Reaction mass of 2,4-bis(xylylazo)resorcinol and 2,4-bis[(4-dodecylphenyl)azo]resorcinol and 2-[(4-dodecylphenyl)azo]-4-(2,4-xylylazo)resorcinol

Genetic toxicity: in vitro

Administrative data

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:

6-28-1989 to 7-31-1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD guideline study performed in accordance with GLP; exact details of test material (certificate of analysis, Characterisation) are not included in the report. E.Coli was not tested in this study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2,4-bis(xylylazo)resorcinol and 2,4-bis[(4-dodecylphenyl)azo]resorcinol and 2-[(4-dodecylphenyl)azo]-4-(2,4-xylylazo)resorcinol

EC Number:

915-586-1

Molecular formula:

Variable; substance is a UVCB.

IUPAC Name:

Reaction mass of 2,4-bis(xylylazo)resorcinol and 2,4-bis[(4-dodecylphenyl)azo]resorcinol and 2-[(4-dodecylphenyl)azo]-4-(2,4-xylylazo)resorcinol

Test material form:

other: Liquid

Details on test material:

The test article Automate Yellow 8, Lot #7146-266, was received by Pharmakon Research on June 26, 1989 in a metal container and was described as an orange liquid. Normal precautions were used in handling the test article. Information regarding technical aspects of the test article, as provided by the sponsor, was recorded in the sponsor's file. For the purposes of this study, the test article was stored at room temperature in the container received from the sponsor. At the time of testing the test article was described as an orange liquid. There was no apparent change in the physical state of the test article during the assay. All required dilutions were made with acetone, Lot #A03611, supplied by J. T. Baker Chemical Company (Phillipsburg, NJ). Dilutions were prepared the day of the test and used within two hours of preparation.

Method

Target gene:

Histidine loci

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

The S9 mixture included 30% (v/v) un-induced male Syrian Golden hamster liver homogenate with the appropriate buffer and cofactors.

Test concentrations with justification for top dose:

Automate Yellow 8 was evaluated in triplicate cultures in strains TA1535, TA1537, TA1538, TA98 and TA100 in the presence and absence of S9 at doses of 1.67, 5.00, 16.7, 50.0, 167 and 500 ug/plate.
Based on the results in the first test, a retest was done with TA1537 using dose groups; 0.05, 0.167, 0.5, 1.67, 5.0, 16.7, 50.0, 167 and 500 ug/plate with S9.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone, Lot #A03611, supplied by J. T. Baker Chemical Company (Phillipsburg, NJ).

Controls

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

congo red

other: 2-Anthramine, benzidine

Details on test system and experimental conditions:

Test Organism/Strains:
Salmonella typhimurium - strains TA1535, TA1537, TA1538, TA98 and TA100

Source:
Dr. Bruce N., Ames Biochemistry Department University of California Berkeley, CA 94720

All strains contain a uvrB deletion mutation (affecting DNA excision repair), as well as a rfa mutation (affecting membrane permeability). In addition, strains TA98 and TA100 contain the plasmid pKM101, which enhances the error-prone DNA repair system normally present in this organism. Strains TA1535 and TA100 detect base pair substitution mutations affecting the hisG locus. In contrast, strains TA1538 and TA98 detect frameshift mutations-affecting the hisD locus, while TA1537 detects frameshift mutations at the hisC locus. All tester strains were checked for the presence of the appropriate genetic markers on approximately a monthly basis.

Test Cultures:
Fresh cultures for mutagenesis testing were prepared by quickly thawing a vial of frozen working stock cultures of each tester strain and transferring the culture to 25 ml of Oxoid Nutrient Broth #2. After growth for approximately 10 hours at 37°C in an orbital shaking incubator, samples of each culture were diluted 1:4 in distilled water and optical densities were determined at 650 nm. Cultures with optical densities of 0.40 to 0.60 (approximately 1-2 x 10^9 cells/ml; representative of cells in late exponential or early stationary phase) were utilized for this study.

