Disodium [6-[(2-anilino-1-naphthyl)azo]-2,4-dinitrophenolato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]naphthalene-1-sulphonato(3-)]chromate(2-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non genotoxic

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 28th to December 15th, 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Principles of method if other than guideline:

The experiments were performed using similar methods to those described in HRC protocol MCB 104 adopting modifications found in 'On concrete techniques for the mutagenicity test using micro-organisms' issued by the Japanese Ministry of Labour.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 100 and TA 98

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Prof. B.N. Ames, University of California, Berkeley, California, U.S.A.
- Storage temperature: - 80 °C
- Storage conditions: amploules containing 0.8 ml of bacterial suspension and 0.07 ml of dimethylsulphoxide.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: rof. National Collection of lndustrial Bacteria, Aberdeen, Scotland.
- Storage temperature: - 80 °C
- Storage conditions: amploules containing 0.8 ml of bacterial suspension and 0.07 ml of dimethylsulphoxide.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Range findig experiment: 0, 100, 1000 and 10000 µg/plate
Main experiment: 0, 50, 100, 500, 1000, 5000 and 10000 µg/plate

Vehicle / solvent:

Solvent: dimethylsulphoxide

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

methylmethanesulfonate

other: 4-notro-o-phanylenediamine // 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: the compound was tested using the poured plate method.

TEST CONDITIONS: 0.1 ml of bacterial suspension; 0.1 ml of test solution, 0.5 ml of sodium phosphate buffer or S9 mix, 2.0 ml top agar.

INCUBATION: 72 hours at 37 °C

PREPARATION OF S9-MIX
- Storage temperature: -80 °C
- Animals: rat (Crl: COBS CD (SD) BR); males, weighting ca 200 grams and approximately 8 weeks old.
- Induction material: Araclor 1254 (a polychlorinoted biphenyl mixture).
- Induction administration: single interperitoneal injection.
- Induction dose level: 500 mg/kg bw
- Induction period: 5 days.

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 100 and TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

With all five tester strains of S. typhimurium, test item, both in the presence and absence of liver microsomal fraction, provoked large dose related increases in revertant colony numbers.
With tester strain E. coli WP2 uvr, test item did not produce any substantial increases in revertant colony numbers.

Main test - Mean revertant colony counts obtained per plate using S. typhimurium strains TA 1535, TA 1597, TA 1538, TA 98 and TA 100 and E. coli strain WP2 uwA

Test concentration µ/plate	S9 mix	Reverse mutation (number of colonies/plate)
		Base pair exchange type			Frame shift type
		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537	TA 1538
Solvent control	-	71	10	25	24	6	14
Test item, 10000	-	307 p	6p	20 p	61 p	3 p	0 p
Test item, 5000	-	259	7	23	67	19	6
Test item, 1000	-	359	17	25	124	21	185
Test item, 500	-	242	20	21	99	23	167
Test item, 100	-	160	-	11	68	13	86
Test item, 50	-	117	11	17	44	12	50
Solvent control	+	88	13	12	22	9	12
Test item, 10000	+	120 p	80 p	14 p	70 p	20 p	30 p
Test item, 5000	+	321	56	22	108	47	76
Test item, 1000	+	369	33	27	129	35	196
Test item, 500	+	283	75	22	107	28	125
Test item, 100	+	162	-	16	58	19	58
Test item, 50	+	112	17	13	40	10	35
Methyl methane sulphonate, 500	-	894
N-ethyl-N'-nitro-N-nitrosoguanidine, 10	-		164
N-ethyl-N'-nitro-N-nitrosoguanidine, 5	-			1636
2-nitrofluorene, 2	-				2737
9-amino-acridine, 10	-					92
4-nitro-o-phenylenediamine, 10	-						861
2-amino-anthracene, 0.5	+	1164			726		51
2-amino-anthracene, 1	+		80			53
2-amino-anthracene, 40	+			224

Dose range finding test - Revertant colony counts obtained

Test concentration µ/plate	S9 mix	Reverse mutation (number of colonies/plate)
		Bose pair exchange type		Frame shift type
		TA 100	TA 1535	TA 98	TA 1537	TA 1538
Solvent control	-	58	5	18	9	10
Test item, 10000	-	218	19	49	3	13
Test item, 1000	-	271	17	102	29	173
Test item, 100	-	87	6	48	9	65
Test item, 10	-	79	14	28	9	21
Solvent control	+	72	4	9	5	9
Test item, 10000	+	203	5	127	38	35
Test item, 1000	+	297	23	93	32	178
Test item, 100	+	132	16	61	17	99
Test item, 10	+	71	6	15	10	26

WP2 uvrA was not tested because strain failed to grow on day of test

Conclusions:

Test item showed evidence of strong mutagenic potential when tested in the bacterial system.

Executive summary:

Microbial metabolic activation test was conducted to assess the potential mutagenic effect of the test item, using Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 100 and TA 98 strains and Escherichia coli WP2 uvrA strain. The experiments were performed using similar methods to those described in HRC protocol MCB 104 adopting modifications found in 'On concrete techniques for the mutagenicity test using micro-organisms' issued by the Japanese Ministry of Labour.

Dimethylsulphoxide was used as solvent. The substance was tested at concentrations of 0 (solvent), 50, 100, 500, 1000, 5000 and 10000 µg/plate. Adequate positive controls were also assayed.

With all five tester strains of S. typhimurium, test item, both in the presence and absence of liver microsomal fraction, provoked large dose related increases in revertant colony numbers.

