Reaction mass of 1,4-bis(methylamino)anthraquinone and 1,4-bis[(2-ethylhexyl)amino]anthraquinone and 1-[(2-ethylhexyl)amino]-4-(methylamino)anthraquinone and 9,10-Anthracenedione, 1,4-bis(pentylamino)-, branched and linear and 9,10-Anthracenedione, 1-(methylamino)-4-(pentylamino)-, branched and linear and 9,10-Anthracenedione, 1-[(2-ethylhexyl)amino]-4-(pentylamino)-, branched and linear

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In Vitro Chromosomal Aberration Assay Utilizing Rat Lymphocytes

Target gene:

NOt applicable

Species / strain / cell type:

lymphocytes: rat

Details on mammalian cell type (if applicable):

Strain of rats: Crl:CD(SD)
Sex: Male
Age at start of study: 10-16 weeks
Whole blood cultures were set up in complete medium (RPMI 1640 medium with 25 mM HEPES, supplemented with 10% heat-inactivated fetal bovine serum, antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone 0.25 µg/ml), and an additional 2 mM L-glutamine) in addition with 30 - 50 µg/ml PHA-P. Cultures were initiated by inoculating approximately 0.5 ml of whole blood into 5 ml of medium.

Cytokinesis block (if used):

Colcemid (1 microgram/culture)

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate prepared from Aroclor 1254-treated (500 mg/kg body weight) male Sprague Dawley

Test concentrations with justification for top dose:

Assay B1 (4hr treatment with and without metabolic activation, and 24 hours in the absence of metabolic activation:
Doses used were: 0 (vehicle control), 2.8, 5.6, 11.2, 22.3, 44.7, 89.3, and 178.6 µg/ml
Based on precipitation at the top two dose levels and the Mitotic indices scores, the following dose levels were selected for analysis of chromosomal aberration frequency for the 4hr treatment with S9 and the 24 hr treatment without S9: 0 (vehicle control), 2.8, 11.2, and 44.7 µg/ml
a technical error resulted in the 4hr treatment in the absence of s( being repeated in Assay C1.
Assay C1: 4hr treatment in the absence of s9 (repeat of this part of B1)
Doses used were: 0 (vehicle control), 2.8, 5.6, 11.2, 22.3, 44.7, 89.3, and 178.6 µg/ml
Dose levels of 0 (vehicle control), 22.3, 89.3, and 178.6 µg/ml were chosen for the determination of chromosomal aberration frequency and incidence of polyploidy in the absence of S9 activation.

Vehicle / solvent:

Ethanol (CAS No.64-17-5)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol (CAS No.64-17-5)

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

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), 5.6, 11.2, 22.3, 44.7, 89.3, 178.6, and 357.2 µg/ml. Cultures were also treated continuously for 24 hour in the absence of S9 with the above concentrations plus and additional lower concentration of 2.8mg/ml. The test material precipitated in the treatment medium at the top two concentrations (i.e., 178.6 and 357.2mg/ml in all treatment conditions as, observed at the end of treatment. Due to poor lymphocyte growth the slides in all conditions were not evaluated for mitotic indices or chromosome aberrations. As a result all conditions of this assay were repeated in a separate assay (Assay B1). Analytically detected concentrations of the test material in the stock solutions (Assay A1) varied from 105.0 to 142.0% of the target and verified that concentrations used for treatment were within the acceptable range. 

Assay B1

Cultures were treated with the test material for 4 hours in the absence and presence of S9 activation at concentrations of 0 (vehicle control), 2.8, 5.6, 11.2, 22.3, 44.7, 89.3, and 178.6 µg/ml. Cultures were also treated continuously for 24 hours in the absence of S9 with the above concentrations plus an additional lower concentration of 1.4mg/ml. The test material precipitated in the treatment medium at the top concentration (i.e., 178.6 µg/ml) in all treatment conditions, as observed at the end of treatment. 

Short Treatment

In the absence of S9, the cultures displayed no toxicity with relative mitotic indices ranging from 86.2 to 120.0% compared to the vehicle control values. However, due to a technical error in the absence of S9 the vehicle controls were unanalyzable for chromosomal aberrations and therefore, this portion of the assay was repeated in an independent assay. In the presence of S9, the mitotic indices of the treated cultures ranged from 17.1 to 75.0% as compared to the vehicle control values. Based upon these results, cultures treated with targeted concentrations of 0 (vehicle control), 2.8, 11.2, and 44.7 µg/ml were chosen for the determination of chromosomal aberration frequency and incidence of polyploidy in the presence of S9 activation. 

Among the cultures treated with the positive control chemicals for 4 hours, 2 µg/ml of CP were selected for evaluation of aberrations in the presence of S9.

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 activation assay, cultures treated with the test material at concentrations of 2.8, 11.2, and 44.7 µg/ml had aberrant cell frequencies of 0.7, 2.7 and 3.0%, 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.

Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemical. Aberrant cell frequencies in CP (+S9, 4-hour treatment) cultures were 31.3%. 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 severe toxicity with relative mitotic indices ranging from 0.9 to 92.7% relative to the vehicle control value. Based upon these results, cultures treated with targeted concentrations of 0 (vehicle control), 2.8, 11.2, and 44.7mg/ml were chosen for the determination of chromosomal aberration frequencies and incidence of polyploidy. Cultures treated with 0.075mg/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 0.3% and the corresponding values at concentration levels of 2.8, 11.2, and 44.7mg/ml were 0.0, 0.0, and 0.8%, respectively. As a result of the decreased relative mitotic index and limited number of mitotic figures present on the slides only 259 metaphase spreads were evaluated from the 44.7 µg/ml concentration and 272 metaphase spreads from the 0.075 µg/ml MMC concentration instead of the standard 300. This minor variation was deemed to be inconsequential to the interpretation of the study. 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 11.8%. All values were within the control limits of the laboratory historical positive control range.

Assay C1

Cultures were treated with the test material for 4 hours in the absence of S9 activation at concentrations of 0 (vehicle control), 2.8, 5.6, 11.2, 22.3, 44.7, 89.3, and 178.6 µg/ml. The test material precipitated in the treatment medium at the top concentration (i.e., 178.6 µg/ml), as observed at the end of treatment. 

Short Treatment

In the absence of S9, the cultures displayed no toxicity with relative mitotic indices ranging from 71.4 to 89.8% compared to the vehicle control values. Based upon these results, cultures treated with targeted concentrations of 0 (vehicle control), 22.3, 89.3, and 178.6 µg/ml were chosen for the determination of chromosomal aberration frequency and incidence of polyploidy in the absence of S9 activation.

Among the cultures treated with the positive control chemicals for 4 hours, 0.5 µg/ml of MMC was selected for evaluation of aberrations in the absence of S9.

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 non-activation assay, cultures treated with the test material at concentrations of 22.3, 89.3, and 178.6 µg/ml had aberrant cell frequencies of 0.0, 0.0 and 0.0%, respectively as compared to the vehicle control value of 1.3%. 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.

Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemical. Aberrant cell frequencies in MMC (-S9, 4-hour treatment) cultures were 27.3%. All values were within the control limits of the laboratory historical positive control range.

Conclusions:

It was concluded that under the experimental conditions used, C.I. Solvent Blue 98 (3 Amine) was negative in this in vitro chromosomal aberration test.

Executive summary:

C.I. Solvent Blue 98 (3 Amine) (1,4-Diamino-9,10-anthracenedione N,N’-mixed 2-ethylhexyl and methyl and pentyl derivatives) was evaluated in anin 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 178.6mg C.I. Solvent Blue 98 (3 Amine) per ml of culture medium. The highest concentration was based on the limit of solubility of the test material in the treatment medium. The analytically determined concentrations of C.I. Solvent Blue 98 (3 Amine) in the dose preparations ranged from 105.0 to 142.0% of the targeted values. The duration of treatment was 4 hours without and with S9 and 24 hours without S9.  Selection of concentrations for the determination of the incidence of chromosomal aberrations was based upon cytotoxicity and the solubility of the test material. In this study cultures treated for 4 hours with targeted concentrations of 0 (vehicle control), 22.3, 89.3, and 178.6 mg/ml in the absence of S9 and 0 (vehicle control), 2.8, 11.2, and 44.7 mg/ml in the presence of S9 and cultures treated for 24 hours with 0 (vehicle control), 2.8, 11.2, and 44.7mg/ml were analyzed.

