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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative results obtained in both in vitro and in vivo studies.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6-28-1989 to 7-31-1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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:

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

Test concentrations with justification for top dose:

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

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

congo red

other: 2-Anthramine, benzidine

Details on test system and experimental conditions:

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

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

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

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

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

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

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

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

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

Evaluation criteria:

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

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

Statistics:

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

Key result

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

All dose groups tested.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

All dose groups tested.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

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

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

All dose groups tested.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

All dose groups tested.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

All dose groups tested.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

All dose groups tested.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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

Conclusions:

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

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

Executive summary:

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

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine auxotrophs of Salmonella typhimurium and tryptophan auxotrophs of Escherichia coli.

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared from the livers of male Sprague-Dawley rats

Test concentrations with justification for top dose:

Five concentrations of the test material (50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: A yellow colour (becoming orange/red with increasing test material concentration) was noted from 50 μg/plate. A red test material precipitate was also observed at and above 150 μg/plate. Neither of these observations prevented the scoring of revertant colonies. The test material was insoluble in both sterile distilled water and dimethyl sulphoxide at 50 mg/ml (the most concentrated stock solution) but was fully soluble in acetone at the same concentration in solubility checks performed in-house. Acetone was therefore selected as the vehicle of choice.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate incorporation method

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard cofactors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate. The experiment was repeated on a separate day using the same dose range as the rangefinding test, fresh cultures of the bacterial strains and fresh test material formulations.

Tester Strains:
Salmonella typhimurium TA1535, TA1537, TA98 and TA100
Escherichia coli WP2uvrA

The Salmonella typhimurium strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 whilst Escherichia coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association on 17 August 1987. All of the strains were stored at -196ºC. Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37ºC for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Preparation of Test and Control Materials:
The test material was insoluble in both distilled water and dimethyl sulphoxide at 50 mg/ml in solubility checks performed in–house but was fully soluble in acetone at the same concentration. Acetone was therefore selected as the vehicle of choice.
The test material was accurately weighed and approximate half-log dilutions prepared in acetone by mixing on a vortex mixer on the day of each experiment. As an aid to dosing, glass (instead of plastic) tubes were employed at and above 1.5 mg/ml to prevent test material adherence. Analysis for concentration, homogeneity and stability of the test material formulations is not a requirement of the test guidelines and was, therefore, not determined. Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) i.e. 2 mm pellets with a nominal pore diameter of 4 x 10^-4 microns.
Vehicle and positive controls were used in parallel with the test material. A solvent treatment group was used as the vehicle control and the positive control materials were as follows:
N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 μg/plate for WP2uvrA-, 3 μg/plate for TA100 and 5 μg/plate for TA1535
9-Aminoacridine (9AA): 80 μg/plate for TA1537
4-Nitroquinoline-1-oxide (4NQO): 0.2 μg/plate for TA98

In addition, 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP), which are non-mutagenic in the absence of metabolising enzymes, were used in the series of plates with S9-mix at the following concentrations:
2AA at 1 μg/plate for TA100
2AA at 2 μg/plate for TA1535 and TA1537
2AA at 10 μg/plate for WP2uvrABP
at 5 μg/plate for TA98

Microsomal Enzyme Fraction:
S9 was prepared in-house on 28 November 2004 from the livers of male Sprague-Dawley rats weighing ~ 250g. These had each orally received three consecutive daily doses of phenobarbitone/β-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on Day 4. Before use, each batch of S9 was assayed for its ability to metabolise appropriate indirect mutagens used in the Ames Test. The S9 was stored at -196ºC.

S9-Mix and Agar:
The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration of the test.
S9: 5.0 ml
1.65 M KCl/0.4 M MgCl2: 1.0 ml
0.1 M Glucose-6-phosphate: 2.5 ml
0.1 M NADPH: 2.0 ml
0.1 M NADH: 2.0 ml
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 ml
Sterile distilled water: 12.5 ml

A 0.5 ml aliquot of S9-mix and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
Top agar was prepared using 0.6% Difco Bacto agar and 0.5% sodium chloride with 5 ml of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 ml of top agar. Vogel-Bonner Minimal agar plates were purchased from ILS Ltd.

Test Procedure Preliminary Toxicity Test:
In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. The assay was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA-), 0.1 ml of test material formulation, 0.5 ml of S9-mix or phosphate buffer and 2 ml of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test material and a vehicle control (acetone) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37ºC the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at and above 500 μg/plate because of excessive test material precipitation.

Test Procedure Mutation Test – Experiment 1 (Range-finding Test):
Five concentrations of the test material (50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37ºC for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter. Manual counts were performed at and above 500 μg/plate because of excessive test material precipitation. Manual counts were performed by hand by marking individual colonies with a marker pen and recording the count.

Test Procedure Mutation Test – Experiment 2 (Main Test):
The second experiment was performed using methodology as described for the range-finding test, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as the range-finding test (50 to 5000 μg/plate).

Evaluation criteria:

Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method section 3 with historical control ranges for 2002 and 2003 presented in Appendix 2.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.

All tester strain cultures should be in the approximate range of 1 to 9.9 x 10^9 bacteria per ml. Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation Criteria:
The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression (5)) significant increase in the revertant count in at least one strain of bacteria.

Statistics:

The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression (5)) significant increase in the revertant count in at least one strain of bacteria.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Negative at all dose levels for cytotoxicity, Precipitate formed at 150, 500, 1500 and 5000 ug/plate

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Negative at all dose levels for cytotoxicity, Precipitate formed at 150, 500, 1500 and 5000 ug/plate

Key result

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:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Negative at all dose levels for cytotoxicity, Precipitate formed at 150, 500, 1500 and 5000 ug/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels.