With tester strain E. coli WP2 uvrA, test item did not produce any substantial increases in revertant colony numbers.

Conclusion

Test item showed evidence of strong mutagenic potential when tested in the bacterial system.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

June from 13th to 17th, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

Details on the read-across are available in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted July 21,1997

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS
- Proliferation rate: 12 - 14 h doubling time of the testing laboratory stock cultures.
- Storage: the V79 cells were stored over liquid nitrogen (vapour phase) in the cell bank of testing labopratory. This allows the repeated use of the same cell culture batch in experiments.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Properly maintained: thawed stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM).
- Periodically checked for Mycoplasma contamination: each cell batch was routinely checked for mycoplasma infections.
- Periodically 'cleansed' against high spontaneous background: for purifying the cell population of pre-existing HPRT- mutants cells were exposed to HAT medium containing 100 µM hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine and 10.0 µM glycine for several cell doublings (2-3 days).
- Complete Culture Medium: MEM medium supplemented with 10 % fetal bovine serum (FBS), 100 U/100 µg/ml penicillin/streptomycin, 2 mM L-glutamine, 25 mM HEPES and 2.5 µglml amphotericin B.

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Sprague Dawley phenobarbital and ß-naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

All concentrations used referred to the active components of test item.
Pre-experiment for experiment I without metabolic activation: 3.16, 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/ml
Pre-experiment for experiment I with metabolic activation: 1.0, 2.5, 5.0, 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/ml
Experiment I without metabolic activation: 0.025, 0.05, 0.10, 0.25, 0.50, 1.0, 2.5, 5.0, 10 and 20 µg/ml
Experiment I with metabolic activation: 10, 25, 50, 100, 150, 175, 200, 225, 250, and 275 µg/ml
Experiment II without metabolic activation: 10, 20, 40, 50, 60, 70, 80, 90 and 100 µg/ml
Experiment II with metabolic activation: 30, 60, 140, 220, 240, 250, 260, 270, 280 and 290 µg/ml

Vehicle / solvent:

- Vehicle: the test item was dissolved in cell culture medium (MEM + 0 % FBS 4h treatment; MEM + 10 % FBS 20h treatment) and diluted prior to treatment.
- Justification for choice of vehicle: the solvent was compatible with the survival of cells and the S9 activity.

Untreated negative controls:

yes

Remarks:

treatment medium, duplicate cultures

Negative solvent / vehicle controls:

no

Remarks:

not necessary

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

TREATMENT MEDIUM
MEM medium supplemented with 0 % fetal bovine serum (FBS) for short-term exposure, 10 % fetal bovine serum (FBS) for long-term exposure, 100 U/100 µg/ml penicillin/streptomycin, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/ml amphotericin B.

DURATION
Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as 4 h short-term exposure assay.
Experiment II without metabolic activation was performed as a 20 h long-term exposure assay.

SELECTIVE MEDIUM
MEM complete culture medium supplemented with thioguanine (11 µg/ml).

SEEDING OF THE CULTURES
Prior to use, cultures have been cleansed of pre-existing cells. Two or three days old exponentially growing stock cultures (more than 50 % confluent) were trypsinised at 37 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsine concentration for all subculturing steps was 0.05 %.
Approximately 1.0 × 10^6 cells per concentration, solvent/negative and positive control, were seeded in complete culture medium (MEM supplemented with 10 % FBS) in a culture flask, respectively.

TREATMENT
Approx. 24 h after seeding, cells were exposed to designated concentrations of test item either in presence or absence of metabolic activation in the mutation experiment. After 4 h (short time exposure) or 20 h (long time exposure) the treatment medium containing the test item was removed and the cells were washed twice with PBS. Subsequently complete medium (MEM supplemented with 10 % FBS) was added.
During the following expression period the cells of the logarithmic growing culture were subcultured 48 to 72 h after treatment. Additionally the cell density was measured (for toxicity criteria) and adjusted to 1 × 10^6 cells/ml.
At the end of the expression period for selection the mutants, about 4 × 10^5 cells from each treatment group, were seeded in cell culture petri dishes (diameter 90 mm) with selection medium containing lI µg/ml thioguanine (TG) for further incubation (about one week). At the end of the selection period, colonies were fixed and stained for counting.
The cloning efficiencies were determined in parallel to the selection of mutants.
For each treatment group two 25 cm2 flasks were seeded with approx. 200 cells to determine cloning efficiencies. After incubation for an appropriate time colonies were fixed, stained and counted.

DETERMINATION OF CYTOTOXICITY
The toxicity of the test item was determined in pre-experiments.
Eight concentrations (i.e. 3.16, 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/ml) were tested without metabolic activation. Ten concentrations 1.0, 2.5, 5.0, 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/ml were tested with metabolic activation The experimental conditions in these pre-experiments were the same as described for the main experiment with short-term exposure.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
The S9 liver microsomal fraction was obtained from Trinova Biochem GmbH, Giessen, Germany. Male Sprague Dawley rats were induced with phenobarbital / β-naphthoflavone.
The following quality control determinations were performed: Alkoxyresorufin-0-dealkylase activities and test for the presence of adventitious agents. Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene).
The following additional quality control determination was performed by testing laboratory: biological activity in the mouse lymphoma assay using benzo[a]pyrene; biological activity of the HPRT assay using 7,12-dimethylbenz[a]anthracene.
A stock of the supernatant containing the microsomes is frozen in aliquots of 5 ml and stored at ≤ 75 °C.
The protein concentration in the S9 preparation was 36.4 mg/ml.
An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/ml in the cultures.
Cofactors were added to the S9 mix to reach the concentrations: 8 mM MgCl2, 33 mM KCI, 5 mM Glucose-6-phosphate, 5 mM NADP in 100 mM sodium-phosphate-buffer pH7.4. During the experiment the S9 mix was stored on ice.

