies) and the in vivo micronucleus assay were clearly negative indicating the lack of genotoxic potential for the structural ADPODS analogues differing only in the alkyl side chain structure and length (refer to full read-across ADPODS category justification document). Genotoxicity is not typically associated with surfactant compounds and, together with the negative results obtained with its structural analogue, it is not concluded likely that Dowfax 2A1 would be a genotoxicant. This conclusion can be further reinforced by considering the lack of carcinogenicity observed in the two carcinogenicity studies available for this substance.


Justification for selection of genetic toxicity endpoint
in vivo study with the ADPODS category member containing sufficient information for interpretation of study results and genotoxicity assessment.

Justification for classification or non-classification

No classification required.