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Negative at all dose levels for cytotoxicity, Precipitate formed at 150, 500, 1500 and 5000 ug/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

Mutation Test:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The S9-mix used in both experiments of the main test was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation for Experiment 1 (Range-Finding Test), are presented in Tables 2 and 3; the same results and information for Experiment 2 (Main Test) are presented in Tables 4 and 5.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A yellow colour (becoming orange/red with increasing test material concentration) was noted from 50 μg/plate. A red test material precipitate was also observed at and above 150 μg/plate. Neither of these observations prevented the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: other: Preliminary Toxicity Test

Remarks:

Migrated from field 'Test system'.

Preliminary Toxicity Test:The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9- mix	Strain	Dose (μg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	113	131	106	133	134	135	126	117P	102P	98P	104P
+	TA100	108	107	132	100	120	117	108	116P	115P	108P	95P
-	WP2uvrA	25	22	35	26	22	26	22	23P	16P	20P	16P
+	WP2uvrA	25	31	37	34	33	26	34	38P	30P	27P	25P

P: Precipitate

Table 1 Spontaneous Mutation Rates (Concurrent Negative Controls)

Range-finding Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA-		TA98		TA1537
106	(100)	24	(26)	38	(31)	21	(22)	9	(7)
102		31		31		19		3
93		22		23		26		8

 

Main Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA-		TA98		TA1537
75	(71)	16	(16)	31	(27)	30	(29)	7	(10)
71		16		26		29		9
66		16		23		29		13

 

Table 2 Test Results: Range-Finding Test– Without Metabolic Activation

Test Period		From: 21 January 2005					To: 24 January 2005
With or without S9-Mix	Test substance concentration (μg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA-		TA98		TA1537
-	0(V)	106	(107) 4.0#	22	(25) 5.5	25	(24) 1.2	17	(23) 5.5	15	(13) 2.5
		103		31		23		27		10
		111		21		25		26		13
-	50	87	(89) 11.7	37	(32) 5.0	19	(22) 8.5	19	(21) 2.1	11	(9) 1.5
		79		31		16		22		9
		102		27		32		23		8
-	150	103P	(99) 10.6	36P	(30) 6.0	21P	(19) 3.5	21P	(21) 2.0	14P	(15) 1.2
		87P		24P		21P		19P		16P
		107P		31P		15P		23P		14P
-	500	104P	(102) 9.6	17P	(19) 7.8	17P	(18) 2.3	21P	(20) 5.1	9P	(11) 1.5
		111P		28P		17P		14P		11P
		92P		13P		21P		24P		12P
-	1500	117P	(106) 10.5	25P	(26) 3.1	23P	(21) 2.1	19P	(22) 3.1	11P	(12) 1.0
		105P		23P		19P		25P		12P
		96P		29P		22P		21P		13P
-	5000	106P	(106) 6.5	27P	(21) 4.9	14P	(20) 5.3	14P	(19) 4.6	11P	(10) 3.1
		113P		18P		24P		23P		13P
		100P		19P		22P		20P		7P
Positive controls S9-Mix -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG		4NQO		9AA
		3		5		2		0.2		80
		600	(690) 79.6	812	(906) 100.5	977	(997) 19.5	102	(106) 4.0	812	(818) 10.1
		721		894		998		110		813
		750		1012		1016		107		830

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA 9-Aminoacridine

P Precipitate

V Vehicle (acetone) control

# Standard deviation

Table 3 Test Results: Range-Finding Test– With Metabolic Activation

Test Period		From: 21 January 2005					To: 24 January 2005
With or without S9-Mix	Test substance concentration (μg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA-		TA98		TA1537
+	0(V)	102	(111) 17.7#	13	(15) 2.1	38	(36) 3.8	34	(37) 9.3	20	(20) 5.5
		131		17		32		29		15
		99		16		39		47		26
+	50	130	(115) 13.3	12	(14) 2.1	41	(33) 8.5	35	(36) 5.1	20	(25) 5.0
		104		13		24		32		30
		112		16		34		42		25
+	150	110P	(116) 7.2	17P	(14) 3.6	29P	(26) 3.1	33P	(36) 3.1	22P	(20) 2.6
		124P		15P		27P		39P		21P
		114P		10P		23P		35P		17P
+	500	105P	(108) 4.6	13P	(16) 4.4	41P	(24) 15.5	28P	(29) 1.7	15P	(20) 4.6
		105P		21P		11P		31P		21P
		113P		14P		19P		28P		24P
+	1500	103P	(102) 2.1	11P	(15) 3.2	36P	(33) 2.5	35P	(28) 8.3	18P	(22) 6.1
		104P		17P		33P		31P		19P
		100P		16P		31P		19P		29P
+	5000	108P	(109) 11.5	15P	(14) 1.5	27P	(29) 2.0	30P	(31) 2.3	15P	(20) 4.4
		121P		12P		31P		34P		23P
		98P		14P		29P		30P		22P
Positive controls S9-Mix +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA		BP		2AA
		1		2		10		5		2
		360	(334) 35.9	463	(441) 20.6	545	(520) 46.2	261	(296) 30.4	363	(403) 48.7
		293		422		549		311		388
		349		439		467		316		457

BP Benzo(a)pyrene

2AA 2-Aminoanthracene

P Precipitate

V Vehicle (acetone) control

# Standard deviation

Conclusions:

negative with metabolic activation All strains at all dose levels.
negative without metabolic activation All strains at all dose levels.

The test material, Automate (™) Yellow 8-Solvent Stripped, was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction:

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471" Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guideline No.870.5100.