There were no significant increases in the frequency of cells with aberrations administered C.I. Solvent Blue 98 (3 Amine) 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, C.I. Solvent Blue 98 (3 Amine) was considered to be negative in this in vitro chromosomal aberration assayutilizing rat lymphocytes.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

9-21-1983 to 10-7-1983

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 the experiment. OECD guideline followed was not specified.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

E. coli not tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine loci

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 fraction

Test concentrations with justification for top dose:

50, 166.6, 500, 1666.6 and 5000 ug/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine

Details on test system and experimental conditions:

Rationale for Test System:
Chemicals capable of inducing mutations have been shown to increase the reversion frequency at the histidine locus in selected tester strains of Salmonella typhimurium with and without the addition of a metabolic activation system.

Test organism storage and maintenance:
Frozen working stock cultures were prepared by scraping a wooden applicator stick over the surface of frozen Master cultures and inoculating the scrapings into 50 ml of Oxoid Broth #2 and grown for approximately 16 hours at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker. Following the 16 hour growth period, 1 ml aliquots of the culture was dispensed into Nunc vials marked with the particular strain and quick frozen in an ethanol-dry ice bath before being stored at a minimum of -60°C.
In order to avoid the effect of surface thawing and re-freezing of frozen permanent vials of bacterial stock, frozen working stock cultures are employed as a source of inoculum for mutagenesis testing.
Fresh cultures for mutagenesis testing were prepared by quick thawing a vial of frozen working stock cultures of each tester strain and transferring the culture to 50 ml of Oxoid Nutrient Broth #2 and grown for approximately 16 hours (1-2 x 10E9 cells/ml) at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker. After the 16 hour incubation, samples of culture suspensions were diluted 1:4 in distilled water and optical densities were observed at 650 nm using a Beckman Model 35 Spectrophotometer.
Historical data has shown that optical densities of 0.400 or greater are representative of cells in late exponential or early stationary phase of growth. Tester strains were checked concurrently for the presence of the appropriate genetic markers at the time of the assay.

Top Agar:
Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5 mM histidine and 0.5 mM biotin at a volume of 0.1 ml per ml of agar, and maintained at 45°C until used. Sterile tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath at 45°C. All negative and positive control tubes and plates were done in triplicate. All compound-treated tubes and plates were done in triplicate. Using sterile technique, the following were added to each tube in the following order: 2 ml aliquots of top agar solution, 0.1 ml of tester strain, and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden.

Metabolic Activation System:
Tubes requiring metabolic activation have, in addition to the preceding top agar ingredients, an S-9 fraction of rat liver homogenate obtained from Aroclor 1254-treated Sprague Dawley rats. The activation system (S-9 mix) contained the following per ml:
0.4 M MgC12; 1.65 M KCI 20 ul
1 M Glucose - 6 - Phosphate 5 ul
0.1 M NADP 40 ul
0.2 M Phosphate buffer pH 7.4 500 ul
Sterile distilled H2O 355 ul
S-9 Fraction 80 ul
The S-9 fraction was thawed on the day of use and kept cold on ice. 0.5 ml of the S-9 mix was added to the tubes which were then vortexed and poured onto minimal glucose plates. The plates were allowed to harden for several minutes. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for 48 - 72 hours, checked for uniform background lawn, and scored by counting revertant colonies on an electronic colony counter interfaced with a computer.

Bacterial Contaminant Control:
To insure the quality of aseptic technique during the assay and also the sterility of solvents, compounds and equipment, standard contamination checks were conducted with the assay. These contamination checks included the top level of test substance, solvent, top agar and S-9 mix at the same volumes as in the assay. The test substance or S-9 mix were added to 2 ml of molten top agar supplemented with 0.5mM histidine - 0.5mM biotin and poured onto minimal glucose plates. 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 (± 2°C) and then scored for contamination.

Test Article Purity:
The identity, purity, quality, and strength of the test article is the responsibility of the sponsor.

Test Article Stability:
Test article Automate Blue 8, Lot #1658-79, produced precipitates when added to an aqueous top agar solution at all levels tested. There was no apparent change in the physical state of the control articles during the assay.

Evaluation criteria:

Data Reporting:
In scoring the assay, the positive and negative controls were first evaluated. If the negative control values did not fall within the acceptable historical mean values, the remaining plates were not scored and the assay was repeated. A summary of the data are presented in the summary data sheet contained·in this report.

Evaluation Criteria:
In most tests with the Salmonella/Microsome Assay, results are either clearly positive or clearly negative. A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within one standard deviation of the historical mean (See Historical Data) for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical is considered positive. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Statistics:

A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within one standard deviation of the historical mean (See Historical Data) for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical is considered positive. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

50 and 5000 ug/plate

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

50, 166.6, 1666.6 and 5000 ug/plate

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

166.6, 500 and 1666.6 ug/plate

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

500 ug/plate

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Screen:
The preliminary toxicity screen for the Ames Assay used two of the histidine auxotrophs of Salmonella typhimurium TA1538 and TA100 at dose levels of 100, 333, 1000, 3333 and 10,000 ug/plate. The preliminary toxicity screen was designed to determine at which levels the compound exhibits toxic effects to the Salmonella typhimurium tester strains. The test compound was prepared to a concentration of 100 mg/ml. Logarithmic dilutions of this stock solution were made in the appropriate solvent to give the following concentrations: 1.0, 3.33, 10.0 and 33.33 mg/ml. Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5mM histidine - 0.5mM biotin at a volume of 0.1 ml/ml of agar, and maintained at 45°C until used. Sterile glass tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath at 45°C. All control and treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube: 2 ml aliquots of top agar solution, 0.1 ml of tester strain and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden. The same procedure was repeated for each tester strain. within an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for 48 hours following which the background lawn and spontaneous revertants were observed and scored as normal growth, inhibited growth or no growth. Inhibition was scored by the presence of pindot colonies and the absence of a confluent lawn of bacteria. Normal growth was observed in both strains tested at all levels. All levels formed a precipitate when added to the top agar.

Re-test of strains with metabolic activation.
In the original assay, the S-9 metabolic activation preparation was contaminated. Therefore, test article Automate Blue 8, Lot #1658-79, was re-evaluated in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium with metabolic activation. Increased mutation frequencies were observed in strains TA1537, TA1538 and TA98 of Salmonella typhimurium at doses of 166.6, 500 and 1666.6 ug/plate with metabolic activation. The results of a second re-evaluation exhibited an increase in revertant frequency three-fold that of the solvent controls in strains TA1538 and TA98 at the 166.6, 500 and 1666.6 ug/plate levels and at the 500 ug/plate with metabolic activation preparation respectively.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation All strains at all doses.
negative with metabolic activation TA1535, TA1537 and TA100 at all doses.
negative with metabolic activation TA1538 at 50 and 5000 ug/plate and TA98 at 50, 166.6, 1666.6 and 5000 ug/plate
positive with metabolic activation TA1538 at 166.6, 500 and 1666.6 ug/plate and TA98 at 500 ug/plate

The results for test article Automate Blue 8, Lot #1658-79 were negative in all strains at all dose levels without metabolic activation and negative in the TA1535, TA1537 and TA100 strains with metabolic activation at all does levels. The test article was negative in strain TA1538 at 50 and 5000 ug/plate and in strain TA98 at 50, 166.6, 1666.6 and 5000 ug/plate with metabolic acticvation. The test article was positive in strain TA1538 at 166.6, 500 and 1666.6 ug/plate and in TA98 at 500 ug/plate with metabolic activation. All solvent and positive controls used in the evaluation of the test article were within the acceptable limits of mean historical limits.

Executive summary:

Test article, Automate Blue 8, Lot #1658-79, was received as a dark blue solid and was determined to be soluble in DMF. Dose levels in a preliminary Toxicity Screen were 100, 333, 1000, 3333 and 10,000 ug/plate. Strains TA1538 and TA100 of Salmonella typhimurium showed no inhibition at the levels tested. Test article Automate Blue 8 was evaluated in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at doses of 50, 166.6, 500, 1666.6 and 5000 ug/plate. There were 0.08 ml of S-9 supernatant (32.3 mg protein/ml) per 1.0 ml of S-9 mix used in the rat liver metabolic activation preparation. The test article was re-evaluated in strains TA1537, TA1538 and TA98 of Salmonella typhimurium with metabolic activation preparation at doses of 50, 166.6, 500, 1666.6 and 5000 ug/plate in order to confirm a greater than three-fold response.The results for test article Automate Blue 8, Lot #1658-79 were negative in all strains at all dose levels without metabolic activation and negative in the TA1535, TA1537 and TA100 strains with metabolic activation at all does levels. The test article was negative in strain TA1538 at 50 and 5000 ug/plate and in strain TA98 at 50, 166.6, 1666.6 and 5000 ug/plate with metabolic acticvation. The test article was positive in strain TA1538 at 166.6, 500 and 1666.6 ug/plate and in TA98 at 500 ug/plate with metabolic activation. All solvent and positive controls used in the evaluation of the test article were within the acceptable limits of mean historical limits.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

4-18-1983 to 5-2-1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted similar to a guideline study and 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 the experiment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

E. coli not tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine loci

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 fraction

Test concentrations with justification for top dose:

Strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at 100, 333, 1000, 3333 and 10,000 ug/plate.