ACCEPTABILITY OF THE ASSAY
A mutation assay is considered acceptable if it meets the following criteria:
- negative and/or solvent controls fall within the performing laboratories historical control data range, i.e. 1-39 mutants/10^6 cells;
- the absolute cloning efficiency ([number of positive cultures × 100] I total number of seeded cultures) of the negative and /or solvent controls is > 50 %;
- the positive controls (EMS and DMBA) induce significant increases (at least 3-fold increase of mutant frequencies related to the comparable negative control values and higher than the historical range of negative controls) in the mutant frequencies.

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
- a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
- a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of the mutant frequency is not observed;
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In experiment I without metabolic activation, the mutant value of one negative control was within the historical control data of test facility (about 1-39 mutants per 10^6 cells), the other negative control showed a mutant value which is slightly higher than this data range (40.43 mutants per 10^6 cells). All mutant values of the test item concentrations found were within the historical control data of the test facility. No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies with the negative control were found to be 38.43 and 40.43 mutants/10^6 cell and in the range of 3.20 to 31.45 mutants/10^6 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 0.80 was found at a concentration of 0.025 µg/ml with a relative growth of 76.4 %. Due to the overall integrity of the data the experiment is still considered as valid.

With metabolic activation all values of the negative controls and most values of the test item concentrations found were within the historical control data of the test facility (about 2-39 mutants per 10^6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.
Mutation frequencies with the negative control were found to be 22.07 and 24.67 mutants/10^6 cell and in the range of 13.99 to 60.77 mutants/10^6 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.60 was found at a concentration of 250 µg/ml with a relative growth of 31.7 %.
At this concentration an increased number of mutant colonies (60.77 mutants per 10^6 cells) was observed, exceeding the historical data range, but the critical mutation factor of 3.0 was not reached. Due to the fact, that this elevated mutant value is enclosed from concentrations with normal mutant values, this finding is considered to be not biologically relevant.

In experiment II without metabolic activation the mutant value of one negative control was within the historical control data of the test facility (about 1-39 mutants per 10^6 cells), the other negative control showed a mutant value which is slightly higher than this data range (42.69 mutants per 10^6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.
Mutation frequencies with the negative control were found to be 42.69 and 31.47 mutants/10^6 cells and in the range of 19.48 to 49.36 mutants/10^6 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.33 was found at a concentration of 100 µg/ml with a relative growth of 20.1 %.
However, the experiment is still considered as valid as mutant values of the negative controls and test item concentrations are all within the same data range.

In experiment II with metabolic activation most mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility (about 2-39 mutants per 10^6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to then negative controls.
Mutation frequencies with the negative control were found to be 34.65 and 21.52 mutants/10^6 cells and in the range of 10.67 to 55.64 mutants/10^6 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.98 was found at a concentration of 140 µg/ml with a relative growth of 83.%.

POSITIVE CONTROLS
DMBA (1.0 and 1.5 µg/ml) and EMS (300 µg/ml) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

TOXICITY
Biologically relevant growth inhibition (reduction of relative growth below 70 %) was observed after the treatment with the test item in experiment I and II with and without metabolic activation.
In experiment I without metabolic activation the relative growth was 5.1 % for the highest concentration (20 µg/ml) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 275 µg/ml with a relative growth of 19.5 %.
In experiment II without metabolic activation the relative growth was 20.1 % for the highest concentration (100 µg/ml) evaluated. The highest concentration evaluated with metabolic activation was 290 µg/ml with a relative growth of 10 %.

Conclusions:

Under test conditions, test item was non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

The test item was assessed for the potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster. The selection of concentrations was based on data from pre-experiments. In experiment I 20 µg/ml (without metabolic activation) and 275 µg/ml (with metabolic activation) were selected as the highest concentrations. In experiment II 100 µg/ml (without metabolic activation) and 290 µg/ml (with metabolic activation) were selected as the highest concentrations. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h long time exposure assay.

The test item was investigated at the following concentrations: for experiment I without metabolic activation were used 0.025, 0.05, 0.10, 0.25, 0.50, 1.0, 2.5, 5.0, 10 and 20 µg/ml, while with metabolic activation were tested 10, 25, 50, 100, 150, 175, 200, 225, 250 and 275 µg/ml; for the experiment II without metabolic activation 10, 20, 40, 50, 60, 70, 80, 90 and 100 µg/ml were used, while with metabolic activation were assessed 30, 60, 140, 220, 240, 250, 260, 270, 280 and 290 µg/ml.

No precipitation of the test item was noted in the experiments. Biologically relevant growth inhibition (reduction of relative growth below 70 %) was observed after the treatment with test item in experiment I and II with and without metabolic activation. In experiment I without metabolic activation, the relative growth was 5.1 % for the highest concentration (20 µg/ml) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 275 µg/ml with a relative growth of 19.5 %. In experiment II without metabolic activation, the relative growth was 20.1 % for the highest concentration (100 µg/ml) evaluated. The highest concentration evaluated with metabolic activation was 290 µg/ml with a relative growth of 10.1 %.

