4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

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:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study did not include testing on Escherichia coli.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Principles of method if other than guideline:

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 (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.

TOXICITY PRESCREEN:
Toxicity of the test article was determined in a preliminary toxicity prescreen by evaluating the growth of the background lawn and/or frequency of
spontaneous revertants.

Treatment with Test and Control Articles: Treatments were performed by combining 0.1 ml tester strain, 0.5 ml 0.2M NaH P04 (pH 7.4) and 0.1 ml test article or solvent in sterile glass tubes preheated to 30°C. The tubes were vortexed and incubated at 30°C for 30 minutes. Following the 30-minute incubation, 2 ml top agar (supplemented with 0.5mM histidine/0.5mM biotin) was added, and the mixture was poured onto minimal glucose plates, evenly distributed, and allowed to solidify. Within an hour the plates were inverted and incubated in the dark at 37°C for 48 hours.

Scoring: Following the 48-hour incubation, the background lawn and spontaneous revertants were scored for normal, inhibited or no growth. Inhibited growth was characterized by the absence of a confluent bacterial lawn and/or the presence of pindot colonies.

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.

Treatment with Test and Control Articles: Treatment for the mutation assay was performed exactly as described in the toxicity pre screen, 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). For cultures treated in the presence of S9, 0.5 ml of the S9 mixture replaced the 0.5 ml phosphate buffer. The S9 mixture contained 8mM MgCI , 33mM KCl, 4mM NADP, 20mM glucose-6- phosphate, 2.8 U/ml G6PDH, 2mM NADH, 2mM FMN, 100mM NaH2 Po4 (pH 7.4) and 30% (v/v) uninduced 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.

Scoring: Revertant colonies were enumerated on an Artek electronic colony counter interfaced with an Apple 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 (see below). A summary of the results is presented in the Summary Data Tables (pages 8-10).

Deviations:
Working Cultures:
Original Statement: Fresh cultures for mutagenesis testing are prepared by quick thawing a vial of frozen working stock cultures of each tester strain and transferring the culture to 25 ml of Oxoid Nutrient Broth #2 for tester strains TA1537, TA1538 and TA98 and minimal glucose medium for strains TA1535 and TA100 in 125 ml screw-capped Erlenmeyer flasks.

Corrected Statement: Fresh cultures for mutagenesis testing are prepared by quick thawing a vial of frozen working stock cultures of each tester strain and transferring the culture to 25 ml of Oxoid Nutrient Broth #2.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

MORTRACE SB CONC.

IUPAC Name:

MORTRACE SB CONC.

Constituent 2

Reference substance name:

4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

EC Number:

410-610-2

EC Name:

4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

Cas Number:

111850-24-9

Molecular formula:

C20 H25 N3 O3

IUPAC Name:

2,6-bis(butan-2-yl)-4-[2-(4-nitrophenyl)diazen-1-yl]phenol

Constituent 3

Reference substance name:

4-(4-Nitrophenylazo)-2,6-di- sec.butylphenol

IUPAC Name:

4-(4-Nitrophenylazo)-2,6-di- sec.butylphenol

Constituent 4

Reference substance name:

4-(4-Nitrophenylazo)-2,6-di- sec.butylphenol

IUPAC Name:

4-(4-Nitrophenylazo)-2,6-di- sec.butylphenol

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Marker SB
- Physical state: Liquid
- Storage condition of test material: Room temperature

Method

Target gene:

Specific histidine loci.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not appplicable

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mixture includiing 30% (v/v) uninduced male Syrian Golden hamster liver homogenate with the appropriate buffer and cofactors.

Test concentrations with justification for top dose:

Marker SB was evaluated in triplicate cultures in strains TA1535, TA1537, TA1538, TA98 and TA100 in the presence and absence of S9 at doses of 0.0500, 0.167, 0.500, 1.67, 5.00, 16.7, 50.0 and 167 ug/plate. Based on these results, Marker SB was re-evaluated in all five strains at doses of 1.00, 5.00, 10.0, 25.0, 50.0, 75.0, 100 and 150 ug/plate with S9.