Control Articles:
Triplicate cultures of each strain were evaluated with the appropriate solvent in the presence and absence of S9 to serve as negative solvent controls. In order to validate the integrity of the test system, triplicate cultures of each tester strain were also evaluated with known positive control chemicals.
Positive controls evaluated in the absence of S9 were specific for each strain and included: TA1535 and TA100 - sodium azide (10.0 ug/plate; Sigma Chemical Company, Lot #56C-0263); TA1537 - 9-aminoacridine (150 ug/plate; Aldrich Chemical Company, Lot #030567); and TA1538 and TA98 - 2-nitrofluorene (5.00 ug/plate; Aldrich Chemical Company, Lot #2610PE). 2-Anthramine (2.50 ug/plate; Sigma Chemical Company, Lot #33F-0816) was evaluated in strains TA1535, TA1537 and TA100 in the presence of S9, while Congo Red (200 ug/plate; Sigma Chemical Company, Lot #123F-6116) and benzidine (40.0 ug/plate; Sigma Chemical Company, Lot #18C-0024) were evaluated in strains TA1538 and TA98 in the presence of S9.

Mutation Assay:
Salmonella which have undergone reversion to his+ form colonies in the absence of histidine. In contrast, his- Salmonella can only undergo a limited number of doublings (due to the histidine supplement in the top agar) and form the typical background lawn. Following incubation for 48 hours, revertant colonies are enumerated with an automated colony counter. All mutation assays are performed in triplicate cultures in all five tester strains for each test article dose, as well as positive and solvent controls. Automate Yellow 8 was evaluated at doses of 1.67, 5.00, 16.7, 50.0, 167 and 500 ug/plate in the presence and absence of S9. Six dose levels of Automate Yellow 8 were evaluated with and without S9 in the event of unacceptably high toxicity at the highest dose levels. However, statistically significant, dose-dependent increases in revertant frequencies, to approximately 1.8-fold control values, were observed in strain TA1537 with S9. Therefore, Automate Yellow 8 was re-evaluated in strain TA1537 at doses of 0.0500, 0.167, 0.500, 1.67, 5.00, 16.7, 50.0, 167 and 500 ug/plate with S9.

Treatment with Test and Control Articles:
Treatment for the mutation assay was performed exactly as described in the toxicity prescreen, except that the test and control articles were evaluated in triplicate cultures in all five strains in the presence and absence of an exogenous metabolic activation system (S9), and 0.1 ml dose volumes were used for the positive controls. For cultures treated in the presence of S9, 0.5 ml of the S9 mixture replaced the 0.5 ml phosphate buffer described for the toxicity prescreen. The S9 mixture contained 8mM MgCl2, 33mM KCl, 4mM NADP, 20mM glucose-6-phosphate, 2.8 U/m1 G6PDH, 2mM NADH, 2mM FMN, 100mM Na2 HPO4 (pH 7.4) and 30% (v/v) un-induced male Syrian Golden hamster liver homogenate.

Bacterial Contaminant Evaluation:
To ensure the sterility of solvents, compounds and equipment, standard contamination evaluations were performed with the assay. The solvent, top agar, S9 mix, and top dose of the test article were evaluated at the same volumes used in the assay. The test article, solvent and S9 mix were evaluated as in the mutation assay, but in the absence of added Salmonella. Top agar alone was also plated on minimal glucose plates. All plating was done in triplicate. Plates were incubated for 48 hours at 37°C and then scored for bacterial growth.

Positive Control preparation:
The following solvents were used for each of the positive controls:
Sodium Azide: dH2O
9-aminoacridine: DMSO
2-nitrofluorene: DMSO
Congo red: DMSO
2-Anthramine: DMSO
Benzidine: DMSO

Evaluation criteria:

Scoring:
Revertant colonies were enumerated on an Artek electronic colony counter interfaced with an IBM PC/XT computer for data acquisition. Solvent and positive controls were scored first, and test article treated cultures were scored only if the average negative control values were within historical ranges.

Evaluation Criteria:
A positive result is defined as a statistically significant, dose-dependent increase in the number of histidine-independent revertants with at least one dose level inducing a revertant frequency that is two-fold the spontaneous solvent control value. Statistical analyses were performed using the program developed by Snee and Irr (1981), with significance established at the 95% confidence limit. If the test article does not induce a statistically significant, dose-dependent increase in revertant frequency but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value, the result is considered equivocal. A negative result is defined as the absence of a statistically significant or dose-dependent increase in the number of histidine-independent revertants.

