[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 30 November 2010; Experiment completion date - 23 December 2010; Study completion date - 21 February 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: "Kanpoan No. 287 -- Environment Protection Agency" "Eisei No. 127 -- Ministry of Health &Welfarew "Heisei 09110131 Kikyoku No. 2 -- Ministry of International Trade & Industry"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Substance type: reactive dyestuff
- Physical state: orange powder
- Analytical purity: 69.9% of all colored components
- Lot/batch No.: TZ 5891 / BOP 02-09
- Expiration date of the lot/batch: 2014-07-31
- Storage condition of test material: At room temperature at about 20 °C

Specific details on test material used for the study:

Identification: FAT 40851/A TE
Batch Number: TZ 5891 / BOP 02-09
Purity: 69.9 % all coloured components
Appearance: Orange powder
Expiry Date: July 31, 2014
Storage Conditions: At room temperature at about 20 °C

Method

Target gene:

Histidine locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA were obtained from Trinova Biochem GmbH (35394 Gießen, Germany).

Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in Harlan CCR according to B. Ames et al. and D. Maron and B. Ames. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

Storage The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 %DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

Metabolic activation:

with and without

Metabolic activation system:

S9 (Preparation by Harlan C C R) Phenobarbital/ -Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old male Wistar rats (Hsd Cpb: WU, Harlan Laboratories GmbH, 33178 Borchen, Germany), weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335 Hamburg) and β -Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at -80 °C. Small numbers of the ampoules can be kept at -20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo(a)pyrene. The protein concentration in the S9 preparation was 34.1 mg/mL (lot no. R 100909) in both experiments.

S9 Mix: Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 10 % v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCl2; 33 mM KCl ; 5 mM Glucose-6-phosphate; 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

Deionised water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation - TA 1535, TA 100 - 10 µg/plate

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

Without metabolic activation - TA 1537 (50 µg/plate), TA 98 (10 µg/plate.-

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation - E.coli WPA uvrA (3 µl/plate)

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation - 2.5 µg/plate (TA 1535, TA 1537, TA 98, TA 100), 10 µg/plate (WP2 uvrA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Two

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: approx. 72 h

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants (below the indication factor of 0.5)

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

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 examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

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 examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

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 examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

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 examined

Positive controls validity:

valid

Test results 5

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Any other information on results incl. tables

Table 1: Summary of results – pre-test and experiment 1

Metabolic activation	Test group	Dose level [µg/plate]	Revertant colony counts (mean ± SD)
			TA1535	TA1537	TA98	TA100	WP2uvrA
without	water		18±	11±1	31±3	136±21	57±5
	untreated		17±4	12±7	26±5	122±14	47±6
	test substance	3	17±2	13±4	30±4	125±6	59±2
		10	18±7	12±3	32±4	123±12	52±7
		33	15±3	14±2	32±3	131±4	52±10
		100	14±3	12±2	33±9	141±11	60±8
		333	15±2	11±4	29±3	129±17	47±7
		1000	13±5	9±2	28±0	143±6	57±5
		2500	13±1	11±2	28±4	133±13	50±11
		5000	9±1	8±2	24±4	146±10	44±16
	NaN3	10	2058±37 *			2263±17 *
	4-NOPD	10			264±15 *
	4-NOPD	50		93±20 *
	MMS	3.0					1296±55 *
with	water		19±4	15±1	44±4	135±4	62±3
	untreated		18±8	20±3	44±	136±16	53±2
	test substance	3	19±4	17±2	49±40	126±5	49±9
		10	17±2	16±2	37±4	143±19	61±9
		33	19±3	13±3	36±4	152±21	63±7
		100	15±4	13±4	41±9	149±18	70±10
		333	14±1	21±1	36±9	135±12	60±9
		1000	13±2	15±2	39±4	149±15	63±8
		2500	14±2	9±4	33±5	138±17	66±4
		5000	18±6	11±4	31±4	136±11	59±3
	2-AA	2.5	462±10 *	347±62 *	2268±122 *	2101±19 *
	2-AA	10					298±13 *

NaN3: sodium azide, 4-NOPD: 4-nitro-o-phenylene-diamine, MMS: methyl methane sulfonate

2-AA: 2-aminoanthracene

* Value exceeds the respective threshold of twice (TA98, TA100, WP2uvrA) or thrice (TA1535, TA1537) the respective solvent control

 

Table 2: Summary of results –experiment 2

Metabolic activation	Test group	Dose level [µg/plate]	Revertant colony counts (mean ± SD)
			TA1535	TA1537	TA98	TA100	WP2uvrA
without	water		17±4	11±3	35±5	133±12	56±5
	untreated		14±1	14±4	28±3	139±6	56±8
	test substance	33	17±3	12±1	25±4	132±3	50±11
		100	18±6	11±5	31±4	125±11	45±7
		333	19±2	14±1	33±6	130±7	52±10
		1000	16±1	10±3	28±4	117±3	54±11
		2500	15±2	10±2	29±4	131±1	48±
		5000	13±3	9±1	28±7	118±9	404
	NaN3	10	1759±98 *			1825±106 *
	4-NOPD	10			309±12 *
	4-NOPD	50		127±6 *
	MMS	3.0					354±93 *
with	water		15±2	18±3	39±9	140±16	65±3
	untreated		17±4	17±2	42±7	137±11	70±6
	test substance	33	18±7	15±1	42±11	135±14	69±2
		100	18±3	18±5	41±14	122±21	65±1
		333	14±2	17±3	32±5	142±14	59±3
		1000	15±	17±3	28±6	129±11	60±5
		2500	14±2	14±3	27±3	133±17	59±5
		5000	10±3	9±3	26±6	135±8	58±2
	2-AA	2.5	349±2 *	258±23 *	1742±149 *	2668±419 *
	2-AA	10					411±39 *

NaN3: sodium azide, 4-NOPD: 4-nitro-o-phenylene-diamine, MMS: methyl methane sulfonate

2-AA: 2-aminoanthracene

* Value exceeds the respective threshold of twice (TA98, TA100, WP2uvrA) or thrice (TA1535, TA1537) the respective solvent control.

Applicant's summary and conclusion

Conclusions:

In conclusion, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of test substance to induce gene mutations in the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA according to OECD Guideline 471 and EC method B13/14 under GLP. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

 

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.