In both experiments no biologically relevant increase of mutants was found after treatment with test item (with and without metabolic activation). No dose-response relationship was observed. DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Conclusion

Under test conditions, the test item can be considered as non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

From September 27th to October 17th, 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

Details on read-across are available in section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted on 24 May 1983

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial gene mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
Source of cells
The original strains were obtained from Prof. Bruce Ames and arrived at the testing laboratory on December 12, 1986.

Production of stock cultures
Immediately upon receipt, the samples were inoculated on nutrient agar plates, to which ampicillin had been added for the TA 100 and TA 98 strains. These plates were incubated at 37 °C fa; approximately 24 hours. Samples were taken from individual colonies with a sterile inoculation loop, and transferred to nutrient broth. In the case of TA 100 and TA 98, amphcillin had also been added to this broth. The samples were again incubated overnight at 37 °C. New samples of these cultures were inoculated onto nutrient agar plates, which had again been provided with ampicillin for TA 100 and TA 98. After an incubation period of approximately 24 hours at 37 °C, new samples of individual colonies from these plates were transferred to flasks containing approximately 30 ml normal nutrient broth. The inoculum was incubated overnight at 37 °C, after which a small sample was taken to check the genotype. At the same time, the remaining cultures were treated with DMSO to protect against the effects of freezingflg and immediately frozen at -80 °C in 1 ml portions.
No ampicillin-resistance test was done on the samples used for testing genotype since the cultures had already been sufficiently selecte by the ampicillin.
In addition to the test for crystal-violet sensitivity (deep rough character), a test was done for UV sensitivity (uvrB).

Whenever new stock cultures needed to be produced, individual cultures grown on nutrient agar were used, to which ampicillin had been added for the TA 100 and TA 98 strains. Samples of these individual colonies were then transferred to approximately 30 ml nutrient broth, incubated, divided up, and checked for crystal-violet and UV sensitivity.
One 1 ml-portion was thawed for each test and strain, and quantities of 0.2 ml of the thawed culture were added to 10 ml nutrient broth. The culture was incubated overnight at 37 °C and used only on the same day. A new, small stock culture, which had been checked for its properties directly before freezing, was thus available for each individual test.
In general this obviated any need to re-check the genotype for each Salmonella/microsome test. The procedure is in accprdance with the methods described by Ames et al. (1975) and Maron and Ames (1983).

CHECK OF GENOTYPE
- Histidine Requirement: In each individual test, histidine dependence of the cultures was automatically checked by the accompanying negative controls.
- Ampicillin Resistance (pKM 101): a special test for ampicillin resistance was not necessary since strains TA 100 and TA 98 were incubated on ampicillin containing nutrient agar and formed individual colonies. Consequently surviving bacteria were ampicillin resistant.
- Crystal-Violet Sensitivity (deep rough): a quantity of 0.1 ml was taken frgm the samples of individual stocks and spread onto nutrient gar, using four plates per strain. After a few minutes, fidlter papers, to which 10 µl of an aqueous, crystal-violet solution had been added at a concentration of 1 mg/ml, were placed in the middle of the plates. The plates were than incubated overnight at 37 °C. The diameters of the inhibition zones that had formed were then measured. The inhibition zones of all stock batches used indicated an adequate sehsitivity to crystal-violet.
- UV Sensitivity (uvrB): samples were spread onto nutrient agar plates. One half of each plate was covered with aluminium foil and irradiated without a lid for six seconds (TA 1535 and TA 1537) or eight seconds (TA 100 and TA 98) with UV light of a wavelength of 254 nm at a distance of 33 cm. The irradied plates were incubated and checked. To demonstrate adequate sensitivity in the test, cultures had to show an inhibition of growth over half their area, i.e. no bacteria should have grown on the irradiated half. This was the case with all the stock batches used.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 mix

Test concentrations with justification for top dose:

First test: 8, 40, 200, 1000 and 5000 µg/plate
I repeatition: 0, 125, 250, 500, 1000, 2000 and 4000 µg/plate
II repeatition: 0, 400, 600, 800, 1000, 1200, 1400 and 1600 µg/plate

Vehicle / solvent:

- Vehiclee used: water.
- Amount: 0.1 ml/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: nitrofurantoin and 4-nitro-1,2-phenylene diamine // 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION
- Bacteria suspension: the bacterial suspensions were obtained from 17-hour cultures in nutrient broth, which had been incubated at 37 °C and 90 rpm.
- Titre: the dilution of bacterial suspensions used for the determination of titers was 1:1000000. Titers were determined under the same conditions as mutations, except that the histidine concentration in the soft agar was increased from 0.5 mM to 2.5 mM to permit the complete growth of bacteria.
- Viability: the numbers of viable cells were established in a parallel procedure when determining the titers.
- Incubation: the count was made after the plates had been incubated for 48 hours at 37 °C. If no immediate count was possible, plates were temporarily stored in a refrigerator.

NUMBER OF REPLICATIONS: 4 plates were used, both with and without S9 mix, for each strain and dose.

DETERMINATION OF CYTOTOXICITY
The doses for the first trial were routinely determined on the basis of a standard protocol: 5000 µg or 5 Ml per plate were used as the highest dose, if not limited by solubility. At least four additional doses were routinely assayed. The results of the first experiment were then considered as a pre-test for toxicity. Doses of repeats were chosen on the basis of the results obtained in the first experiment.
The toxicity of the substance was assessed in three ways: the first was a gross appraisal of background growth on the plates for mutant determination; secondly, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls; thirdly, the titer was determined. Total bacterial counts were taken on two plates for each concentration studied with S9 mix. However, if an evaluattion was performed only without S9 mix, the bacterial count was taken without S9 mix.