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

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:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Three

Evaluation criteria:

Evaluation Criteria:
A positive result is defined as a reproducible, statistically significant two-fold dose-dependent increase in the number of histidine-independent colonies, with at least one dose point inducing an average revertant frequency that is two-fold that of the solvent control. Significance is determined
using the program developed by Snee and Irr (1981). This program applies a linear regression analysis to the data points and any p value less than 0.05 is considered significant. Alternatively, if the test article produces an increase greater than or equal to three times the solvent control value (in the absence of a dose-dependent increase), the test chemical is considered positive. A negative result is defined as the absence of a dose-dependent two-fold increase in the number of histidine-independent revertants (or three-fold increase in the absence of any dose-dependency).

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

positive

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Marker SB (non-volatile) was evaluated in a toxicity pre screen by treating duplicate cultures of strains TA1538 and TAI00 with five doses of Marker SB in the absence of S9. Results of the pre screen indicated Marker SB produced inhibited growth (characterized by a reduced background lawn and/or the presence of pindot colonies) at doses of 50'.0 and 167 ug/plate in strain TA1538, and at a dose of 50.0 ug/plate in strain TAlOO. Complete toxicity was observed at doses of 500, 1670 and 5000 ug/plate in strain TA1538, and at doses of 167, 500, 1670 and 5000 ug/plate in strain TAlOO. In addition, the test article was found to precipitate from solution at the 1670 and 5000 ug/plate dose levels.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Marker SB was re-evaluated in all five strains at doses of 1.00, 5.00, 10.0, 25.0, 50.0, 75.0, 100 and 150 ug/plate with S9. Similar increases in revertant frequencies of approximately two- to five-fold were again observed in strains TA1538, TA98 and TAl00 in the retest. Although the revertant frequencies generally decreased at the highest dose level in each assay (150 or 167 ug/plate; due to toxicity), the increases observed over the remainder of the dose range were linear. All positive and negative control values in both assays were within acceptable limits.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

The results for Marker SB (non-volatile) were positive 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:

Marker SB (non-volatile) 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 typhimurium in the presence and absence of an exogenous metabolic activation system (S9). Toxicity of Marker SB was first evaluated in a prescreen by treating duplicate cultures of strains TA1538 and TA100 with five doses of Marker SB in the absence of S9. Results of the prescreen indicated Marker SB produced inhibited growth (characterized by a reduced background lawn and/or the presence of pindot colonies) at doses of 50.0 and 167 ug/plate in strain TA1538, and at a dose of 50.0 ug/plate in strain TA100. Complete toxicity was observed at doses of 500, 1670 and 5000 ug/plate in strain TA1538, and at doses of 167, 500, 1670 and 5000 ug/plate in strain TA100. In addition, the test article was found to precipitate from solution at the 1670 and 5000 ug/plate dose levels.

Based upon these findings, Marker SB was evaluated in triplicate cultures in strains TA1535, TA1537, TA1538, TA98 and TA100 in the presence and absence of S9 at doses of 0.0500, 0.167, 0.500, 1.67, 5.00, 16.7, 50.0 and 167 ug/plate. Three extra dose levels of Marker SB 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) uninduced male Syrian Golden hamster liver homogenate with the appropriate buffer and cofactors.

Revertant frequencies for all doses of Marker SB in all strains without S9, and strain TA1535 with S9, approximated or were less than those observed in the concurrent negative control cultures. However, increased revertant frequencies of approximately two- to four-fold were observed in strains TA1537, TA1538, TA98 and TA100 in the presence of S9. Therefore, Marker SB was re-evaluated in all five strains at doses of 1.00, 5.00, 10.0, 25.0, 50.0, 75.0, 100 and 150 ug/plate with S9. Similar increases in revertant frequencies of approximately two- to five-fold were again observed in strains TA1538, TA98 and TA100 in the retest.

Although the revertant frequencies generally decreased at the highest dose levels in each assay (150 or 167 ug/plate; due to toxicity), the increases observed over the remainder of the dose range were linear. All positive and negative control values in both assays were within acceptable limits.

Therefore, the results for Marker SB (non-volatile) were positive in the Prival Modification of the Ames/Salmonella Liquid Pre-incubation Assay under the conditions, and according to the criteria, of the test protocol.