Leucophor 1111X

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

From August 03rd to 19th, 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted according to internationally accepted testing guidelines and performed in compliance with Good Laboratory Practice. The OECD recommended combination of strains was tested. Justification for read across approach is given in the endpoint summary and in the read across justification report attached to the Section 13 of this dossier.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Method

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 histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-rninus". In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.
Strain WP2 and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis, Tryptophan-independent (Trp*) mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagen which substitute one base for another. Additionally, the uvrA derivative is deficient in the DNA repair process (excision repair damage). Such a repair-deficient strain may be more readily mutated by agents.
When summarised the mutations of the TA strains and the E. coli strain, used in this study can be described as follows:
S. typhimurium TA 1537 genotype his C 3076; rfa-; uvrB-: frame shift mutations
S. typhimurium TA 98 genotype his D 3052; rfa-; uvrB-;R-factor: frame shift mutations
S. typhimurium TA 1535 genotype his G 46; rfa-; uvrB-: base- pair substitutions
S. typhimurium TA 100 genotype his G 46; rfa-; uvrB-;R-factor: base- pair substitutions
Escherichia coli WP2 uvrA genotype trp-; uvrA-: base- air substitutions and others
Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in the tetsing laboratory according to B. Ames etal. and D. Maron and B. Ames. in this way it was ensured that the experimental conditions set down by Ames were fulfilled.
The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA were obtained from Trinova Biochem GmbH (35394 Giessen, Germany).

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

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

Vehicle / solvent:

On the day of the experiment, the test item was dissolved in deionised water. The solvent was chosen because of its solubility properties.
No precipitation of the test item occurred up to the highest investigated dose.

Controls

Untreated negative controls:

yes

Remarks:

concurrent untreated

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Remarks:

Without metabolic activation: TA1535 and TA100 NaN3, TA1537 and TA98 4-NOPD, WP2 uvrA MMS. With metabolic activation: 2-AA for all the strains.

Details on test system and experimental conditions:

PRECULTURES
From the thawed ampoules of the strains 0.5 ml suspension was transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 µl ampicillin (25 µg/ml) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre: 8 g Merck Nutrient Broth; 5 g NaCl.
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C.

SELECTVE AGAR
The plates with the selective agar were obtained from E. Merck, D-64293 Darmstadt.

OVERLAY AGAR
The overlay agar contains per litre:
for Salmonella strains: 6.0 g MERCK Agar Agar; 6.0 g NaCl; 10.5 mg L-Histidine x HCl x H2O; 12.2 mg Biotin
for Escherichia coli: MERCK Agar Agar; 6.0 g NaCl; 2.5 mg Tryptophan
Sterilisations were performed at 121 °C in an autoclave.

METHOD OF APPLICATION
Plate incorporation test (experiment l) and the pre-incubation test (experiment ll) .

NUMBER OF REPLICATIONS
For each strain and dose level including the controls, three plates were used.

EXPERIMENTAL PERFORMANCE
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
500 µl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
100 µl bacteria suspension (cf. test system, pre-cuIture of the strains)
2000 µl overlay agar
ln the pre-incubation assay 100 µl test solution, 500 µl S9 mix / S9 mix substitution buffer and 100 µl bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

DATA RECORDING
The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software program Ames Study Manager. The counter was connected to an lBM AT compatible PC with printer to print out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates.

MAMMALIAN MICROSOMAL FRACTION S9 Mix
Preparation
Phenobarbital/β-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old male Wistar Hanlbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p.and β-Naphthoflavone p.o. 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 KCI solution (1+3) followed by centrifugation at 9000 g. Aliquotes 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 34.7 mg/ml in the pre-experiment and in experiment l and 36.5 mg/ml in experiment Il.

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 15% 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 KCI
5 mM Glucose-6-phosphate
5 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.

PRE-EXPERIMENT for TOXICITY
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment l, if the following criteria are met:
Evaluable plates (> 0 colonies) at five concentrations or more in all strains used.

