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Toxicological information

Genetic toxicity: in vitro

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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 Nov. 04, 2003 to Nov. 07, 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented study, followed guideline, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(according to German and OECD principles of GLP)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material: INCl-Toluene-2,5-diamine sulfate (2,5-diamino toluene sulfate) (Code # A000165)- TSIN: 23005- Substance type: Pure active substance- Physical state: White-grey powder- Stability under test conditions: The substance is considered to be stable for 5 years if stored in dryness and darkness.- Stability in solution: The solutions of the test substance in water (99.9% and 109%), water/acetone 4:1 v/v ((99.7% and 108%) confirm a good stability of the substance. The stability of Haarbraun in water and in water/acetone (4:1 v/v) was monitored over a time period of 8 days using HPLC-chromatography at the 240 nm. During the procedure the stock solutions were stored at ambient temperature in the absence of light.- Storage condition of test material: At room temperature, protected from light and moisture- Solubility: Solubility in different solvents is as follows: 9.6 g/L in water (1 weight% in water) <0.1 weight% in acetone/water 1:1

Method

Target gene:
Histidine locus serves as a specific target gene of following Salmonella Typhimurium tester strains :TA 1537: his C 3076 (Frame shift mutation)TA98: his D 3052 (Frame shift mutation)TA 1535 and TA 100: his G 46 (Base – pair substitution)TA 102: his G 428 (Base – pair substitution)
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
other: Faulty lipopolysaccharide envelope, nitrate reductase and biotin deficient (uvrB-). Error prone repair and ampicillin resistance marker (R-factor plasmid in TA 98 and TA 100 only)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/B-Naphthoflavone induced rat liver S9
Species / strain:
S. typhimurium TA 102
Additional strain characteristics:
other: Faulty lipopolysaccharide envelope, nitrate reductase and biotin deficient, excision repair proficient (uvrB+) and error prone repair with ampicillin resistance marker (R-factor plasmid). Multicopy plasmid pAQ1 (hisG428 mutation gene) and tetracycline res
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/B-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-Experiment and Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plateThe pre-experiment was reported as main Experiment I since no toxic effects were observed and 5000 µg/plate was chosen as maximal concentration.
Vehicle:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide DMSO (MERCK, D-64293 Darmstadt; purity > 99%)- Justification for choice of solvent/vehicle: The solvent was chosen based on its solubility properties and its relative nontoxicity to the bacteria.
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
Remarks:
(DMSO)
True negative controls:
yes
Positive controls:
yes
Remarks:
(Without metabolic activation)
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: (Prepared by dissolving in deionized water and used at a concentration of 10 µg/plate for strain TA 100 and TA 1535)
Negative controls:
yes
Solvent controls:
yes
Remarks:
(DMSO)
True negative controls:
yes
Positive controls:
yes
Remarks:
(without metabolic activation)
Positive control substance:
other: 4-nitro-o-phenylene-diamine solution prepared by dissolving in DMSO for strain TA98 at 10 µg/plate and strain TA1537 at 50 µg/plate
Negative controls:
yes
Solvent controls:
yes
Remarks:
(DMSO)
True negative controls:
yes
Positive controls:
yes
Remarks:
(without metabolic activation)
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: (Prepared by dissolving in deionized water for strain TA102 at 4.0 µL/plate)
Negative controls:
yes
Solvent controls:
yes
Remarks:
(DMSO)
True negative controls:
yes
Positive controls:
yes
Remarks:
(with metabolic activation)
Positive control substance:
other: 2-aminoanthracene solution prepared by dissolving in DMSO (2-AA) at 2.5 µg/plate for strains TA1535, TA1537, TA98 and TA100 and at 10.0 µg/plate for strain TA102
Details on test system and conditions:
MAINTAINENCE OF TESTER STRAIN: Tester strains (bacterial strains TA 1535, TA 1537, and TA 100 were obtained from Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 98 and TA 102 was obtained from E. Merck (D-64293 Darmstadt) and RCC Ltd.(CH-4332 Stein) respectively. The strains were precultured and stored as follows:- Storage: The strain cultures were stored as stock cultures in ampoules with nutrient broth +5% DMSO in liquid nitrogen.- Periodic checking: Regular checking of the properties of the strains regarding the membrane permeability, ampicillin and tetracycline-resistance as well as spontaneous mutation rates was performed in RCC Cytotest Cell Research according to B. Ames et al and D Maron and B Ames to ensure the experimental conditions set down by Ames.- Precultures: From the thawed ampoules of the strains 0.5 mL bacterial 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, TA 100, and TA 102. Additionally 20 µL tetracycline (2 µg/mL) was added to strain TA 102. This nutrient medium contains per litre: 8 g Merck Nutrient Broth (MERCK, D-64293) 5 g NaCl (MERCK, D-64293)The bacterial cultures were incubated in a shaking water bath for 4 h at 37°C.METHOD OF APPLICATION: in agar (Direct plate incorporation)EXPERIMENTAL DURATION AND PROCEDURE:- Plate incorporation method (Experiment 1): 100 µL test solution at each dose level/solvent (negative control)/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-culture of the strains) and 2000 µL overlay agar were mixed in a test tube and poured onto the selective agar plates. After solidification the plates were incubated upside down for at least 48 h at 37°C in the dark.NUMBER OF REPLICATIONS: 3 plates/strain/dose levelDETERMINATION OF CYTOTOXICITY - Pre-experiment: Toxicity of test material was tested in triplicates/strain/dose group with all strains used. The experimental conditions of pre-experiment were the same as described for the Experiment I above (plate incorporation test) - Method for evaluation: Reduction in the number of spontaneous revertants or clearing of bacterial background lawn
Evaluation criteria:
The test substance was considered a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100, and TA102) or three times (strains TA1535 and TA1537) the colony count of the corresponding solvent control was observed.A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment.A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls such an increase was not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not required