Strain TA1537 of Salmonella typhimurium with metabolic activation at 333, 500, 1000, 1666 and 5000 ug/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine

Details on test system and experimental conditions:

Rationale for Test System:
Chemicals capable of inducing mutations have been shown to increase the reversion frequency at the histidine locus in selected tester strains of salmonella typhimurium with and without the addition of a metabolic activation system.

Test organism storage and maintenance:
The tester strains were maintained in quaduplicate at a minimum of -60°C, and served as a master and stock culture.
In order to avoid the effect of surface thawing and re-freezing of frozen permanent vials of bacterial stock, master plates were employed as a source of inoculum for mutagenesis testing. Master Plates were prepared by spreading 0.1 ml of 0.1 M histidine and 0.5 mM biotin on the surface of minimal glucose agar plates using a sterile glass spreader. For the R-factor strains, 0.1 ml of ampicillin (8 mg/ml) was spread in addition to the histidine and biotin as pressure for retaining the plasmid. The plates were prepared from frozen stock cultures and were then streaked onto the surface of the plates. The plates were incubated overnight at 37°C and were then refrigerated. These plates can be used as a source of inoculum for mutagenesis testing for 2-3 months. New Master Plates were always made from frozen stock cultures.
Fresh cultures for mutagenesis testing were prepared by inoculating a loop of inoculum from Master Plates into 50 ml of oxoid broth and grown overnight at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker. Tester strains were routinely checked for the presence of the appropriate genetic markers.

Top Agar:
Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5 mM histidine and 0.5 mM Biotin at a volume of 0.1 ml per ml of agar, and maintained at 45°C until used. Sterile tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath at 45°C. All negative and positive control tubes and plates were done in triplicate. All compound-treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube in the following order: 2 ml aliquots of top agar solution, 0.1 ml of tester strain, and 0.1 ml of the
appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden.

Metabolic Activation System:
Tubes requiring metabolic activation have, in addition to the preceding top agar ingredients, an S-9 fraction of rat liver homogenate obtained from Aroclor 1254-treated Sprague Dawley rats. The activation system (S-9 mix) contained the following per ml:
0.4 M MgC12; 1.65 M KCI 20 ul
1 M Glucose - 6 - Phosphate 5 ul
0.1 M NADP 40 ul
0.2 M Phosphate buffer pH 7.4 500 ul
Sterile distilled H2O 355 ul
S-9 Fraction 100 ul
The S-9 fraction was thawed on the day of use and kept cold on ice. 0.5 ml of the S-9 mix was added to the tubes which were then vortexed and poured onto minimal glucose plates. The plates were allowed to harden for several minutes. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for 48 - 72 hours, checked for uniform background lawn, and scored by counting revertant colonies on an electronic colony counter interfaced with a computer.

Test Article Purity:
The identity, purity, quality, and strength of the test article is the responsibility of the sponsor.

Test Article Stability:
Test article 7633B, Lot #83-0302, produced a blue precipitate when added to the aqueous top agar solution at the 100, 50, 33.33, 16.6 and 10 mg/ml.

Evaluation criteria:

Data Reporting:
In scoring the assay, the positive and negative controls were first evaluated. If the control values did not fall within the acceptable historical values,
the remaining plates were not scored and the assay was repeated. A summary of the data are presented in the summary data sheet contained in this report.

Evaluation Criteria:
In most tests with the Salmonella/Microsome Assay, results are either clearly positive or clearly negative. A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within one standard deviation of the historical mean (See Historical Data) for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical is considered mutagenic. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Statistics:

A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within one standard deviation of the historical mean (See Historical Data) for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical is considered mutagenic. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Dose levels: 100, 333, 1000, 3333 and 10,000 ug/plate.

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Dose levels: 100, 333, 1000, 3333 and 10,000 ug/plate.

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

Dose levels: 333, 500, 1000, 1666 and 5000 ug/plate

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Screen:
The preliminary toxicity screen for the Ames Assay used two of the histidine auxotrophs of Salmonella typhimurium TA1538 and TA100. The preliminary toxicity screen was designed to determine at which levels the compound exhibits toxic effects to the Salmonella typhimurium tester strains. The test compound was prepared to a concentration of 100 mg/ml. Logarithmic dilutions of this stock solution were made in the appropriate solvent to give the following concentrations: 33.33, 10, 3.33 and 1.0 mg/ml. Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5mM histidine - 0.5mM biotin at a volume of 0.1 ml/ml of agar, and maintained at 45°C until used. Sterile glass tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath at 45°C. All control and treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube: 2 ml aliquots of top agar solution, 0.1 ml of tester strain and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for 48 hours following which the background lawn and spontaneous revertants were observed and scored as normal growth, inhibited growth or no growth. Inhibition was scored by the presence of pindot colonies and the absence of a confluent lawn of bacteria. Dose levels in the Preliminary Toxicity Screen were 100, 333, 1000, 3333 and 10,000 ug/plate. Tester strains TA1538 and TA100 showed no inhibition at all levels tested.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation All strains at all doses.
negative without metabolic activation All strains at all doses.

The results for test article 7633B, Lot #83-0302, Automate Blue 8, Purified, were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at 100, 333, 1000, 3333 and 10,000 ug/plate. In addition, the results were negative in strain TA1537 of Salmonella typhimurium with metabolic activation at 333, 500, 1000, 1666 and 5000 ug/plate. All solvent and positive controls used in the evaluation of the test article were within the acceptable range of historical data.

Executive summary:

Test article 7633B, Lot #83-0302, Automate Blue 8, Purified, was soluble in DMF at 100 mg/ml. Dose levels in the Preliminary Toxicity Screen with tester strains TA1538 and TA100 were 100, 333, 1000, 3333 and 10,000 ug/plate. Tester strains TA1538 and TA100 showed no inhibition at all levels tested. The test article was evaluated in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium with and without metabolic activation preparation in the plate assay at doses of 100, 333, 1000, 3333 and 10,000 ug/plate. The test article produced an increase in the reversion frequency of Salmonella typhimurium tester strain TA1537 with metabolic activation preparation at the 3333 ug/plate level. Due to this aberrant value, it was retested in strain TA1537 of Salmonella typhimurium with metabolic activation at doses of 333, 500, 1000, 1666 and 5000 ug/plate. There were 0.10 ml of S-9 supernatant (42 mg protein/ml) per 1.0 ml of S-9 mix used in the rat liver microsomal activation system.

The results for test article 7633B, Lot #83-0302, Automate Blue 8, Purified, were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at 100, 333, 1000, 3333 and 10,000 ug/plate. In addition, the results were negative in strain TA1537 of Salmonella typhimurium with metabolic activation at 333, 500, 1000, 1666 and 5000 ug/plate. All solvent and positive controls used in the evaluation of the test article were within the acceptable range of historical data.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

3-9-1983 to 3-21-1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted similar to a guideline study and 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 the experiment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

E. coli not tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine loci

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 fraction

Test concentrations with justification for top dose:

Dose levels for test article 7317C, Lot #82-0268 in the plate assay were 100, 333, 1000, 3333 and 10,000 ug/plate. There were 0.1 ml of S-9 supernatant (42 mg protein per ml) per 1.0 ml of S-9 mix used in the rat liver microsomal activation system.

A second study was conducted to verify results obtained for test article 7317C, Lot #82-0268 in strains TA1538 and TA98 of Salmonella typhimurium with metabolic activation preparation. There were 0.1 ml of S-9 supernatant (42 mg protein per ml) per 1.0 ml of S-9 mix used in the rat liver microsomal activation system.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF (Dimethylformamide)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine

Details on test system and experimental conditions:

Organism: Salmonella typhimuriurn

Tester Strains: TA1535, TA1537, TA1538, TA98 and TA100

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

Asceptic Technique: All asceptic techniques, where possible, were carried out in the Baker NCB6 Hood.

Storage: The tester strains were maintained in quadruplicate at a minimum of -60°C, and served as a master and stock culture.