Methods:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

Results:

The vehicle (acetone) control plates gave counts of revertant colonies within the historical control range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A yellow colour (becoming orange/red with increasing test material concentration) was noted from 50 μg/plate. A red test material precipitate was also observed at and above 150 μg/plate. Neither of these observations prevented the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 September 2018 - 09 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Identification: Black solid with orange tinge
Batch Number: TE2245
EC Number: 915-586-1
Purity: 100% (UVCB*)
Physical State/Appearance: Black viscous liquid with orange tinge
Expiry Date: 06 September 2020
Storage Conditions: Room temperature, in the dark
No correction for purity was required.

Target gene:

The tk (thymidine kinase) locus in mouse lymphoma L5178Y cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the
MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were
originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and
were frozen in liquid nitrogen at that time.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

Lot No. PB/βNF S9 29/03/18 was used in this study, and was pre-prepared in-house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test.
S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0. 20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and Mutagenicity Test.

Test concentrations with justification for top dose:

0.31, 0.63, 1.25, 2.5, 5.0, 10 μg/mL

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test.

Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10% are excludedfrom the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.

Vehicle / solvent:

Solvent: Acetone, Acros batch 1845786, purity treated as 100%, expiry 22/08/19

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

positive controls were formulated in DMSO

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Test Item Preparation
The test item was a UVCB* therefore the maximum proposed dose level in the solubility test was initially set at 5000 μg/mL, the maximum recommended dose level, and no correction for the purity of the test item was applied. The test item was found to be insoluble in R0 medium at 50 mg/mL, and dimethyl sulfoxide (DMSO) at 500 and 250 mg/mL. The test item formed a solution in acetone at 500 mg/mL considered acceptable for dosing in the solubility checks. Acetone is toxic to L5178Y cells at dose volumes greater than 0.5% of the total culture volume. Therefore, the test item was formulated at 500 mg/mL and dosed at 0.5% to give the maximum achievable dose level of 2500 μg/mL. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm at the concentration levels investigated (Scott et al., 1991).

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Control Preparation
Vehicle and positive controls were used in parallel with the test item in the Mutagenicity Test. Solvent (acetone, Acros batch 1845786, purity treated as 100%, expiry 22/08/19) exposure groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) (Sigma batch BCBV9352, purity treated as 100%, expiry 10.05.23) at 400 μg/mL and 150 μg/mL, respectively, was used as the positive control in the 4-hour and 24-hour exposure groups in the absence of metabolic activation. Cyclophosphamide (Acros Organics batch A0373263, purity 97%, Expiry 22.02.19) at 1.5 μg/mL was used as the positive control in the presence of metabolic activation. The positive controls were formulated in DMSO.

Test procedures
Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24-hour exposure period without S9. Due to the high levels of precipitate observed in the solubility test, the dose range used in the preliminary toxicity test was 0.63 to 160 μg/mL for all three of the exposure groups. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained. The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiment. Maximum dose levels were selected using the following criteria:
i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 μL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.
ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

Mutagenicity Test
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (0.31 to 40 μg/mL for all three of the exposure groups), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 0.1 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group). The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Rationale for test conditions:

In accordance with OECD guidelines.

Evaluation criteria:

The Historical Vehicle and Positive Control data is generated by the Mutant 240C program on a rolling system of the last twenty sets of archived data. The program combines the 4-hour and 24-hour data in the absence of metabolic activation as the acceptability criteria is the same for all three of the exposure groups.

An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, which is based on the analysis of the distribution of the vehicle control MF data from participating
laboratories.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system. Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly
negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Preliminary Cytotoxicity Test
There was evidence of modest dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in the 4-hour exposure group in the presence of metabolic activation, and more marked reductions in the 24-hour exposure group in the absence of metabolic activation. Precipitate of the test item was observed at and above 10 μg/mL in the 4-hour and 24-hour exposure groups in the absence of metabolic activity, and at and above 20 μg/mL in the 4-hour exposure group in the presence of metabolic activation, at the end of the exposure periods. Therefore, following the recommendations of the OECD 490 guideline, the maximum dose levels in the Mutagenicity Test were limited by the onset of test item precipitate in all three of the exposure groups.

Mutagenicity Test
There was no evidence of any marked reductions in %RSG or RTG values in the 4-hour exposure groups in the absence or presence of metabolic activation, and evidence of very modest reductions in the 24-hour exposure group in the absence of metabolic activation. There was no evidence of any reductions in viability (%V) in any of the three exposure groups, indicating that residual toxicity had not occurred. At the end of the exposure periods, precipitate of the test item was observed at and above 10 μg/mL in all three of the exposure groups. Therefore, following the recommendations of the OECD 490 guideline, the lowest precipitating dose level of 10 μg/mL was plated for viability and 5-TFT resistance in each of the exposure groups and the subsequent dose levels were discarded as they were surplus to requirements. Acceptable levels of toxicity were seen with the positive control substances. The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional. The test item did not induce any toxicologically significant or dose related increases in the mutant frequency x 10-6 per viable cell at any of the dose levels, including the lowest precipitating dose level in all three of the exposure groups.

Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 0.63 to 160 μg/mL.