S9 MIX
S9 mix was made from the livers of at least six adult male Sprague Dawley rats, of approximately 200 to 300 g in weight. For enzyme induction, the animals received a single intraperitoneal injection of Aroclor 1254, dissolved in corn oil, at a dose of 500 mg/kg body weight, five days before sacrifice. The animals were prepared unfasted, following the directions of Ames et al. (1975) and Maron and Ames (1983).
The rats were killed by cergical dislocation. Livers were removed under sterile conditions immediately after sacrifice and kept at 4 °C until all animals had been prepared. All the remaining steps were carried out under sterile conditions at 4 °C.
The livers were washed with cold (4 °C), 0.15 M KCl solution (approximately 1 ml KCl per 1 g liver), and then homogenized in fresh, cold (4 °C), 0.15 M KCl (approximately 3 ml KCl per 1 g liver). The homogenate was then centrifuged in a cooling centrifuge at 34 °C and 9000 g for 10 minutes. The supernatant (the S9 fraction) was stored at -80 °C in small portions. These portions were slowly thawed before using. The S9 mix was freshly prepared (Ames et al., 1973) and used only on the same day. It was placed in a vessel with a double glass wal‘ until used. The hollow wall was filled with ice to keep the S9 mix permanently cold.

TRUE NEGATIVE CONTROL
No "untreated" negative control was set up for deionized water, since sufficient evidence was available in the literature (i.e. Maron and Ames, 1983) and from testing laboratory own experience, indicating that this solvent had no influence in the spontaneous mutant counts of the bacterial strais used.

ACCEPTANCE OF THE ASSAY
The following criteria determined the acceptance of an assay:
a) the negative controls had to be within the expected range, as defined by published data (i.e. Maron and Ames, 1983) and the laboratories own historical data;
b) the positive controls had to show sufficient effects, as defined by the laboratories experience;
c) titer determinations had to demonstrate sufficient bacterial density in the suspension.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result.
For TA 1535, TA 100 and TA 98 this increase should be about twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.
In case of questionable results, investigatios should continue, possibly with modifications, until a final evaluation is possible.

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

reproducibile findings at precipitating concentrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

up to and including 800 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

up to and including 800 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 1535 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

up to and including 800 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Two of the four strains concerned revealed a dose-related increase in mutant counts to double those of negative controls. Strains TA 1537 and TA 98 were affected.
The findings for Salmonella typhimurium TA 1537 were confirmed by further repeat tests.
The findings for Salmonella tyghimurium TA 98 were confirmed by one further repeat test. However, another repeat failed to demonstrate mutagenic effects. Due to positive results in two of three experiments, the effect was considered relevant.
The lowest dose at which this finding was reproducible was approximately 1000 µg per plate for Salmonella typhimurium TA 1537 and approximately 600 µg per plate for Salmonella typhimurium TA 98. For TA 1537 positive findings were obtained only without S9 mix. Positive finfijngs for TA 98 were obtained with and without S9 mix, the effects being comparable in both cases.

The Salmonella/microsome test, employing doses up to 5000 µg per plate, showed test item to produce bacteriotoxic effects only at 800 µg per plate and above. Substance flocculation in soft agar occurred at 1000 µg per plate and above. Therefore doses from 4000 µg per plate and above could not be used for assessment.
Evaluation of individual dose groups, with response relevant assessment parameters (dose effect, reproducibility), revealed biologically relevant variations from the respective negative controls for TA 1537 and TA 98. These were regarded as mutagenic effects of test item. Since the lowest effective doses at which this finding were reproducible were in the medium, respectively high, dose range and only slight increases of mutant counts were noted the Salmonella/microsome test showed substance to be a weak mutagen.
In spite of the low doses used, positive controls increased the mutant counts to well over those of the negative controls, and thus demonstrated the system's high sensitivity. Due to the sensitivity, indications of weak mutagenic effects of test item could be found at assessable doses up to 2000 µg pe plate in Salmonella typhimurium TA 1537 and TA 98.

TOXICITY AND SOLUBILITY
There was no indication of a bacteriotoxic effect of test item at doses of up to and including 800 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. Nor was any inhibition of growth noted. Higher doses had weak, strain-specific bacteriotoxic effect. At 1000 µg per plate, the substance started to flocculate in soft agar so that from 4000 µg per plate no further evaluation was possible.
Due to the substance's precipitation and mutagenicity, doses ranging from 125 µg to 4000 µg per plate were chosen for the repeat tests.

Summary of mean values without S9 mix

µg plate	Strain
	TA 1535	TA 100	TA 1537	TA 98
0	15	110	12	28
8	15	117	11	34
40	13	118	14	38
200	14	112	17	50
1000	13	125	32	68
5000	P	P	P	P
Na-azid	537	243	-	-
NF	-	-	-	-
4-NPDA	-	-	45	65
0	12	102	9	32
125	10	92	19	37
250	10	98	17	37
500	14	108	19	38
1000	7	115	30	46
2000	11	109	23	43
4000	P	P	P	P
Na-azid	747	-	-	-
NF	-	460	-	-
4-NPDA	-	-	74	129
0	-	-	6	33
400	-	-	22	52
600	-	-	20	56
800	-	-	24	56
1000	-	-	34	66
1200	-	-	32	65
1400	-	-	31	59
1600	-	-	21	62
4-NPDA	-	-	83	291