Working Cultures: Fresh cultures for mutagenesis testing were prepared by inoculating a loop of inoculum from Master Platesinto 50 ml of oxoid broth and grown for 16 hours at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker

Master Plates: In order to avoid the effect of surface thawing and re-freezing of frozen permanent vials of bacterial stock, master plates were employed as a source of inoculum for mutagenesis testing.
Master Plates were prepared by spreading 0.1 ml of 0.1 M histidine and 0.5 mM biotin on the surface of minimal glucose agar plates using a sterile glass spreader. For the R-factor strains, 0.1 ml of ampicillin (8 mg/ml) was spread in addition to the histidine and biotin as pressure for retaining the plasmid. The plates were prepared from frozen stock cultures were then streaked onto the surface of the plates. The plates were incubated over night at 37°C and were then refrigerated. These plates can be used as a source of inoculum for mutagenesis testing for 2-3 months. New Master Plates were always made from frozen stock cultures.
Tester strains were routinely checked for the presence of the appropriate genetic markers.

Top Agar: Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5 mM histidine and 0.5 mM Biotin at a volume of 0.1 ml per ml of agar, and maintained at 45°C until used. Sterile tubes (13 x 100 mm) with kaputs were labeled and placed into a Fisher Isotemp Dry Bath (No. 145) at 45°C. All negative and positive control tubes and plates were done in triplicate. All compound-treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube in the following order: 2 ml aliquots of top agar solution, 0.1 ml of tester strain, and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed Dn the plate, and the top agar overlay was allowed to harden.

Metabolic Activation System: Tubes requiring metabolic activation have, in addition to the preceding top agar ingredients, an S-9 fraction of rat liver homogenate obtained from Aroclor 1254- treated Sprague Dawley rats. The activation system (S-9 mix) contained the following per ml:
0.4 M MgCl2; 1.65 M KCl 20 ul
1 M Glucose - 6 - Phosphate 5 ul
0.1 M NADP 40 ul
0.2 M Phosphate buffer pH 7.4 500 ul
Sterile distilled H2O 335 - 395 ul
S-9 Fraction 40 - 100 ul
The S-9 fraction was thawed on the day of use and kept cold on ice. 0.5 ml of the S-9 mix was added to the tubes which were then vortexed and poured onto minimal glucose plates. The plates were allowed to harden for several minutes. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator (GCA/Precision Scientific Thelco 32M). The plates were incubated for 48 - 72 hours, checked for uniform background lawn, a~d scored by counting revertant colonies. (Artek Counter Model 880).

Test Article: The identity, purity, quality, and strength of the test article is the responsibility of the sponsor.

Evaluation criteria:

Data Reporting: Standard form (Data Summary Sheet). In scoring the assay, the positive and negative controls were first evaluated. If the control values did not fall within the acceptable historical values, the remaining plates were not scored and the assay was repeated.

Evaluation Criteria: In most tests with the Salmonella/microsome assay, results are either clearly positive or clearly negative. A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within one standard deviation of the historical mean (See Historical Data) for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical will be considered mutagenic. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Statistics:

A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within one standard deviation of the historical mean (See Historical Data) for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical will be considered mutagenic. A negative result is defined as the absence of a reproducible increase in the number
of histidine-independent colonies.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

10,000 ug/ml

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

100, 333, 1000, 3333 ug/ml

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

3333 and 10,000 ug/ml

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

100, 333 and 1000 ug/ml

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Screen:
The preliminary toxicity screen for the Ames Assay used two of the histidine auxotrophs of Salmonella typhimurium TA1538 and TA100. The preliminary toxicity screen was designed to determine at which levels the compound exhibits toxic effects to the Salmonella typhimurium tester strains. The test compound was prepared to a concentration of 100 mg/ml. Logarithmic dilutions of this stock solution were made in the appropriate solvent to give the following concentrations: 33.3, 10, 3.3 and 1 mg/ml. Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5mM histidine - 0.5mM biotin at a volume of 0.1 ml/ml of agar, and maintained at 45°C until used. Sterile glass tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath (No. 145) at 45°C. All control and treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube: 2 ml aliquots of top agar solution, 0.1 ml of tester strain and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator (GCA/Precision Scientific Thelco Model 32M or Model 4). The plates were incubated for 48 hours following which the background lawn and spontaneous revertants were observed and scored as normal growth, inhibited growth or no growth. Inhibition was scored by the presence of pindot colonies and the absence of a confluent lawn of bacteria. Tester strains TA1538 and TA100 showed normal growth at 100, 333, 1000, 3333 and 10,000 ug/ml. There was a heavy bluish precipitate when added to the top agar observed in the 3333 and 10,000 ug/ml groups.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation All strains at all doses.
negative with metabolic activation Strains TA1535, TA1537 and TA100 at all dose levels.
negative with metabolic activation TA1538 at 10,000 ug/ml
negative with metabolic activation TA98 at 3333 and 10,000 ug/ml
positive with metabolic activation TA1538 at 100, 333, 1000 and 3333 ug/ml and TA98 at 100, 333 and 1000 ug/ml

The results for test article 7317C, Automate Blue 8, Lot #82-0268 were positive in strain TA1538 of Salmonella typhimurium with metabolic activation preparation at 100, 333, 1000 and 3333 ug/ml levels and in strain TA98 of Salmonella typhimurium with metabolic activation at the 100, 333 and 1000 ug/ml levels. All solvent and positive controls used in the evaluation of the test article were within the acceptable range of mean historical data.

Executive summary:

Test article 7317C, Automate Blue 8, Lot #82-0268, was evaluated in the Ames Salmonella/Microsome Plate Test. The test article was soluble in Dimethylformamide. Dose levels in the Preliminary Toxicity Screen with tester strains TA1538 and TA100 were 100, 333, 1000, 3333 and 10,000 ug/plate (Solvent DMF). Tester strains TA1538 and TA100 showed normal growth at all dose levels. The test article was evaluated in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium with and without metabolic activation preparation in the plate assay at doses of 100, 333, 1000, 3333 and 10,000 ug/plate. The results for test article 7317C, Automate Blue 8, Lot #82-0268 were positive in strain TA1538 of Salmonella typhimurium with metabolic activation preparation at 100, 333, 1000 and 3333 ug/ml levels and in strain TA98 of Salmonella typhimurium with metabolic activation at the 100, 333 and 1000 ug/ml levels. All solvent and positive controls used in the evaluation of the test article were within the acceptable range of mean historical data.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12-29-1983 to 1-9-1984

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 the experiment.

Qualifier:

according to guideline

Guideline:

other: OECD ISBN 92-64-12221-4

Deviations:

yes

Remarks:

E. Coli not tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine loci

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 fraction

Test concentrations with justification for top dose:

Test article, Blue 8, Lot #1658-129, was evaluated in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at dose levels of 50, 166.6, 500, 1666.6 and 5000 ug/plate. There were 0.07 ml of S-9 supernatant (19.6 mg protein/ml) per 1.0 ml of S-9 mix used in the rat liver metabolic activation preparation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF (Dimethylformamide) analytical grade, Lot #92866, supplied by J. T. Baker Chemical Company, Phillipsburg, New Jersey, 08865.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine

Details on test system and experimental conditions:

Test Article:
The test article, Blue 8, Lot #1658-129, was received by Pharmakon Research on November 9, 1983 in a clear glass bottle and the physical description of the test article upon receipt was described as a dark blue solid. Normal precautions were used in handling the test article. Stability and purity of the test article was the responsibility of the sponsor and information as to the stability, purity, expiration date and other technical aspects of the test article was recorded in the sponsor's file, if provided. For the purposes of this study, the test article was stored at room temperature in the container received from the sponsor. All required dilutions were made with dimethyl formamide , analytical grade, Lot #92866, supplied by J. T. Baker Chemical Company, Phillipsburg, New Jersey, 08865. Dilutions were prepared the day of the test. At the time of testing the test article was described as a dark blue solid. Precipitation of the test article was observed at all concentrations upon addition of the dosing solution to the top agar overlay. There was no apparent change in the physical state of the control articles during the assay. Details of the test article preparation are contained in the experimental data section of this report. Dosing solutions were used within four hours of preparation.

Rationale for Test System:
Chemicals capable of inducing mutations have been shown to increase the reversion frequency at the histidine locus in selected tester strains of salmonella typhimurium with and without the addition of a metabolic activation system.

Test organism storage and maintenance:
Frozen working stock cultures were prepared by scraping a wooden applicator stick over the surface of frozen Master cultures and inoculating the scrapings into 50 ml of Oxoid Broth #2 and grown for approximately 16 hours at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker. Following the 16 hour growth period, 1 ml aliquots of the culture was dispensed into Nunc vials marked with the particular strain and quick frozen in an ethanol-dry ice bath before being stored at a minimum of -60°C.
In order to avoid the effect of surface thawing and re-freezing of frozen permanent vials of bacterial stock, frozen working stock cultures are employed as a source of inoculum for mutagenesis testing.
Fresh cultures for mutagenesis testing were prepared by quick thawing a vial of frozen working stock cultures of each tester strain and transferring the culture to 50 ml of Oxoid Nutrient Broth #2 and grown for approximately 16 hours (1-2 x 10E9 cells/ml) at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker. After the 16 hour incubation, samples of culture suspensions were diluted 1:4 in distilled water and optical densities were observed at 650 nm using a Beckman Model 35 Spectrophotometer. Historical data has shown that opticai densities of 0.400 or greater are representative of cells in late exponential or early stationary phase of growth. Tester strains were checked concurrently for the presence of the appropriate genetic markers at the time of the assay.