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA1535, TA1537, TA98, TA100, TA102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
yes (in TA 1535, TA1537 and TA98: at 2500 - 5000 µg/plate; in TA 100 and TA 102: at 5000 µg/plate)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
(at 2500-5000 µg/plate)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
PRE-EXPERIMENT TOXICITY RESULTS: Toxicity was measured as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. . In this study, evaluable plates (>0) with colonies were observed at five concentrations or more in all strains used. Based on these results, pre-experiment was reported as main Experiment I and 5000 µg/plate was chosen as maximal concentration as no relevant toxic effects were observed up to 5000 µg/plate.COMPARISON WITH CONCURRENT AND HISTORICAL CONTROL DATA: Negative (solvent) controls did not result in mutagenic activity. Positive controls showed a distinct increase of induced revertant colonies. The historical range of positive controls was exceeded in strains TA1535 without metabolic activation and in strain TA1537 with metabolic activation. This effect indicated the sensitivity of the strains rather than compromising the assay.ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects evident as a reduction in the number of revertants were observed in all the test groups with and without metabolic activation except TA 102 (with metabolic activation).For details please refer to ‘Table 1’ under ‘Any other information on results incl. tables’ section.MUTAGENIC ACTIVITY: Substantial and dose dependent increases in revertant colony numbers were observed following treatment with the test substance in strains TA1535, TA1537, TA98, and TA100 with metabolic activation. The number of colonies reached or exceeding the threshold of twice (strain TA98 and TA100) and three times (strain TA1535 and TA1537) the number of the corresponding solvent control was noted at the following concentrations with activation only: TA1535 (10-2500 µg/plate), TA1537 (33-2500 µg/plate), TA98 (10-5000 µg/plate) and TA100 (333-1000 µg/plate). At the higher concentrations (2500 and 5000 µg/plate) the number of colonies was reduced due to overlapping toxic effects.

Any other information on results incl. tables

Table 1: Toxic effects of test material on all tested strains

Strain

Without S9 mix

With S9 mix

TA1535

2500-5000 µg/plate

2500-5000 µg/plate

TA1537

2500-5000 µg/plate

2500-5000 µg/plate

TA98

2500-5000 µg/plate

2500-5000 µg/plate

TA100

5000 µg/plate

2500-5000 µg/plate

TA102

5000 µg/plate

no toxic effects

The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 activation in all strains used.

Table 2:Reverse mutation assay ofToluene-2, 5-diamine sulfate (Haarbraun)

Strain

Experiment I

without 59 mix

with S9 mix

TA 1535

-

10-2500

TA 1537

-

33-2500

TA98

-

10-5000

TA 100

-

333-5000

TA 102

-

-

- =Threshold was not reached or exceeded

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):negative without metabolic activation (Strains TA1535, TA1537, TA98, TA100, TA102)positive with metabolic activation (Strains TA1535, TA1537, TA98, TA100)negative with metabolic activation (Strain TA102)Toluene-2, 5-diamine sulfate (Haarbraun) was considered mutagenic in Salmonella typhimurium reverse mutation assay.
Executive summary:

The bacterial reverse mutation test of Toluene-2, 5-diamine sulfate (Haarbraun) was determined following OECD guideline 471 (Bacterial Reverse Mutation Test.

The study was performed to investigate the potential of Toluene-2,5 -diamine sulfate to induce gene mutations according to the direct plate incorporation test using the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102. The assay was performed using plate incorporation with and without liver microsomal activation (Phenobarbital/B-Naphthoflavone induced rat liver S9). Each concentration, including the controls, was tested in triplicate. The test substance was tested at the following concentrations:

3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate.

Plates were incubated for at least 48 hat 37° C in the dark.

Toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation at higher concentrations.The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

Substantial and dose dependent increases in revertant colony numbers were observed following treatment with the test substance in strains TA1535, TA1537, TA98 and TA100 with metabolic activation. The number of colonies reached or exceeded the threshold of twice (strain TA98 and TA100) and three times (strain TA1535 and TA1537) the number of the corresponding solvent control at concentrations of 10 µg/plate and above but not in TA 102 (with metabolic activation). Whereas at the higher concentrations (2500 and 5000 µg/plate) the number of colonies was reduced due to overlapping toxic effects.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

The test substance did induce gene mutations by base pair exchanges and frameshifts in the genome of the strains used (except TA102), in the presence of metabolic activation.

Based on the above, Toluene-2, 5-diamine sulfate (Haarbraun) was considered mutagenic in Salmonella typhimurium reverse mutation assay.

This bacterial reverse mutation test is classified as acceptable, and satisfies the guideline requirements of the OECD 471 method.