Summary of mean values with S9mix

µg plate	Strain
	TA 1535	TA 100	TA 1537	TA 98
0	22	159	15	34
8	29	165	11	46
40	25	182	14	38
200	23	172	15	57
1000	18	192	23	85
5000	P	P	P	P
2-AA	152	663	75	464
0	15	126	13	36
125	17	145	14	54
250	14	153	13	55
500	15	124	18	53
1000	15	109	23	50
2000	11	107	22	42
4000	P	P	P	P
2-AA	253	394	443	1304
0	-	-	13	52
400	-	-	12	71
600	-	-	14	83
800	-	-	15	83
1000	-	-	26	87
1200	-	-	22	83
1400	-	-	18	82
1600	-	-	24	65
2-AA	-	-	116	586

Conclusions:

The Salmonella/microsome test showed test item to have a weak mutagenic effect.

Executive summary:

The substance was investigated using the Salmonella microsome test for point mutagenic effects in doses up to 5000 µg per plate on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98. Doses up to and including 800 µg per plate did not cause any bacteriotoxic effects: total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a weak, strain-specific bacteriotoxic effect. Substance flocculation in soft agar occurred at the dose 1000 µg per plate and above, such that from 4000 µg per plate, the results were no longer interpretable.

Evidence of mutagenic activity of test item was seen. On Salmonella typhimurium TA 1537 and TA 98, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Both with and without S9 mix, there was a positive response for TA 98 and the effect was comparable. For TA 1537 a positive response was found only without S9 mix. The lowest reproducible effective dose was 1000 µg per plate for Salmonella typhimurium TA 1537 and 600 µg per plate for TA 98. The Salmonella/microsome test thus showed test item to have a weak mutagenic effect.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Conclusion

The Salmonella/microsome test thus showed test item to have a weak mutagenic effect.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Non genotoxic

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From October 26th to November 24th, 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

Details on the read-across are available in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann Borchen Germany.
- Age at study initiation: 8 to 12 weeks, young adult male and virgin female mice.
- Weight at study initiation: 28 to 43 g.
- Assigned to test groups randomly: yes.
- Housing: the females were kept in groups of a maximum of three mice in Makrolon type I cages. Males were kept singly in type I cages.
- Diet: Altromin 1324 Standard Diet, ad libitum.
- Water: tap water and feed were available ad libitum.
- Acclimation period: at least one week.
- Health check: the breed's state of health was regularly spot-checked for the major specific pathogens.

ENVIRONMENTAL CONDITIONS
- Temperature: 22.5 - 23 °C
- Humidity: 44 - 52 %
- Air changes: ten times per hour.
- Photoperiod: 12 hrs dark / 12 hrs light, electrical lighting daily of about 500 lux.

Route of administration:

intraperitoneal

Vehicle:

- Vehicle used: physiol. saline.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test item was dissolved in physiological saline solution, using a magnetic stirrer for 30 minutes, stirred with a magnetic mixer during administration and injected intraperitoneally.
Administration volume was 10 ml/kg bw.

Duration of treatment / exposure:

Administration at 16, 24, or 48 hours

Frequency of treatment:

Single dose

Dose / conc.:

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

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: cyclophosphamide.
- Justification for choice of positive control: routinely used positve control.
- Vehicle: physiological saline.
- Route of administration: intraperitoneal.
- Doses / concentrations: 20 mg/kg bw.

Tissues and cell types examined:

Bone marrow (femur)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
The selection of test item dose was based on a pilot test, in which groups of five animals, including both males and females, were intraperitoneally administered 100 mg/kg, 150 mg/kg, 250 mg/kg and 500 mg/kg.
Based on these results, 150 mg/kg test item was chosen for the main test.

DETAILS OF SLIDE PREPARATION
At least one intact femur was prepared from each sacrificed animal (not pretreated with a spindle inhibitor). A suitable instrument was used to sever the pelvic bones and lower leg. The femur was separated from muscular tissue.
The lower-Ieg stump, including the knee and all attached soft parts, was separated in the distal epiphyseal cartilage by a gentle pull at the distal end.
The proximal end of the femur was opened at its extreme end with a suitable instrument, e.g. fine scissors, making visible a small opening in the bone-marrow channel. The femur was then completely immersed in the calf serum and pressed against the wall of the tube, to prevent its slipping off.

A suitable tube was filled with sufficient fetal calf serum.
A small amount of serum was drawn from the tube into a suitable syringe with a thin cannula. The cannula was pushed into the open end of the marrow cavity. The femur was then completely immersed in the calf serum and pressed against the wall of the tube, to prevent its slipping off.
The contents were then flushed several times and the bone marrow was passed into the serum as a fine suspension.
Finally, the flushing might be repeated from the other end, after it had been opened.

The tube containing the serum and bone marrow was centrifuged in a suitable centrifuge at approximately 1000 rpm for five minutes.
The supernatant was removed with a suitable pipette (e.g. Pasteur pipette), leaving only a small remainder.
The sediment was mixed to produce a homogeneous suspension.

One drop of the viscous suspension was placed on a well cleaned slide and spread with a suitable object, to allow proper evaluation of the smear.
The labeled slides were dried overnight. If fresh smears needed to be stained, they needed to be dried with heat for a short period.

The staining of smears.
The smears were stained automatically with an Ames HemaTek Slide Stainer from the Miles Company. The slides were then "destained" with methanol, rinsed with deionized water and left to dry.