Top Agar:
Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5 mM histidine and 0.5 mM biotin at a volume of 0.1 ml per ml of agar, and maintained at 45°C until used. Sterile tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath at 45°C. All negative and positive control tubes and plates were done in triplicate. All compound-treated tubes and plates were done in triplicate. Using sterile technique, the following were added to each tube in the following order: 2 ml aliquots of top agar solution, 0.1 ml of tester strain, and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden.

Metabolic Activation System:
Tubes requiring metabolic activation had, in addition to the preceding top agar ingredients, an S-9 fraction of rat liver homogenate obtained from Aroclor 1254-treated Sprague Dawley rats. The activation system (S-9 mix) contained the following per ml:
0.4 M MgCl2; 1.65 M KCI 20 ul
1 M Glucose - 6 - Phosphate 5 ul
0.1 M NADP 40 ul
0.2 M Phosphate buffer pH 7.4 500 ul
Sterile distilled H2O 365 ul
S-9 Fraction 70 ul
The S-9 fraction was thawed on the day of use and kept cold on ice. 0.5 ml of the S-9 mix was added to the tubes which were then vortexed and poured onto minimal glucose plates. The plates were allowed to harden for several minutes. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for 48 - 72 hours, checked for uniform background lawn, and scored by counting revertant colonies on an electronic colony counter interfaced with a computer.

Bacterial Contaminant Control:
To insure the quality of aseptic technique during the assay and also the sterility of solvents, compounds and equipment, standard contamination checks were conducted with the assay. These contamination checks included the top level of test substance, solvent, top agar and S-9 mix at the same volumes as in the assay. The test substance or S-9 mix were added to 2 ml of molten top agar supplemented with 0.5mM histidine - 0.5mM biotin and poured onto minimal glucose plates. 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 (± 2°C) and then scored for contamination.

Test Article Purity:
The identity, purity, quality, and strength of the test article are the responsibility of the sponsor.

Evaluation criteria:

Data Reporting:
In scoring the assay, the positive and negative controls were first evaluated. If the negative control values did not fall within the acceptable historical mean values, the remaining plates were not scored and the assay was repeated. A summary of the data are presented in the summary data sheet contained in this report.

Evaluation Criteria:
In most tests with the Salmonella/Microsome Assay, results are either clearly positive or clearly negative. A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within one standard deviation of the historical mean (See Historical Data) for control
values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical is considered positive. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Statistics:

A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within one standard deviation of the historical mean (See Historical Data) for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical is considered positive. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Screen:
The preliminary toxicity screen for the Ames Assay performed without metabolic activation used two of the histidine auxotrophs of Salmonella typhimurium, TA1538 and TA100. The preliminary toxicity screen was designed to determine at which levels the compound exhibits toxic effects to the Salmonella typhimurium tester strains. The test compound was prepared to a concentration of 50 mg/ml and five different levels tested for toxicity; 50, 166, 500, 1666 and 5000 ug/plate. Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5mM histidine - 0.5mM biotin at a volume of 0.1 ml/ml of agar, and maintained at 45°C until used. Sterile glass tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath at 45°C. All control and treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube: 2 ml aliquots of top agar solution, 0.1 ml of tester strain and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for 48 hours following which the background lawn and spontaneous revertants were observed and scored as normal growth, ,inhibited growth or no growth. Inhibition was scored by the presence of pindot colonies and the absence of a confluent lawn of bacteria. Normal growth was observed at all dose levels. A precipitate was observed at all dose levels when added to the top agar.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation All strains at all doses.
negative without metabolic activation All strains at all doses.

The results for test article, Blue 8, Lot #1658-129, of Salmonella typhimurium were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 both with and without metabolic activation at concentrations of 50, 166.6, 500, 1666.6 and 5000 ug/plate.

Executive summary:

Test article, Blue 8, Lot #1658-129, was supplied for testing as a dark blue solid and was determined to be soluble in dimethylformamide (DMF). Test article, Blue 8, Lot #1658-129, was tested for cytotoxic effects in Ames/Salmonella tester strains TA1538 and TA100 at concentrations of 50, 166, 500, 1666 and 5000 ug/plate without metabolic activation. No toxic effects were observed at any of the concen tra tions evaluated. Based upon these findings, a dose of 5000 ug/plate was chosen as the highest concentration to be employed in the plate incorporation mutation assay. Test article, Blue 8, Lot #1658-129, was evaluated in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at dose levels of 50, 166.6, 500, 1666.6 and 5000 ug/plate. There were 0.07 ml of S-9 supernatant (19.6 mg protein/ml) per 1.0 ml of S-9 mix used in the rat liver metabolic activation preparation. The results for test article, Blue 8, Lot #1658-129, of Salmonella typhimurium were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 both with and without metabolic activation at concentrations of 50, 166.6, 500, 1666.6 and 5000 ug/plate. All solvent and positive controls used in the evaluation of the test article were wi thin the acceptable range of mean historical data.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1984

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 the experiment.

Qualifier:

according to guideline

Guideline:

other: OECD ISBN 92-64-12221-4, EPA 560/6-82-001, EPA 560/6-83-001

Deviations:

yes

Remarks:

E. Coli was not tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine loci

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 fraction

Test concentrations with justification for top dose:

Test article, Blue #8 NV Dye, Lot# 84-9389, was evaluated in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without rat liver metabolic activation preparation at doses of 50, 166, 500, 1666 and 5000 ug/plate. The test article was re-evaluated in strain TA1538 with activation at doses of 10, 25, 50, 100 and 200 ug/plate in order to confirm an increased mutation frequency that was at least three times greater than the solvent controls in the original assay. The rat liver metabolic activation preparation contained 0.08 ml of S-9 supernatant (34.0 mg
protein/ml) per 1.0 ml of S-9 mix.
Test article was also re-evaluated in strain TA98 of Salmonella typhimurium with activation at doses of 25, 100, 250, 500 and 750 ug/plate to confirm an increased mutation frequency that was at least three times greater than the negative controls in the original assay. There were 0.08 ml of S-9
supernatant (38.3 mg protein/ml) per 1.0 ml of S-9 mix in the rat liver metabolic activation preparation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF (Dimethylformamide)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine

Details on test system and experimental conditions:

TEST ARTICLE:
The test article Blue #8 NV Dye, Lot# 84-9389, was received by Pharmakon Research on July 27, 1984 in a clear glass container and the physical description of the test article upon receipt was described as a purple solid. Normal precautions were used in handling the test article. Stability and purity of the test article were the responsibility of the sponsor and information as to the stability, purity, expiration date and other technical aspects of the test article was recorded in the sponsor's file, if provided. For the purposes of this study, the test article was stored at room temperature in the container received from the sponsor. All required dilutions were made with dimethylformamide (DMF) , Lot# KTJZ, supplied by Mallinckrodt Inc, Paris, Kentucky, 40361. Dilutions were prepared the day of the test. At the time of testing the test article was described as a purple solid. There was no apparent change in the physical state of the test or control articles during the assay. Details of the test article preparation are contained in the experimental data section of this report. Dosing solutions were used within four hours of preparation.

Rationale for Test System:
Chemicals capable of inducing mutations have been shown to increase the reversion frequency at the histidine locus in selected tester strains of
salmonella typhimurium with and without the addition of a metabolic activation system.

Test organism storage and maintenance:
Frozen working stock cultures were prepared by scraping a wooden applicator stick over the surface of frozen Master cultures and inoculating the scrapings into 50 ml of Oxoid Broth #2 and grown for 10-12 hours at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker. Following the 10-12 hour growth period, 1 ml aliquots of the culture was dispensed into Nunc vials marked with the particular strain and quick frozen in an ethanol-dry ice bath before being stored at a minimum of -60°C.
In order to avoid the effect of surface thawing and re-freezing of frozen permanent vials of bacterial stock, frozen working stock cultures are employed as a source of inoculum for mutagenesis testing. Fresh cultures for mutagenesis testing were 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 and grown for approximately 10 hours at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker. After the 10 hour incubation, samples of culture suspensions were
diluted 1:4 in distilled water and optical densities were observed at 650 nm using a Beckman Model 35 Spectrophotometer. Historical data has shown that optical densities of 0.400 or greater are representative of cells in late exponential or early stationary phase of growth. Tester strains were checked for the presence of the appropriate genetic markers on a monthly basis.