DOSE SELECTION
ln the pre-experiment the concentration range of the test item was 3 - 5000 pg/plate. The pre-experiment is reported as experiment l since no relevant toxic effects were observed and 5000 pg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of 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

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.
No toxic effects, evident as a reduction in the number of revertants, 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 item 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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of results Pre-experiment and experiment I

Test group	Dose level (µg/plate)	Relevant colony counts (mean± SD)
		Ta 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without metabolic activation
Deionised water		19 ± 5	14 ± 4	36 ± 10	152 ± 25	62 ± 6
Untreated		18 ± 5	15 ± 4	29 ± 3	128 ± 9	53 ± 5
Test item	3	18 ± 4	11 ± 3	28 ± 2	134 ± 3	65 ± 5
	10	16 ± 5	16 ± 1	28 ± 2	136 ± 12	68 ± 8
	33	19 ± 3	15 ± 1	33 ± 8	142 ± 14	62 ± 4
	100	21 ± 2	10 ± 4	34 ± 8	139 ± 3	69 ± 4
	333	16 ± 2	17 ± 4	32 ± 3	142 ± 12	53 ± 3
	1000	22 ± 2	15 ± 5	27 ± 4	130 ± 6	64 ± 2
	2500	21 ± 8	15 ± 5	31 ± 6	133 ± 18	64 ± 7
	5000	18 ± 3	9 ± 3	27 ± 9	135 ± 11	54 ± 8
NaN3	10	1063 ± 43			1790 ± 84
4-NOPD	10			453 ± 65
4-NOPD	50		100 ± 9
MMS	4					1510 ± 59
With metabolic activation
Deionised water		26 ± 4	25 ± 3	40 ± 7	157 ± 12	63 ± 7
Untreated		24 ± 6	25 ± 4	41 ± 8	152 ± 29	58 ± 4
Test item	3	18 ± 3	25 ± 5	46 ± 7	150 ± 20	59 ± 4
	10	21 ± 6	24 ± 13	43 ± 1	150 ± 14	55 ± 8
	33	23 ± 3	22 ± 5	38 ± 2	158 ± 5	59 ± 6
	100	24 ± 5	25 ± 9	40 ± 7	146 ± 7	53 ±15
	333	24 ± 3	26 ± 5	46 ± 8	158 ± 13	56 ± 8
	1000	30 ± 7	25 ± 5	41 ± 1	161 ± 14	65 ± 10
	2500	21 ± 1	18 ± 3	32 ± 3	166 ± 3	68 ± 12
	5000	23 ± 7	23 ± 6	43 ± 8	148 ± 9	63 ± 2
2-AA	2.5	485 ± 27	261 ± 32	1998 ± 85	2711 ± 192
2-AA	10					434 ± 26

Summary of results experiment II

Test group	Dose level (µg/plate)	Relevant colony counts (mean± SD)
		Ta 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without metabolic activation
Deionised water		21 ± 2	24 ± 5	26 ± 3	132 ± 12	34 ± 8
Untreated		26 ± 4	23 ± 2	23 ± 3	106 ± 17	38 ± 6
	33	20 ± 3	21 ± 1	27 ± 5	109 ± 10	39 ± 3
	100	14 ± 8	20 ± 1	27 ± 7	119 ± 18	43 ± 11
	333	18 ± 3	16 ± 5	34 ± 7	103 ± 15	41 ± 9
	1000	20 ± 3	20 ± 11	27 ± 5	109 ± 9	39 ± 13
	2500	20 ± 4	19 ± 7	23 ± 28	134 ± 3	41 ± 7
	5000	29 ± 11	28 ± 7	26 ± 4	120 ± 14	45 ± 9
NaN3	10	1422 ± 26			2099 ± 41
4-NOPD	10			458 ± 41
4-NOPD	50		121 ± 9
MMS	4					1443 ± 39
With metabolic activation
Deionised water		31 ± 6	20 ± 3	32 ± 6	152 ± 16	45 ± 3
Untreated		22 ± 1	19 ± 3	42 ± 8	139 ± 3	46 ± 9
	33	21 ± 4	21 ± 7	38 ± 3	129 ± 13	48 ± 6
	100	30 ± 6	26 ± 9	32 ± 8	135 ± 20	61 ± 8
	333	28 ± 10	26 ± 4	32 ± 2	159 ± 13	68 ± 6
	1000	26 ± 8	20 ± 6	37 ± 11	146 ± 7	56 ± 2
	2500	28 ± 3	29 ± 10	32 ± 3	149 ± 6	58 ± 3
	5000	27 ± 7	26 ± 6	35 ± 6	114 ± 5	62 ± 5
2-AA	2.5	221 ± 21	173 ± 12	1504 ± 544	1772 ± 154
2-AA	10					326 ± 22

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Substance is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment ll) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. 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 and Experiment I 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Experiment ll 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, 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 the test item 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 in- crease of induced revertant colonies.

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, substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.