The results for the Relative Suspension Growth (%RSG) were as follows:

 

Dose (µg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
0.63	95	103	102
1.25	95	97	116
2.5	81	87	102
5	83	87	108
10	102	97	105
20	79	89	125
40	93	71	108
80	90	72	70
100	90	70	53

 

Main experiment

Concentration(µg/mL)	4-hours-S9			Concentration(µg/mL)	4-hours+S9
	%RSG	RTG	MF§		%RSG	RTG	MF§
0	100	1.00	154.45	0	100	1.00	165.53
0.31	103	1.10	157.57	0.31	98	0.98	157.31
0.63	112	1.23	145.89	0.63	98	0.90	160.80
1.25	94	1.08	151.53	1.25	103	0.94	157.54
2.5	109	1.08	174.61	2.5	102	1.09	129.28
5	103	1.03	163.30	5	97	0.95	156.97
10	103	1.08	177.48	10	107	1.02	147.07
20Ø	113			20Ø	95
40Ø	115			40Ø	103
MF threshold for a positive response = 280.45				MF threshold for a positive response = 291.53
Positive control				Positive control
EMS				CP
400	89	0.66	1051.6	1.5	93	0.71	741.54

 

Concentration(µg/mL)	24-Hours-S9
	%RSG	RTG	MF§
0	100	1.00	164.09
0.31	84	1.03	122.97
0.63	106	1.04	151.47
1.25	103	1.02	156.97
2.5	87	0.86	161.98
5	74	0.78	172.59
10	78	0.83	166.20
20Ø	67
40Ø	80
MF threshold for a positive response = 290.09
Positive control
EMS
150	43	0.43	1783.83

 

 

Cell and 96-Well Plate Counts: Mutagenicity Test (-S9) 4-Hour Exposure

Concentration (µg/mL)		Cell counts $			Viability § after day 2 2 cells/well				Resistant mutants § after day 2 2000 cells/well
		0 h	24 h	48 h
0	A	8.51	6.22	6.52	78	73	80	80	25	21	21	22
	B	9.75	6.38	6.06	73	78	75	74	17	19	18	24
0.31	A	9.86	5.81	6.15	73	78			22	24
	B	9.90	6.58	5.98	82	82			26	19
0.63	A	10.93	5.63	6.99	83	76			16	19
	B	9.59	6.08	6.49	79	80			27	25
1.25	A	11.04	5.62	5.53	82	82			18	23
	B	9.40	6.11	5.83	75	82			25	26
2.5	A	10.21	5.24	6.75	77	78			25	26
	B	9.95	6.24	6.87	72	77			21	20
5	A	9.91	6.16	5.57	77	78			24	21
	B	9.88	5.99	6.83	71	80			20	23
10	A	10.47	4.91	7.44	73	81			24	24
	B	9.56	6.07	6.18	82	75			23	27
20	A	10.31	7.05	6.25	NP	NP			NP	NP
	B	9.88	5.20	6.97	NP	NP			NP	NP
40	A	9.34	6.31	6.49	NP	NP			NP	NP
	B	10.29	5.84	7.47	NP	NP			NP	NP
Positive control EMS (µg/mL)
400	A	10.29	5.36	6.19	59	62			74	63
	B	10.28	4.93	6.04	68	74			63	70

 

Summary Analysis: Mutagenicity Test (-S9) 4-Hour Exposure

Concentration(µg/mL)	SG	%RSG	%V	RTG	MF§
0	9.91	100	79.37	1.00	154.45
0.31	9.39	103	85.83	1.10	157.57
0.63	9.87	112	88.05	1.23	145.89
1.25	8.33	94	90.38	1.08	151.53
2.5	9.77	109	78.43	1.08	174.61
5	9.42	103	79.70	1.03	163.30
10	9.35	103	83.01	1.08	177.48
20Ø	10.12	113
40Ø	10.60	115
Positive control EMS
400	7.87	89	57.74	0.66	1051.60

GEF =126, therefore MF threshold for a positive response = 280.45

 

Large and Small Colonies Analysis: Mutagenicity Test (-S9) 4-Hour Exposure

Concentration(µg/mL)		Viability # after day 2				Small colonies # after day 2				Larger colonies # after day 2
0	A	78	73	80	80	10	8	10	10	15	13	11	12
	B	73	78	75	74	9	8	8	11	8	11	10	13
0.31	A	73	78			8	12			14	12
	B	82	82			10	9			16	10
0.63	A	73	76			8	10			8	9
	B	79	80			13	12			14	13
1.25	A	82	82			6	9			12	14
	B	75	82			12	14			13	12
2.5	A	77	78			10	8			15	18
	B	72	77			9	7			12	13
5	A	77	78			9	11			15	10
	B	71	80			10	9			10	14
10	A	73	81			8	7			16	17
	B	82	75			9	12			14	15
400 EMS	A	59	62			36	36			38	27
	B	68	74			42	25			21	42

 

Mutation frequencies

Concentration (µg/mL)	Viable		Small colonies			Large colonies			Proportionsmall colony mutants
			Mutants			Mutants
	Yv	Nv	Ym	Nm	MF§	Ym	Nm	MF§
0	157	768	694	768	63.8	675	768	81.3	0.44
0.31	69	384	345	384	62.4	332	384	84.8	0.42
0.63	66	384	341	384	67.4	340	384	69.1	0.49
1.25	63	384	343	384	62.5	333	384	78.8	0.45
2.5	80	384	350	384	59.1	326	384	104.4	0.37
5	78	384	345	384	67.2	335	384	85.6	0.44
10	73	384	348	384	59.3	322	384	106.1	0.37
400 EMS	121	384	242	384	399.8	256	384	351.1	0.53

 