Top Agar:
Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5 mM histidine and 0.5 mM biotin at a volume of 0.1 ml per ml of agar, and maintained at 45°C until used. Sterile tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath at 45°C. All negative and positive control tubes and plates were done in triplicate. All compound-treated tubes and plates were done in triplicate. Using sterile technique, the following were added to each tube in the following order: 2 ml aliquots of top agar solution, 0.1 ml of tester strain, and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden.

Metabolic Activation System:
Tubes requiring metabolic activation had, in addition to the preceding top agar ingredients, an S-9 fraction of rat liver homogenate obtained from
Aroclor 1254-treated Sprague Dawley rats. The activation system (S-9 mix) contained the following per ml:
0.4 M M9Cl2; 1.65 M KCI 20 ul
1 M Glucose - 6 - Phosphate 5 ul
0.1 M NADP 40 ul
0.2 M Phosphate buffer pH 7.4 500 ul
Sterile distilled H2O 355 ul
S-9 Fraction 80 ul
The S-9 fraction was thawed on the day of use and kept cold on ice. 0.5 ml of the S-9 mix was added to the tubes which were then vortexed and poured onto minimal glucose plates. The plates were allowed to harden for several minutes. The same procedure was repeated for each tester strain. within an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for 48 - 72 hours, checked for uniform background lawn, and scored by counting revertant colonies on an electronic colony counter interfaced with an Apple computer for data acquisition.

Bacterial Contaminant Control:
To insure the quality of aseptic technique during the assay and also the sterility of solvents, compounds and equipment, standard contamination checks were conducted with the assay. These contamination checks included the top level of test substance, solvent, top agar and S-9 mix at the same volumes as in the assay. The test substance or S-9 mix were added to 2 ml of molten top agar supplemented with 0.5mM histidine - 0.5mM biotin and poured onto minimal glucose plates. 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 (± 2°C) and then scored for contamination.

Test Article Purity:
The identity, purity, quality, and strength of the test article are the responsibility of the sponsor.

Evaluation criteria:

Data Reporting:
In scoring the assay, the positive and negative controls were first evaluated. If the negative control values did not fall within the acceptable historical mean values, the remaining plates were not scored and the assay was repeated. A summary of the data are presented in the summary data sheet contained in this report.

Evaluation Criteria:
In most tests with the Salmonella/Microsome Assay, results are either clearly positive or clearly negative. A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within the 95% confidence limits of the historical mean (See Historical Data) for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical is considered positive. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Statistics:

A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within the 95% confidence limits of the historical mean (See Historical Data) for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical is considered positive. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Species / strain:

other: S. typhimurium TA1535, 1537 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

other: S. typhimurium TA1538 and TA98

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

10 ug/plate

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

25, 50, 100 and 200 ug/plate

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

25 and 100 ug/plate

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

250, 500 and 750 ug/plate

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Screen:
The preliminary toxicity screen for the Ames Assay performed without metabolic activation used two of the histidine auxotrophs of Salmonella typhimurium, TA1538 and TA100. The preliminary toxicity screen was designed to determine at which levels the compound exhibits toxic effects to the Salmonella typhimurium tester strains. The test compound was prepared to a concentration of 50 mg/ml and five different levels tested for toxicity. Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5mM histidine - 0.5mM biotin at a volume of 0.1 ml/ml of agar, and maintained at 45°C until used. Sterile glass tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath at 45°C. All control and treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube: 2 ml aliquots of top agar solution, 0.1 ml of tester strain and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden. The same procedure was repeated for each tester strain. within an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for 48 hours following which the background lawn and spontaneous revertants were observed and scored as normal growth, inhibited growth or no growth. Inhibition was scored by the presence of pindot colonies and the absence of a confluent lawn of bacteria. Strains TA1538 and TA100 were tested at 50, 166, 500, 1666 and 5000 ug/plate. Normal growth was observed at these dose levels.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation All strains at all dose levels.
negative with metabolic activation TA1535, TA1537 and TA100 at all dose levels.
negative with metabolic activation TA1538 at 10 ug/plate and TA98 at 25 and 100 ug/plate.
positive with metabolic activation TA1538 at 25, 50, 100 and 200 ug/plate and TA98 at 250, 500 and 750 ug/plate.

There was a dose-related increase in mutation frequency for test article Blue #8 NV Dye, Lot# 84-9389, in strain TA1538 of Salmonella typhimurium with metabolic activation preparation at doses of 25, 50, 100 and 200 ug/plate and in strain TA98 with activation at doses of 250, 500 and 750 ug/plate.

Executive summary:

Test article, Blue #8 NV Dye, Lot# 84-9389, was received as a purple solid. The solvent used throughout the assay was dimethylformamide (DMF). Dose levels in a Preliminary Toxicity screen were 50, 166, 500, 1666 and 5000 ug/plate. Strains TA1538 and TA100 of Salmonella typhimurium exhibited no inhibition of bacterial lawn growth at any of the levels tested. Based on

these findings the top dose selected for the Plate Incorporation Mutation Assay was 5000 ug/plate.

Test article, Blue #8 NV Dye, Lot# 84-9389, was evaluated in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without rat liver metabolic activation preparation at doses of 50, 166, 500, 1666 and 5000 ug/plate. The test article was re-evaluated in strain TA1538 with activation at doses of 10, 25, 50, 100 and 200 ug/plate in order to confirm an increased mutation frequency that was at least three times greater than the solvent controls in the original assay. The rat liver metabolic activation preparation contained 0.08 ml of S-9 supernatant (34.0 mg protein/ml) per 1.0 ml of S-9 mix.

Test article was also re-evaluated in strain TA98 of Salmonella typhimurium with activation at doses of 25, 100, 250, 500 and 750 ug/plate to confirm an increased mutation frequency that was at least three times greater than the negative controls in the original assay. There were 0.08 ml of S-9 supernatant (38.3 mg protein/ml) per 1.0 ml of S-9 mix in the rat liver metabolic activation preparation.

There was a positive dose-related increase in mutation frequency for test article Blue #8 NV Dye, Lot# 84-9389, in strain TA1538 of Salmonella typhimurium with metabolic activation preparation at doses of 25, 50, 100 and 200 ug/plate and in strain TA98 with activation at doses of 250, 500 and 750 ug/plate. All solvent and positive controls used in the evaluation of the test article were within the acceptable limits of mean historical data.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo mutagenicity assay (Pig A) integrated into the OECD 422 assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

The in vivo Pig-a assay uses flow cytometry to measure phenotypic variants for antibody binding to cell surface glycosylphosphatidylinositol (GPI)-anchored proteins. There is good evidence suggesting that the absence of antibody binding is the result of a mutation in the endogenous X-linked Pig-a gene, which forms the rationale for the assay.

The study was integrated into the OECD 422 guideline study described in section 7.8 and 7.5.
Gollapudi, B. B., Lynch, A. M., Heflich, R. H., et al. (2014). The in vivo Pig-a assay: A report of the International Workshop on Genotoxicity Testing (IWGT) Workgroup. Mutation Research: Genetic Toxicology and Environmental Mutagenesis, http://dx.doi.org/10.1016/j.mrgentox.2014.09.007

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vivo pig A

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male

Route of administration:

oral: gavage

Vehicle:

Corn Oil

Duration of treatment / exposure:

dosed daily for 14 days prior to mating and continuing throughout the mating period for at least 34 days

Frequency of treatment:

once daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

6 male animals were included into the OECD 422 study as a satellite group, serving as the positive control for the pig A portion of the study. These animals were exposed to N-Ehtyl-N-nitroso-urea (759-73-9) on test days 1-3 of the study at a dose level of 20 mg/kg bw/day via gavage in an acidic buffer solution (KH2PO4, Na2HPO4).