Statistics:

A positive result is defined as a statistically significant, dose-dependent increase in the number of histidine-independent revertants with at least one dose level inducing a revertant frequency that is two-fold the spontaneous solvent control value. Statistical analyses were performed using the program developed by Snee and Irr (1981), with significance established at the 95% confidence limit. If the test article does not induce a statistically significant, dose-dependent increase in revertant frequency but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value, the result is considered equivocal. A negative result is defined as the absence of a statistically significant or dose-dependent increase in the number of histidine-independent revertants.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

All dose groups tested.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed in 167 and 500 ug/plate dose groups

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

All dose groups tested.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed in 167 and 500 ug/plate dose groups

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

All dose groups tested.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

All dose groups tested.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

All dose groups tested.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

All dose groups tested.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Presecreen Toxicity Results:
Automate Yellow 8 was evaluated in a toxicity prescreen by treating duplicate cultures of strains TA1538 and TA100 with five doses of Automate Yellow 8 in the absence of S9. Results of the prescreen indicated Automate Yellow 8 was not toxic to either strain at a dose of 50.0 ug/plate. Inhibited growth (characterized by a reduced background lawn and/or the presence of pindot colonies) was observed in both strains at a dose of 167 ug/plate and in strain TA100 at a dose of 500 ug/plate. Complete toxicity was observed in strain TA1538 at a dose of 500 ug/plate and in both strains at doses of 1670 and 5000 ug/plate. In addition, the test article precipitated from solution/formed oily droplets at all doses.

Applicant's summary and conclusion

Conclusions:

Negative with metabolic activation All dose levels.
Negative without metabolic activation All dose levels.

The results for Automate Yellow 8 were negative in the Prival modification of the Ames/Salmonella Liquid Pre-incubation Assay under the conditions, and according to the criteria, of the test protocol.

Executive summary:

Automate Yellow 8 was evaluated in the Prival modification of the Ames/Salmonella Liquid pre-incubation Assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of Salmonella typhimuriurn in the presence and absence of an exogenous metabolic activation system (S9). Toxicity of Automate Yellow 8 was first evaluated in a prescreen by treating duplicate cultures of strains TA1538 and TA100 with five doses of Automate Yellow 8 in the absence of S9. Results of the prescreen indicated Automate Yellow 8 was not toxic to either strain at a dose of 50.0 ug/plate. Inhibited growth (characterized by a reduced background lawn and/or the presence of pindot colonies) was observed in both strains at a dose of 167 ug/plate and in strain TA100 at a dose of 500 ug/plate. Complete toxicity was observed in strain TA1538 at a dose of 500 ug/plate and in both strains at doses of 1670 and 5000 ug/plate. In addition, the test article precipitated from solution/formed oily droplets at all doses. Based upon these findings, Automate Yellow 8 was evaluated in triplicate cultures in strains TA1535, TA1537, TA1538, TA98 and TA100 in the presence and absence of S9 at doses of 1.67, 5.00, 16.7, 50.0, 167 and 500 ug/plate. Six dose levels of Automate Yellow 8 were evaluated with and without S9 in the event of unacceptably high toxicity at the highest dose levels. The S9 mixture included 30% (v/v) un-induced male Syrian Golden hamster liver homogenate with the appropriate buffer and cofactors. The test article was found to be incompletely soluble at doses of greater than or equal to 50.0 ug/plate. Inhibited growth was observed in all tester strains at doses of 167 and 500 ug/plate without S9. Revertant frequencies for all doses of Automate Yellow 8 in all strains without S9, and in strains TA1535, TA1538, TA98 and TA100 with S9, approximated or were less than those observed in the concurrent solvent control cultures. In contrast, statistically significant and apparently dose-dependent increases in revertant frequencies, to approximately 1.8-fold control values, were observed in strain TA1537 with S9. However, the increases observed were within historical control values, and dose-dependent decreases in revertant frequencies were observed over the dose range of 1.67-500 ug/plate. Therefore, Automate Yellow 8 was re-evaluated in strain TA1537 at doses of 0.0500, 0.167, 0.500, 1.67, 5.00, 16.7, 50.0, 167 and 500 ug/plate with S9. Revertant frequencies for all doses of Automate Yellow 8 in strain TA1537 in the re-test approximated those observed in the concurrent solvent control values. Thus, the slight increases observed in strain TA1537 in the original assay are considered to be statistical abberations due to random fluctuation of the spontaneous revertant frequency. All positive and negative control values in both assays were within acceptable limits. Therefore, the results for Automate Yellow 8 were negative in the Prival modification of the Ames/Salmonella Liquid Pre-incubation Assay under the conditions, and according to the criteria, of the test protocol.