Cell and 96-Well Plate Counts: Mutagenicity Test (+S9) 4-Hour Exposure

Concentration(µg/mL)		Cell counts $			Viability§after day2 2cells/well				Resistant mutants § after day 2 2000 cells/well
		0h	24h	48h
0	A	10.60	5.24	6.94	81	77	81	79	26	23	23	23
	B	10.61	5.69	8.19	76	74	73	74	17	22	22	19
0.13	A	11.32	5.67	6.46	72	80			24	24
	B	11.31	4.73	8.16	78	79			19	20
0.63	A	10.41	4.72	7.85	79	73			22	22
	B	11.39	4.48	9.33	74	72			17	20
1.25	A	11.28	5.34	8.11	77	74			19	17
	B	10.90	4.40	8.66	70	72			20	23
2.5	A	11.39	4.91	6.93	79	83			19	21
	B	11.42	4.80	9.22	80	77			17	20
5	A	10.55	5.20	7.48	76	80			24	24
	B	10.38	4.54	9.16	72	74			19	18
10	A	12.00	4.99	6.45	80	78			22	24
	B	10.91	5.00	9.92	81	70			17	15
20	A	9.81	5.16	7.65	NP73	NP			NP	NP
	B	9.64	5.67	8.23	NP	NP			NP	NP
40	A	10.02	5.21	8.29	NP	NP			NP	NP
	B	9.999	4.52	10.26	NP	NP			NP	NP
Positive control CP (µg/mL)
1.5	A	11.50	4.21	8.93	65	70			53	60
	B	10.71	4.02	8.98	69	68			59	58

 

Summary Analysis: Mutagenicity Test (+S9) 4-Hour Exposure

Concentration(µg/mL	SG	%RSG	%V	RTG	MF§
0	10.34	100	80.67	1.00	165.53
0.13	9.50	98	81.66	0.98	157.31
0.63	9.88	98	73.67	0.90	160.80
1.25	10.21	103	73.10	0.94	157.54
2.5	9.80	102	86.56	1.09	129.28
5	10.13	97	79.70	0.95	156.97
10	10.22	107	77.20	1.02	147.07
20	10.75	95
40	11.28	103
Positive control CP (µg/mL)
1.5	9.21	93	61.61	0.71	741.54

GEF =126, therefore MF threshold for a positive response = 291.53

 

 

Large and Small Colonies Analysis: Mutagenicity Test (+S9) 4-Hour Exposure

Concentration(µg/mL)		Viability # after day 2				Small colonies # after day 2				Large colonies # after day 2
0	A	81	77	81	79	11	13	7	11	15	15	16	12
	B	76	74	73	74	5	6	7	10	12	16	15	9
0.13	A	72	80			11	12			13	12
	B	78	79			9	6			10	14
0.63	A	74	73			5	10			17	12
	B	70	72			8	11			9	9
1.25	A	77	74			3	4			16	13
	B	79	72			11	10			9	13
2.5	A	80	83			5	9			14	12
	B	76	77			3	6			14	14
5	A	72	80			11	12			13	12
	B	80	74			7	6			12	12
10	A	81	78			9	9			13	15
	B	73	70			6	4			11	11
1.5 CP	A	65	70			45	36			8	24
	B	69	68			30	32			29	26

Mutation frequencies

Concentration(µg/mL)	Viable		Small colonies			Large colonies			Proportionsmall colony mutants
			Mutants			Mutants
	Yv	Nv	Ym	Nm	MF§	Ym	Nm	MF§
0	153	768	698	768	59.2	685	768	95.8	0.39
0.31	75	384	346	384	63.8	335	384	83.6	0.44
0.63	88	384	350	384	62.9	337	384	88.6	0.42
1.25	89	384	356	384	51.8	333	384	97.5	0.35
2.5	68	384	361	384	35.7	330	384	87.5	0.30
5	78	384	348	384	61.8	335	384	85.6	0.42
10	82	384	356	384	49.0	334	384	90.4	0.36
1.5 CP	112	384	241	384	378.1	297	384	208.5	0.62

 

 

Cell and 96-Well Plate Counts: Mutagenicity Test (-S9) 24-Hour Exposure

Concentration(µg/mL)		Cell counts $			Viability§after day2 2cells/well				Resistant mutants § after day 2 2000 cells/well
		0h	24h	48h
0	A	7.37	8.17	6.45	73	80	75	73	18	21	22	23
	B	7.61	7.44	5.43	77	75	81	80	24	25	23	22
0.13	A	7.19	6.61	6.29	81	82			18	21
	B	7.06	7.60	5.84	83	79			20	20
0.63	A	8.33	7.11	6.30	77	72			23	20
	B	7.26	8.70	5.16	82	81			20	23
1.25	A	7.29	8.96	5.68	83	72			21	22
	B	7.70	8.98	4.98	73	78			21	21
2.5	A	7.71	7.09	5.82	72	75			22	21
	B	7.21	8.84	4.34	80	80			22	23
5	A	7.59	7.62	4.91	82	77			23	25
	B	6.74	8.04	4.70	72	76			25	20
10	A	7.51	8.63	4.69	79	73			16	24
	B	6.80	8.66	4.53	78	77			25	25
20	A	6.72	9.23	5.44	NP	NP			NP	NP
	B	5.40	9.43	4.73	NP	NP			NP	NP
40	A	7.70	7.57	5.22	NP	NP			NP	NP
	B	6.77	9.11	4.37	NP	NP			NP	NP
Positive control EMS (µg/mL)
150	A	5.78	7.92	4.11	70	68			86	81
	B	6.08	7.45	4.12	69	72			90	89

 

Summary Analysis: Mutagenicity Test (-S9) 24-Hour Exposure

Concentration(µg/mL)	SG	%RSG	%V	RTG	MF§
0	57.87	100	80.34	1.00	164.09
0.31	51.28	84	93.66	1.03	122.97
0.63	58.85	106	98.70	1.04	151.47
1.25	59.72	103	79.70	1.02	156.97
2.5	50.31	87	80.34	0.86	161.98
5	44.93	74	80.34	0.78	172.59
10	47.53	78	80.34	0.83	166.20
20 Ø	47.92	67
40 Ø	48.22	80
Positive control EMS
150	31.25	43	64.83	0.43	1783.83

GEF =126, therefore MF threshold for a positive response = 290.09

 