Tissues and cell types examined:

red blood cells

Statistics:

The Pig-a assay data were analyzed by the following statistical methods as recommended in Gollapudi et al. (2014). A Bartlett’s test (alpha=0.01; Winer, 1971) was used to test for homogeneity of variance, the test was also sensitive to normality. Transformations of mutant frequencies (e.g., a log (10) transformation) were conducted to meet these assumptions when necessary. Since zeroes may have been observed occasionally, prior to the log-transformation, an offset of +0.1 (i.e., addition of 0.1 to each mutant cell frequency expressed as mutants × 10−6) may have been required. An analysis of variance (ANOVA) followed by pair-wise comparisons of mutant frequencies in treated groups to the vehicle control group was performed utilizing a one-sided (upper to detect an increase in mutant cells) Dunnett’s test at alpha=0.05 for mutant RBC and mutant RET and a 2-sided Dunnett’s test at alpha=0.05 for percent RET. If any of the pairwise tests were significant, evidence of dose-related increases were evaluated with a linear trend test. The positive control was compared to the vehicle control with an analysis of variance followed by a one-sided (upper) Dunnett’s test at alpha=0.05 for mutant RBC and mutant RET and a 2-sided Dunnett’s test at alpha=0.05 for percent RET.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

general toxixity - increased liver weights, and histopathology in the pancreas - effects decribed in Repeat dose tox section

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Two animals with pre-exposure frequency of CD59-negative reticulocytes (RET^CD59-) exceeding the reported background range of 0-5 × 10^-6(Gollapudiet al.,2014), (animal 2458 in the 300 mkd test material group and animal 2472 in the 1000 mg/kg/day test material group) were excluded from the post-exposurePig-aassay. Six male animals from eachC.I. Solvent Blue 98 (3Amine)treated group and the vehicle control group were selected for the post-exposurePig-aassay by the ascending order of animal ID among surviving animals with pre-exposure frequency of RET^CD59-not exceeding
5 × 10^-6. 

The frequencies(×10^-6)of RET^CD59-and RBC^CD59-in the vehicle control animals were within the reported background range (Gollapudiet al.,2014). The frequencies of RET^CD59-and RBC^CD59-in the positive control (ENU) group were significantly higher than those in the vehicle control group (Text Table 12). The normal frequencies of RET^CD59-and RBC^CD59-in the vehicle control animals and the significantly increased frequencies of RET^CD59-and RBC^CD59-in the positive control animals indicated acceptability of the test system for identifying a mutagen. There were no significant differences in the frequency of RET^CD59-or RBC^CD59-among the vehicle control group and theC.I. Solvent Blue 98 (3Amine)treated groups (Text Table 12). 

There were no significant differences in the percentage of reticulocytes (% RET) among the vehicle control group and theC.I. Solvent Blue 98 (3Amine)treated groups (Text Table 12); however, the gross pathology observation of blue discoloration of tissue observed at necropsy demonstrated that theC.I. Solvent Blue 98 (3Amine)and/or its metabolites were absorbed and systemically bioavailable after oral gavage (see adult gross pathology) . 

Therefore,C.I. Solvent Blue 98 (3Amine)was negative in thisin vivogene mutationPig-aassay under the experimental conditions used.

Conclusions:

C.I. Solvent Blue 98 (3Amine) was negative in this in vivo gene mutation Pig-a assay under the experimental conditions used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial Mutagenicity:

Five bacterial mutagenicity assays are available for this substance. These studies were performed over the course of a two year period on five different lots of test material. All studies were conducted according to an older version of the Ames test guideline using only strains TA1535, TA1537, TA1538, TA98, and TA100.

A summary of the findings is presented in the table below:

Ames (1983)	DR-0273-5866-007 (3/29/1983) Lot # 82-0268 Tester strains: TA1535, TA1537, TA1538, TA98, and TA100 Doses: 100 – 10000 µg/plate with and without S9 Results: STRONG positive in strains TA1538 and TA98 with S9
Ames (1983)	DR-0273-5866-006 (5/9/1983) Lot # 83-0302 Tester strains: TA1535, TA1537, TA1538, TA98, and TA100 Doses: 100 – 10000µg/plate with and without S9 Results: negative
Ames (1983)	DR-0273-5866-005 (10/10/1983) Lot # 1658-79 Tester strains: TA1535, TA1537, TA1538, TA98, and TA100 Doses: 50, 166.6, 500, 1666.6, and 5000µg/plate with and without S9 Results: positive in strains TA1537, TA1538 and TA98 with S9
Ames (1983)	DR-0273-5866-008 (1/24/1984) Lot # 1658-129 Tester strains: TA1535, TA1537, TA1538, TA98, and TA100 Doses: 50, 166.6, 500, 1666.6, and 5000µg/plate with and without S9 Results: negative
Ames (1983)	DR-0273-5866-009 (9/26/1984) Lot # 84-9389 Tester strains: TA1535, TA1537, TA1538, TA98, and TA100 Doses: 50, 166, 500, 1666, and 5000µg/plate with and without S9 Results: STRONG positive in strains TA1538 and TA98 with S9

It is clear from the results that there is substantial variation in mutagenicity outcome (positive, negative, strains) across the 5 batches tested although the 3 out of the 5 assays indicate week to strong positive findings in at least TA1538 and TA98 in the presence of metabolic activation. On this basis it was concluded that this substance may be mutagenic in bacteria. Consequently further in vitro testing of mammalian mutagenicity was waived, and the potential for mutagenicity was followed up in vivo.

In vitro Clastogenicity:

A OECD guideline (473) clastogenicity assay performed using cultured rat lymphocytes is available for C.I. Solvent Blue 98 (3 Amine). In this assay there were no significant increases in the frequency of cells with aberrations administered C.I. Solvent Blue 98 (3 Amine) 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, C.I. Solvent Blue 98 (3 Amine) was considered to be negative in this in vitro chromosomal aberration assay utilizing rat lymphocytes.

In vivo mutagenicity: PigA assay in rats

Due to the positive findings in the Ames assay it was considered appropriate to perform a follow up in vivo assessment of mutagenicity to identify whether classification for mutagenicity is appropriate and whether further assessments of mutagenicity in vivo are needed. Consequently an in vivo assay was added into the conduct of the reproductive screening study required for REACH registration.

The Pig-a assay is an in vivo gene mutation assay. It was first reported in 2008 [1-4] and since then has received extensive interest as a potential assay for regulatory safety assessment [5-9]. A Workgroup, made up of experts from academic, regulatory, and industrial laboratories, was formed in 2012 under the auspices of the International Workshop on Genotoxicity Testing (IWGT) to review the development of the Pig-a assay in the context of safety assessment strategies. The Workgroup consensus on the underlying science of the Pig-a assay, technical considerations for the assay protocol, a view to assay acceptance in a regulatory context, and recommendations on where and how further progress on developing the assay have been published [8]. An OECD test guideline development for the Pig-a assay has been initiated.

Principle of the assay

The Pig-a assay is based on the identification of mutant cells that have an altered repertoire of cell surface markers. The assay was developed from an understanding of the molecular nature of a rare human acquired genetic disorder, paroxysmal nocturnal hemoglobinuria (PNH). The Pig-a gene (phosphatidylinositol glycan, class A gene) codes for the catalytic subunit of an N-acetyl glucosamine transferase that is involved in an early step of glycosylphosphatidylinositol (GPI) biosynthesis. GPI anchors an assortment of protein markers (e. g., CD59, CD24, and CD55) to the exterior surface of the cytoplasmic membranes of higher eukaryotes. In mammals, Pig-a is an X-linked gene present as a single functional copy in cells from both males and females. Other genes involved in GPI biosynthesis are autosomal and have two functional copies. A single inactivating mutation in the Pig-a gene is sufficient to make a cell deficient in GPI anchors and, as a consequence, deficient in surface-bound GPI-anchored markers. Since it is exceedingly unlikely that anchor deficiency would occur due to inactivating mutations in both copies of the autosomal genes involved in GPI synthesis, measuring GPI deficiency is considered ‘virtually equivalent’ to measuring Pig-a mutation [3, 10-12].

Validation of the assay

Multi-laboratory trials initiated by Litron Laboratories [13], the Japanese Research Group [14], and the HESI initiative [7] have contributed to establishing protocols for the assay, testing the inter-laboratory reproducibility of the assay, and in expanding the number of agents tested.

The assay has been investigated extensively in peripheral blood erythrocytes of rats and has been performed with several types of hematopoietic cells and in a variety of mammalian species, including humans. Currently the IWGT Workgroup recommends measuring CD59-deficient erythrocytes in the peripheral blood of rats for use in safety assessment.

Though no studies to date have specifically tested the intra-laboratory reproducibility of the assay beyond assaying technical replicates, there is good evidence for a high degree of inter-laboratory transferability and reproducibility based on the results with several potent and weak mutagens (and low doses of potent mutagens) that were tested in a systematic manner as part of the Litron trial [15-20]. Results from the Japanese trial, using a different staining protocol, have also demonstrate a similar degree of inter-laboratory reproducibility [14]. Therefore, the available data show that the assay is both robust and demonstrably reproducible within and across laboratories for test agents with a range of mutagenic potency.