METHOD OF ANALYSIS
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. They can be distinguished from artifacts by varying the focus.
Normally, 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern.

Evaluation criteria:

A test was considered positive if, at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control.
A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time.
A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of negative controls.
In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. In this case, a second test had to be performed at the most sensitive interval.

An assay was considered acceptable if the figures of negative and positive controls were within the expected range, in accordance with the laboratory's experience and/or the available literature data.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The results with test item gave no relevant indications of clastogenic effects after a single intraperitoneal treatment with 150 mg/kg.

Toleration by the animals
After single intraperitoneal administration of 150 mg/kg test item treated animals showed the following compound-related symptoms until sacrifice: apathy back discoloration of hairless parts of skin, staggering gait, spasm, difficulty in breathing and eyelids stuck together.
Their feeding behavior was normal. One of 40 treated animals died during the test period, due to the acute toxicity of 150 mg/kg test item.
No symptoms were recorded for the control groups. No animals died in these groups.

Microscopic Evaluation
Concerning the assessment of the clastogenic potential of test item, there were no relevant variations in results between males and females. Therefore, they were evaluated jointly.
As animal 12 of the 48 hours group exhibited a pathological change of the ratio polychromatic to normochromatic erythrocytes, it was not included in the evaluation.
The ratio of polychromatic to normochromatic erythrocytes was altered by the treatment with test item, being 1000: 1056 (1s=386) in th gative control, 1000: 2370 (1s=1258) in the 16 hogts group, 1000: 1537 (1s=593) in the 24 hours group and 1000: 1733 (1s=1012) in the 48 hours group. Relevant vaiations were thus noted.
No biologically important or statistically significant variations existed between the negative control and the groups treated intraperitoneally with 150 mg/kg, with respect to the incidence of micronucleated polychromatic erythrocytes. The incidence of these micronucleated cells was 1.3 / 1000 (1s=0.7) in the negative control, and 1.6 / 1000 (1s=1.6), 2.0 / 1000 (1s=1.7) and 1.6 / 1000 (1s=0.9) in the test item groups.
Similarly, there could be no biologically significant variation between the negative control and Ttest item groups in the number of micronucleated normechromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones. As expected, relevant variations were not observed.

POSITIVE CONTROL
The positive control, cyclophosphamigg, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. The incidence of micronucleated cells was 14.9 / 1000 (1s=9.8), which represe ts a biologically relevant increase in comparison to the negative control.
There could not have been a biologically relevant effect on the number of micronuclhated normochromatic erythrocytes in the positive control since, in conjunction with the cell-cycle duration, normochromatic erythrocytes originated from polychromat ones.
No further effect of cyclophosphamide was found concerning the ratio of polychromatic to normochromatic erythrocytes, since the ratio did not vary to a biologically relevant degree [1000: 674 (1s=232), as against 1000: 1056 in the negative control. This clearly demonstrates that an alteration of the ratio of polychromatic to normochromatic erythrocytes is not necessary for the induction of micronuclei.

RESULTS OF RANGE-FINDING STUDY
The following symptoms were recorded for up to 48 hours, starting at 100 mg/kg: apathy, lack discoloration of hairless parts of skin, staggerig gait, sternal recumbency, spasm, difficulty in breathing and eyelids stuck together. In addition, 1 of 5 animals died in the 150 mg/kg group and 5 of 5 animals died in the 250 and 500 mg/kg groups.

Results of micronucleus test with test item after acute peritoneal treatment with 150 mg/kg bw

Group	Number of evaluated PCE	NCE/1000 PCE	Micronucelated cells per 1000 NCE	Micronucleated Cells per 1000 PCE
negative control	10000	1056 ± 386	1.2 ± 1.2	1.3 ± 0.7
test 16 hours	10000	2370 ± 1258	1.2 ± 1.0	1.6 ± 1.6
test 24 hours	10000	1537 ± 593	1.1 ± 0.8	2.0 ± 1.7
test 48 hours	10000	1733 ± 1012	0.5 ± 0.7	1.6 ± 0.9
positive control	10000	674 ± 232	0.4 ± 0.8	14.9 ± 9.8

Conclusions:

The test item did not cause the formation of micronuclei in mice in vivo.

Executive summary:

Test substance was assayed in the micronucleus test in male and female mice for a possible clastogenic effect on the chromosomes of bone-marrow erythroblasts. The known clastogen and cytostatic agent, cyclophosphamide, served as positive control. Treated animals received a single intraperitoneal administration of either test item or cyclophosphamide. The femoral marrow of groups treated with the substance was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours. Doses of test item and positive control, cyclophosphamide, were 150 and 20 mg/kg body weight, respectively.

Animals treated with test item showed symptoms of toxicity after administration. One of forty animals died before the end of the test due to the acute intraperitoneal toxicity of 150 mg/kg. There was an altered ratio between polychromatic and normochromatic erythrocytes.

No indications of a clastogenic effect of test item were found after a single intraperitoneal treatment with 150 mg/kg.

Cyclophosphamide, the positive control, had a clear clastogenic effect, as is shown by the biologically relevant increase in polychromatic erythrocytes with micronuclei.

The ratio of polychromatic to normochromatic erythrocytes was not altered.

Conclusion

Test item did not cause the formation of micronuclei in mice in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Evaluation of the genetic toxicity has been performed with the integrated evaluation of the following studies: in vitro Ames tests, in vitro gene mutation on mammalian cells and in vivo chromosomal aberration.