Large and Small Colonies Analysis: Mutagenicity Test (-S9) 24-Hour Exposure

Concentration(µg/mL)		Viability # after day 2				Small colonies # after day 2				Large colonies # after day 2
0	A	73	80	75	73	10	13	12	9	8	8	10	14
	B	77	75	81	80	10	7	7	11	14	18	16	11
0.13	A	81	82			8	4			10	17
	B	83	79			6	8			14	12
0.63	A	77	72			10	6			13	14
	B	82	81			8	8			12	15
1.25	A	83	72			7	12			14	10
	B	73	78			4	10			17	11
2.5	A	72	75			12	6			10	15
	B	80	80			15	14			7	9
5	A	82	77			9	12			14	13
	B	72	76			11	6			14	14
10	A	79	73			11	12			5	12
	B	78	77			10	8			15	17
150 CP	A	70	68			50	51			36	30
	B	69	72			47	50			43	39

Mutation frequencies

Concentration(µg/mL)	Viable		Small colonies			Large colonies			Proportionsmall colony mutants
			Mutants			Mutants
	Yv	Nv	Ym	Nm	MF§	Ym	Nm	MF§
0	154	768	689	768	67.6	669	768	85.9	0.44
0.31	59	384	358	384	37.4	331	384	79.3	0.33
0.63	72	384	352	384	52.0	330	384	80.5	0.37
1.25	78	384	351	384	56.4	332	384	91.3	0.39
2.5	77	384	337	384	81.3	343	384	70.3	0.53
5	77	384	346	384	64.9	329	384	96.2	0.41
10	77	384	343	384	70.3	335	384	85.0	0.46
150 EMS	105	384	186	384	559.0	236	384	375.4	0.57

 

Historical Vehicle and Positive Control Mutation Frequencies

Experiments -S9

Vehicle control	Positive control (EMS)
MF	MF
119.09	913.73
135.00	1168.73
158.36	1133.94
167.39	1203.85
158.52	1594.01
148.54	950.32
114.37	1353.40
150.65	1427. 33
131.90	1166.45
134.70	1916.01
114.72	1036.64
132.82	2124.87
116.65	1266.71
122.91	1373.76
134.37	1637.45
133.14	1707.56
166.66	1319.01
132.03	1274.43
147.75	1407.64
138.98	1681.70
Mean: 137.93	Mean: 1382.38
SD: 16.52	SD: 315.08
Minimum: 114.37	Minimum: 913.73
Maximum: 167.39	Maximum: 2124.87

 

Historical Vehicle and Positive Control Mutation Frequencies

Experiments +S9

 

Vehicle control	Positive control (EMS)
MF	MF
119.09	913.73
135.00	1168.73
158.36	1133.94
167.39	1203.85
158.52	1594.01
148.54	950.32
114.37	1353.40
150.65	1427. 33
131.90	1166.45
134.70	1916.01
114.72	1036.64
132.82	2124.87
116.65	1266.71
122.91	1373.76
134.37	1637.45
133.14	1707.56
166.66	1319.01
132.03	1274.43
147.75	1407.64
138.98	1681.70
Mean: 141.854	Mean: 996.48
SD: 16.60	SD: 333.61
Minimum: 116.93	Minimum: 534.63
Maximum: 183.21	Maximum: 1972.34

 

Conclusions:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

 

Methods

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (acetone), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows: 0.31, 0.63, 4-hour with S9 (2%) 1.25, 2.5, 5, 10 μg/mL.

 

Results

The maximum dose levels in the Mutagenicity Test were limited by the presence of test item precipitate in all three of the exposure groups, as recommended by the OECD 490 guideline.

The vehicle control cultures had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

 

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted in general conformance with applicable Environmental Protection Agency, Toxic Substances Control Act, Good Laboratory Practice standards with the following exceptions: 1. Test substance characterization and stability data were not developed by SRI International. 2. Assays to verify concentration, stability, and homogeneity of the test substance in the carrier vehicle were not performed. These deviations should not affect the results or conclusions of this study.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test Animal specification:
Swiss-Webster mice, 123 males and 123 females, born on May 16, 1989, were received by the SRI Laboratory Animal Medicine Department (LAMD) from Charles River Breeding Laboratories, Inc. (Colony POI) on June 14, 1989. The weights of 10 male and 10 female mice selected randomly ranged from 16.5 to 19.3 g and 14.2 to 16.1 g, respectively, at the time the animals were received. These animals were used for the range-finding study conducted the week of June 25, 1989.
Swiss-Webster mice, 120 males and 119 females, born on July 18, 1989, were received on August 16, 1989. The weights of 10 male and 10 female mice selected randomly ranged from 13.0 to 14.7 g and 12.0 to 13.2 g, respectively, at the time the animals were received. These animals were used for the definitive study conducted the week of August 27, 1989.

Supplier:
Charles River Breeding Laboratories, Inc., [Colony POI], Shaver Road, Portage, MI 49081.

Test System Identification:
The animals were randomized and uniquely identified by ear punch. Cards attached to the outside of each cage contained the study number, test group and subgroup number, dose, and the ear-punch numbers of the animals housed in that cage.

Quarantine:
Mice received on June 14, 1989, and August 16, 1989, were quarantined for seven days and were released on June 21, 1989, and August 23, 1989, respectively. No sign or evidence of significant clinical disease was observed at any time during the quarantine periods or during the study. No notable gross pathologies were found in either 12 male or 12 female mice that were necropsied during the two quarantine periods. Mice not used for this study were assigned to concurrently conducted studies within the same project.