The IWGT Workgroup compiled a list of 41 agents tested in the rat Pig-a assay in the report [8]. Most of the agents were genotoxic in one or more tests, including 26 that were Ames' test positive. The Pig-a assay identified most of the agents expected to be positive as positive in the assay. Of the 8 agents tested that generally are considered non-genotoxicants, all were negative in the assay with 5 of those tested to the maximum tolerated dose (MTD) or the limit dose using the most sensitive protocols (i. e., using immunomagnetic seperation).

The Pig-a assay is based on the identification of the phenotype alteration resulting from gene mutation. Establishing the mutational basis of the response is a challenge because the assay measures the mutant phenotype in enucleated erythrocytes; however, there is good evidence from nucleated bone marrow and spleen cells from rodents, and extensive experience from human PNH patients, that supports the conclusion that GPI-anchored protein-deficient cells, in fact, have mutations in the Pig-a gene. The IWGT Workgroup concluded that the weight of evidence was consistent with a direct association between Pig-a mutation (rather than gene silencing or enzymatic inactivation) and the phenotype measured in the assay, and indicated that lack of absolute proof should not preclude use of the Pig-a assay for regulatory purposes [8].

Placement of the assay within regulatory genotoxicity testing strategies

Therefore, the IWGT Workgroup recommended a placement of the Pig-a assay within regulatory genotoxicity testing strategies [8]: “The Pig-a assay should be considered an appropriate in vivo follow-up to positive results in bacterial and in vitro mammalian cell gene mutation assays. …... the Workgroup noted the potential of using the Pig-a assay, as a measure of gene mutation, to complement the micronucleus (MN) assay, which measures clastogenicity/aneugenicity. Both can be readily included in routine in vivo safety evaluations, especially when the assays are integrated into subchronic (28 or 90 day) treatment protocols. ”

The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) has accepted the Pig-a assay as one of tests to investigate the in vivo relevance of in vitro mutagens (positive bacterial mutagenicity) in DNA reactive impurities in pharmaceuticals [21].

Scientific guideline for the assay

Detailed technical considerations for the assay protocol have been provided by the IWGT Workgroup [8], e. g., test animals/sex, number of doses, maximum doses, and age and number of animals, prescreening animals, treatment and sampling schedules, sample analysis, data analysis and interpretation.

The IWGT Workgroup also recognized that one of the most attractive features of the Pig-a assay is its potential for integration into repeat dose toxicology studies and with other genetic toxicology assays. The combination of several assays in one set of test animals is also consistent with the 3Rs principles for animal welfare. The assay requires only microliter samples of peripheral blood, which can be obtained in a minimally invasive manner without disturbing the assessment of other endpoints. Unlike the TGR gene mutation test, the Pig-a assay is not limited to specific strains of animals, and the timing of sample collection is not as critical as it is for the in vivo Comet assay. An official OECD test guideline development for the Pig-a assay has been initiated.

In vivo mutagenicity assessment of CI Solvent Blue (3 Amine) using the Pig-a assay

The present Pig-a assay was integrated into a Reproductive/Developmental Toxicity Screening Test to promote the 3Rs principles. All the study parameters were compliant to the protocol provided by the IWGT Workgroup. The animals were prescreened (as described in the IWGT guidance) to maximize the assay sensitivity and specificity. The frequencies (×10-6) of CD59-deficient reticulocytes (RETCD59-) and CD59-deficient red blood cells (RBCCD59-) in the vehicle control animals were within the reported background range (≤5 × 10-6) [8]. The mutant frequencies of RETCD59- and RBCCD59- in the positive control group were significantly higher than those in the vehicle control group. The percentage of reticulocytes (% RET) in the 200 mg/kg/day test material group was significantly higher than that in the vehicle control group and the increase was dose-dependent, indicative of bone marrow exposure to the test material or metabolites. Therefore, this study met the requirement for a valid assay according to the IWGT Workgroup criteria and, under the experimental conditions of this study, indicated that CI Solvent Blue (3 Amine) does not have in vivo mutagenic potential.

References

1. Bryce, S. M., J. C. Bemis, and S. D. Dertinger, In vivo mutation assay based on the endogenous Pig-a locus. Environ Mol Mutagen, 2008. 49(4): p. 256-64.

2. Miura, D., et al., Development of an in vivo gene mutation assay using the endogenous Pig-A gene: I. Flow cytometric detection of CD59-negative peripheral red blood cells and CD48-negative spleen T-cells from the rat. Environ Mol Mutagen, 2008. 49(8): p. 614-21.

3. Miura, D., et al., Development of an in vivo gene mutation assay using the endogenous Pig-A gene: II. Selection of Pig-A mutant rat spleen T-cells with proaerolysin and sequencing Pig-A cDNA from the mutants. Environ Mol Mutagen, 2008. 49(8): p. 622-30.

4. Phonethepswath, S., et al., Erythrocyte-based Pig-a gene mutation assay: demonstration of cross-species potential. Mutat Res, 2008. 657(2): p. 122-6.

5. Dobrovolsky, V. N., et al., The in vivo Pig-a gene mutation assay, a potential tool for regulatory safety assessment. Environ Mol Mutagen, 2010. 51(8-9): p. 825-35.

6. Lynch, A. M., et al., New and emerging technologies for genetic toxicity testing. Environ Mol Mutagen, 2011. 52(3): p. 205-23.

7. Schuler, M., et al., Need and potential value of the Pig-a in vivo mutation assay-a HESI perspective. Environ Mol Mutagen, 2011. 52(9): p. 685-9.

8. Gollapudi, B. B., et al., The in vivo Pig-a assay: A report of the International Workshop On Genotoxicity Testing (IWGT) Workgroup. Mutat Res Genet Toxicol Environ Mutagen, 2015. 783: p. 23-35.

9. Godin-Ethier, J., et al., Characterisation of an in vivo Pig-a gene mutation assay for use in regulatory toxicology studies. Mutagenesis, 2015. 30(3): p. 359-63.

10. Kimoto, T., et al., Manifestation of Pig-a mutant bone marrow erythroids and peripheral blood erythrocytes in mice treated with N-ethyl-N-nitrosourea: direct sequencing of Pig-a cDNA from bone marrow cells negative for GPI-anchored protein expression. Mutat Res, 2011. 723(1): p. 36-42.

11. Miura, D., et al., Analysis of mutations in the Pig-a gene of spleen T-cells from N-ethyl-N-nitrosourea-treated fisher 344 rats. Environ Mol Mutagen, 2011. 52(5): p. 419-23.

12. Mortazavi, Y., et al., The spectrum of PIG-A gene mutations in aplastic anemia/paroxysmal nocturnal hemoglobinuria (AA/PNH): a high incidence of multiple mutations and evidence of a mutational hot spot. Blood, 2003. 101(7): p. 2833-41.

13. Dertinger, S. D. and R. H. Heflich, In vivo assessment of Pig-a gene mutation-recent developments and assay validation. Environ Mol Mutagen, 2011. 52(9): p. 681-4.

14. Kimoto, T., et al., Interlaboratory trial of the rat Pig-a mutation assay using an erythroid marker HIS49 antibody. Mutat Res, 2013. 755(2): p. 126-34.

15. Bhalli, J. A., et al., Report on stage III Pig-a mutation assays using benzo[a]pyrene. Environ Mol Mutagen, 2011. 52(9): p. 731-7.

16. Cammerer, Z., et al., Report on stage III Pig-a mutation assays using N-ethyl-N-nitrosourea-comparison with other in vivo genotoxicity endpoints. Environ Mol Mutagen, 2011. 52(9): p. 721-30.

17. Dertinger, S. D., et al., International Pig-a gene mutation assay trial: evaluation of transferability across 14 laboratories. Environ Mol Mutagen, 2011. 52(9): p. 690-8.

18. Lynch, A. M., et al., International Pig-a gene mutation assay trial (stage III): results with N-methyl-N-nitrosourea. Environ Mol Mutagen, 2011. 52(9): p. 699-710.

19. Shi, J., et al., Assessment of genotoxicity induced by 7,12-dimethylbenz(a) anthracene or diethylnitrosamine in the Pig-a, micronucleus and Comet assays integrated into 28-day repeat dose studies. Environ Mol Mutagen, 2011. 52(9): p. 711-20.

20. Stankowski, L. F., Jr., et al., Integration of Pig-a, micronucleus, chromosome aberration, and Comet assay endpoints in a 28-day rodent toxicity study with 4-nitroquinoline-1-oxide. Environ Mol Mutagen, 2011. 52(9): p. 738-47.

21. ICH, Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk M7. ICH Harmonised Tripartite Guideline, 2014.

Justification for classification or non-classification

Although a positive bacterial mutagenicity assay indicated a potential for mutagenicity in vitro, a follow up in vivo mutagenicity assay (pig A) was negative. Consequently it is concluded that at this time, the substance does not meet the criteria for classification for mutagenicity.