An AMES test, showing positive results, is available on Acid Black 222, however there is no data on the mutagenic potential in mammalian cells. Therefore, available data on a structural analogue Similar Substance 01 was taken into account to deeply investigate the genetic toxicity potential. The read across approach can be considered as appropriate and suitable to assess the property under investigation. Details are available in section 13.

IN VITRO GENE MUTATION IN BACTERIA

Potential mutagenic effects of the Acid Black 222 were assessed on Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 100 and TA 98 strains and Escherichia coli WP2 uvrA strain. The experiments were performed following the poured plate method. Test substance caused dose related increase in revertant colony numbers in all S. typhimurium strains, both in presence and absence of liver microsomal fraction; with E. coli WP2 uvrA strain, test item did not produce any substantial increases in revertant colony numbers.

Similar Substance 01 was investigated using Salmonella TA 1535, TA 100, TA 1537 and TA 98 strains. On Salmonella typhimurium TA 1537 and TA 98, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Both with and without S9 mix, there was a positive response for TA 98 and the effect was comparable. For TA 1537, a positive response was found only without S9 mix. The lowest reproducible effective dose was 1000 µg per plate for Salmonella typhimurium TA 1537 and 600 µg per plate for TA 98. Overall, the substance showed a weak mutagenic effect.

IN VITRO GENE MUTATION IN MAMMALIAN CELLS

Similar Substance 01 was assessed for the potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster. Biologically relevant growth inhibition (reduction of relative growth below 70 %) was observed after treatment with test item in experiments performed, with and without metabolic activation. In both experiments, no biologically relevant increase of mutants was found after treatment with test item. No dose-response relationship was observed.

IN VIVO CHROMOSOMAL ASSAY

The micronucleus test on Similar Substance 01 was employed to investigate test item in mice for a possible clastogenic effect on the chromosomes of bone-marrow erythroblasts. Animals treated with test item showed symptoms of toxicity after administration; one of forty animals died before the end of the test due to the acute intraperitoneal toxicity of 150 mg/kg. No indications of a clastogenic effect of test item were found after a single intraperitoneal treatment with 150 mg/kg. The ratio of polychromatic to normochromatic erythrocytes was not altered.

DISCUSSION AND CONCLUSION

Gene mutation test in bacteria available on Acid Black 222 showed positive results, thus available experiments in mammalian cells using the structural analogue Similar Substance 01 have been taken into consideration to clarify the potential genotoxicity of the substance under investigation.

Different results in non-mammalian system and in mammalian cell tests may be addressed by considering possible differences in substance uptake and metabolism, or in genetic material organisation and ability to repair. In general, results of mammalian tests are regarded as of higher significance.

In the AMES test conducted on Acid Black 222, there are no information about cytotoxicity: it seems that the impact of test item on the viability of bacteria was not specifically investigated.

Similar Substance 01 showed cytotoxicity in the Ames test and in the HPRT assay.

In addition, precipitation was seen in gene mutation tests in bacteria with Acid Black 222 and Similar Substance 01. It is not clear if it might have had an impact on the study outcomes.

It is known that some nitro compounds are positive in AMES test but mostly negative or weakly positive in vivo tests. However, it has been demonstrated in various publications that this is a bacteria-specific effect and that these Ames positive substances are not mutagenic in mammalian assays. This has been explained by the metabolism of bacteria, which has specific enzyme nitro-reductase. The nitroreductase family comprises a group of flavin derivative dependent enzymes that are able to metabolize nitroaromatic and nitroheterocyclic derivatives (nitrosubstituted compounds) using the reducing power of nicotinamide adenine dinucleotide (NAD(P)H). These enzymes can be found in bacterial species and, to a lesser extent, in eukaryotes. The nitroreductase proteins play a central role in the activation of nitrocompounds. Mutant strains of bacteria such as Salmonella typhimurium can non express or overexpress these enzymes (de Oliveira et al. 2010).

Therefore, nitroreductase in bacteria can be considered a metabolic pathway, unlikely relevant for humans (Kirkland et al, 2014).

Similar Substance 01 gave negative results in both in vitro gene mutation assay and in vivo micronucleous test performed in mammalian cells, confirming that the positive outcomes in bacteria cells are likely due to specific organism metabolism.

In conclusion, the review of all the available information suggests that test substance is not expect to be able to induce heritable mutations in mammalian cells.

REFERENCE

De Oliveira IM, Bonatto D, Pega Henriques JA. Nitroreductases: Enzymes with Environmental Biotechnological and Clinical Importance. In Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology; Mendez-Vilas, A., Ed.; Formatex: Badajoz, Spain, 2010:1008–1019.

Kirkland D, Zeiger E, Madia F, Gooderham N, Kasper P, Lynch A, Morita T, Ouedraogo G, Morte J.M. P, Pfuhler S, Rogiers V, Schulz V, Thybaud V, van Benthem J,Vanparys P, Worth A, Corvi R. (2014).Canin vitromammalian cell genotoxicity test results be used tocomplement positive results in the Ames test and help predictcarcinogenic orin vivogenotoxic activity? I. Reports of individualdatabases presented at an EURL ECVAM Workshop. Mutation Research 775–776 (2014) 55–68

Justification for classification or non-classification

According to the CLP Regulation (EC ) No 1272/2008, for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The available information indicate that test substance did not raise any concern from the genotoxicity point of view.

 

In conclusion, the substance does not meet the criteria to be classified for genetic toxicity according to the CLP Regulation (EC) No 1272/2008.