Animal Room Environmental Conditions:
Rooms: Building L, Rooms W110 and W107
Temperature range: 60.8 to 84°F
Humidity range: 38 to 81%
Light cycle: 12 hours light/12 hours dark
Cage specification: Mice were housed no more than 10/cage during quarantine and 5/cage during test period in polycarbonate cages containing hardwood-chip bedding.

Food and water Supply:
Food: Purina certified Rodent Chow #5002 ad libitum, Ralston Purina Co., St. Louis, MO., Lot nos. APR5891C, MAR30891A, MAY23892B, MAY23892A.
Water: Purified tap water ad libitum via automatic watering system. Water-purity analysis on file, currently in Building 203/45.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
Name: Corn oil.
Lot no.: FEB1490L.
storage conditions: stored at 4°C.
Received: December 20, 1988.
Expiration: December 20, 1989.
Supplier: Mazola, Mission Supermarkets, Best Foods CPC International, Inc. Englewood Cliffs, NJ 07632.
Disposition: Retained as vehicle control.

Name: Corn oil.
Lot no.: L12590.
Storage conditions: Stored at 4°C.
Received: July 28, 1989.
Expiration: July 28, 1990.
Supplier: Springfield, PW Supermarkets certified Grocers of Calif., LTD. Los Angeles, CA 90040.
Disposition: Retained as vehicle control.

Details on exposure:

Preliminary Dose-range Assay:
Male and female swiss-Webster mice were given a single dose of the test article by oral intubation (gavage) on Days 1, 2, 3, and 4 and were sacrificed on Day 5. Mice, weighed individually, were dosed (three per group) with Automate Yellow 8 Petroleum at 0, 300, 600, 1200, 2500, or 5000 mg/kg body weight (BW).

Definitive Assay:
The test chemical was suspended in corn oil and administered by gavage at 0, 1200, 2500, or 5000 mg/kg BW. The total volume of test suspension administered per kilogram of body weight was 10 mI. Mice were dosed with Automate Yellow 8 Petroleum for four consecutive days and sacrificed approximately 24 hours after the final dose of Automate Yellow 8 Petroleum.

Duration of treatment / exposure:

Mice were dosed with Automate Yellow 8 Petroleum for four consecutive days and sacrificed approximately 24 hours after the final dose of Automate Yellow 8 Petroleum.

Frequency of treatment:

Mice were given a single dose of the test article by oral intubation (gavage) for four consecutive days and sacrificed approximately 24 hours after the final dose.

Post exposure period:

The mice were sacrificed approximately 24 hours after the final dose.

Remarks:

Doses / Concentrations:
0, 1200, 2500, or 5000 mg/kg BW
Basis:
nominal conc.

No. of animals per sex per dose:

5 animals/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Benzene (500 mg/kg in corn oil) was the positive control. The purpose of the positive control is to ensure proper conduct of the assay procedures. The positive control agent was administered only to male mice.

Name: Benzene (99+% pure, 500 mgjkg BW).
Lot no.: 03825EV.
Received: October 10, 1988.
Expiration: October 10, 1998.
Storage conditions: Stored at room temperature.
Supplier: Aldrich Chemical, 940 W. st. Paul Avenue, Milwaukee, WI 53233.
Disposition: Retained as positive control.

Tissues and cell types examined:

The target cell population tested consists of erythroblasts undergoing their final chromosome replication and mitosis prior to expulsion of the main nucleus.

Cytological Analysis:
Blood smears were evaluated using epifluorescence microscopy. Two parameters were determined: (1) the number of micronucleated RNA-positive erythrocytes among a total of 1000 RNA-positive erythrocytes per animal, which provides an index of chromosomal damage, and (2) the number of RNA-positive erythrocytes among 5000 erythrocytes per animal, which provides an index of cytotoxicity to the nucleated erythrocyte precursors. The criteria for micronuclei are those described by Schmid (1976), with the additional requirement that they exhibit the fluorescent characteristic of the staining combination (i.e., bright yellow in the case of acridine orange stain). The ratio of RNA-containing erythrocytes to mature erythrocytes (RBC) was based on the number of RNA-positive cells among approximately 5000 erythrocytes. Data from a given slide were entered directly into an IBM PC data file while scoring. After analyses were completed, the slides were decoded and the data were summarized using a decoding program on the IBM PC.

Details of tissue and slide preparation:

Peripheral Blood Micronucleus Assay:
Blood samples were obtained by pricking the ventral tail vessel with a 25-gauge needle and drawing 2-3 ul of blood into a capillary tube. The sample was transferred to a clean microscope slide, spread, air-dried, fixed in absolute methanol for 5 minutes, and stored until staining. Three slides were prepared from each animal. Immediately prior to scoring, one of the three coded slides from the test animal was stained with acridine orange (Hayashi et al., 1983).

Data Collection:
Slides for micronucleus evaluation were coded using random letter codes generated by an IBM PC computer program. Slide labels were printed directly from the computer. Slides were coded by an individual not involved in the microscopic evaluation.

Evaluation criteria:

Criteria for a Valid Assay:
The data from this assay were considered acceptable if the frequency of micronucleated cells in the vehicle control group was within the normal historical range, if the positive control article resulted in a statistically significant elevation in the incidence of micronucleated cells, and if there were a minimum of three surviving animals of each sex with a percentage of RNA-positive erythrocytes greater than or equal to 15% of the control value.

Statistics:

Statistical Tests Employed:
Data from each sex were analyzed both separately and combined, unless a statistically significant sex difference was observed between the vehicle control groups. The frequency of micronucleated RNA-containing erythrocytes among RNA-positive erythrocytes (i.e., the frequency of micronucleated PCES) and the percentage of RNA-positive erythrocytes among total erythrocytes were calculated for each animal. The statistical significance of differences in the percentage of RNA-positive erythrocytes among groups was evaluated using the Kruskall-Wallace analysis of variance on ranks. If a significant overall difference was observed, the individual dose groups that differed from the control were determined by using a distribution-free multiple comparison test (Gad and Weil, 1986).
The micronucleus frequency data were analyzed as follows: In experiments in which frequencies of micronucleated cells are based on scoring 1000 cells per animal, data are not expected to be distributed normally. Such data are analyzed using the CochranArmitage test for trend in binomial proportions, to determine if a significant dose-response relationship was present, and the normal test for equality of proportions, to determine if individual dose groups were statistically elevated above controls. These tests and their rationale are discussed in the ASTM Standard Guide for Conduct of Micronucleus Assays in Mammalian Bone Marrow Erythrocytes (ASTM Committee, 1988).

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

All dose levels.

Toxicity:

no effects

Remarks:

All dose levels.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary Dose Range Assay Results:
A preliminary dose-range assay was performed to determine the appropriate dose levels for the definitive micronucleus study. Male and female swiss-Webster mice were given a single dose of the test article by oral intubation (gavage) on Days 1, 2, 3, and 4 and were sacrificed on Day 5. Mice, weighed individually, were dosed (three per group) with Automate Yellow 8 Petroleum at 0, 300, 600, 1200, 2500, or 5000 mg/kg body weight (BW). One male mouse in the 600 mg/kg BW dose group was observed as having rough hair; no animals died as a result of Automate Yellow 8 Petroleum administration. Treatment with the above doses produced polychromatic erythrocyte to red blood cell (PCE/RBC) ratios of 1.30, 1.27, 1.59, 1.67, 1.41, and 1.35%, respectively, in males and 1.57, 1.40, 1.44, 1.51, 1.48, and 0.96%, respectively, in females. The high dose of Automate Yellow 8 Petroleum was greater than 50% of the control PCE ratio (1.35% vs 1.30%, males; 0.96% vs 1.57%, females). Therefore, this dose was selected as the high dose for the definitive assay. No other statistical evaluations of the range-finding data were deemed necessary.

Conclusions:

Interpretation of results: negative
On the basis of the results, it is concluded that Automate Yellow 8 Petroleum does not induce MN in polychromatic erythrocytes from swiss-Webster mice under the conditions of this assay.

Executive summary:

The peripheral erythrocyte micronucleus (MN) assay was used to evaluate the clastogenic potential of Automate Yellow 8 Petroleum. Doses of 0, 1200, 2500, and 5000 mg/kg BW of Automate Yellow 8 Petroleum in males produced PCE/RBC values of 1.68, 1.80, 1.58, and 1.62%, respectively. In females, the same doses produced PCE/RBC values of 1.58, 1.50, 1.37, and 1.54%, respectively. In males, doses of 1200, 2500, and 5000 mg/kg BW yielded 0.06, 0.22, and 0.16% PCE with MN, respectively, compared with a vehicle control value of 0.20%. In females, the same doses yielded 0.22, 0.20, and 0.24% PCE with MN, respectively, compared with a vehicle control value of 0.26%. In contrast, benzene yielded 3.90% PCE with MN in male mice. None of the doses of Automate Yellow 8 Petroleum induced a statistically significant increase in the micronucleus frequency.

On the basis of the results, it is concluded that Automate Yellow 8 Petroleum does not induce MN in polychromatic erythrocytes from swiss-Webster mice under the conditions of this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In vitro Bacterial Mutagenicity

Study 1:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (acetone) control plates gave counts of revertant colonies within the historical control range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A yellow colour (becoming orange/red with increasing test material concentration) was noted from 50 μg/plate. A red test material precipitate was also observed at and above 150 μg/plate. Neither of these observations prevented the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Study 2:

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

in vivo Clastogenicity:

The peripheral erythrocyte micronucleus (MN) assay was used to evaluate the clastogenic potential of Automate Yellow 8 Petroleum. Doses of 0, 1200, 2500, and 5000 mg/kg BW of Automate Yellow 8 Petroleum in males produced PCE/RBC values of 1.68, 1.80, 1.58, and 1.62%, respectively. In females, the same doses produced PCE/RBC values of 1.58, 1.50, 1.37, and 1.54%, respectively. In males, doses of 1200, 2500, and 5000 mg/kg BW yielded 0.06, 0.22, and 0.16% PCE with MN, respectively, compared with a vehicle control value of 0.20%. In females, the same doses yielded 0.22, 0.20, and 0.24% PCE with MN, respectively, compared with a vehicle control value of 0.26%. In contrast, benzene yielded 3.90% PCE with MN in male mice. None of the doses of Automate Yellow 8 Petroleum induced a statistically significant increase in the micronucleus frequency.

On the basis of the results, it is concluded that Automate Yellow 8 Petroleum does not induce MN in polychromatic erythrocytes from swiss-Webster mice under the conditions of this assay.

Summary:

In the two available bacterial mutagenicity assays Automate Yellow 8 was negative in the presence and absence of metabolic activity. In the in vivo micronucleus assay it also failed to produce any evidence of clastogenicity. It is therefore concluded that this substance is not mutagenic or clastogenic and at this time, requires no further assessment of genotoxicity.

In Vitro Mammalian Cell Gene Mutation Test: L5178Y TK +/- Mouse Lymphoma Assay

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

 

Methods

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (acetone), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows: 0.31, 0.63, 4-hour with S9 (2%) 1.25, 2.5, 5, 10 μg/mL.

 

Results

The maximum dose levels in the Mutagenicity Test were limited by the presence of test item precipitate in all three of the exposure groups, as recommended by the OECD 490 guideline.

The vehicle control cultures had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

 

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Justification for classification or non-classification

Does not meet the criteria for classification for mutagenicity.