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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:
experimental study
Adequacy of study:
key study
Study period:
2014-07-03 to 2014-07-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: FAT 40062/C TE
Product: LANASOL RED B CRUDE DRY
Reactive Red 065
Chemical Name: Disodium 6-amino-5-[{4-[(2-bromoacryloyl)amino]-2-sulfonatophenyl}diazenyl]-4-hydroxynaphthalene-2-sulfonate
CAS No.: 70210-40-1
Batch No.: SBL/027/LRB (India)
Physical State: solid
Colour: brown
pH (specify): 5.9 (2%(w/w) aqu. Solution), RT
Melting Point: > 100 °C
Solubility in water: 60 g/L at 20 °C
Active Components: > 94 % (a/a), factor 0.76, > 71 % (w/w)
Unidentified Components: < 6 % (a/a)
Molecular weight of base form: 569.63 g/mol (anion)
Molecular weight of salt form: 615.34 g/mol (salt)
Storage Conditions: ≤ -20 °C
Date of Analysis: 06 May 2014
Expiry Date: 05 May 2019
Hazards and Precautions: gloves, avoid inhalation of dust; safety glasses with side-shields
Safety Precautions: safety gloves, mask and safety glasses were worn while handling the test item.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 274-437-2
- Expiration date of the lot/batch: 274-437-2

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ≤-20 °C
- Solubility of the test substance in the solvent/dispersant/vehicle/test medium: 60 g/L in water at 20 °C

Method

Target gene:
His
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat S9 liver microsomal fraction
Test concentrations with justification for top dose:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Aqua destillata (distilled water)
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 100 and TA 1535 at 10 µg/plate- without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-NOPD (4-nitro-o-phenylene-diamine)
Remarks:
TA 98 at 10 µg/plate and TA 1537 at 40 µg/plate- without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 102 at 1 µL/plate- without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanththracene
Remarks:
TA 98, TA 100, TA 1535 and TA 1537 at 2.5 µg/plate and TA 102 at 10 µg/plate- with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
-For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL Overlay agar.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

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

DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤0.5 in relation to the solvent control
Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

Evaluation of Cytotoxicity:
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤0.5 in relation to the solvent control.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
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 precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). Toxic effects of the test item were noted only in tester strain TA 102 at a concentration of 5000 µg/plate (without metabolic activation). No biologically relevant increases in revertant colony numbers were noted in tester strains TA 98, TA 1535 and TA 102. Biologically relevant increases of revertant colony numbers were observed in tester strain TA 100 at concentrations of 1000 µg/plate and higher (without metabolic activation) and at a concentration of 5000 µg/plate (with metabolic activation). The threshold value of 2.0 was exceeded and a maximum mutation factor of 3.6 was reached at a concentration of 5000 µg/plate (without metabolic activation). Biologically relevant increases of revertant colony numbers were also observed in tester strain TA 1537 at a concentration of 5000 µg/plate (with metabolic activation). The threshold value of 3.0 was exceeded and a maximum mutation factor of 4.0 was reached at a concentration of 5000 µg/plate (with metabolic activation). Moreover, a dose-response relationship was found in tester strain TA 100 (with and without metabolic activation) and in tester strain TA 1537 (with metabolic activation).
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
FAT 40062/C TE is considered to be mutagenic in this bacterial reverse mutation assay.
Executive summary:

FAT 40062/C TE was investigated for its potential to induce gene mutations according to the plate incorporation test with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102. Several concentrations of the test item were used. The assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used:

311.6, 100, 316, 1000, 2500 and 5000 µg/plate. No precipitation of the test item was observed in any tester strain used (with and without metabolic activation). Toxic effects of the test item were noted only in tester strain TA 102 at a concentration of 5000 µg/plate (without metabolic activation). The reduction in the number of revertants down to a mutation factor of 0.5 found in tester strain TA 102 at a concentration of 1000 µg/plate (with and without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship and lack of concomitant clearing of the background lawn. No biologically relevant increases in revertant colony numbers were noted in tester strains TA 98, TA 1535 and TA 102. Biologically relevant increases of revertant colony numbers were observed in tester strain TA 100 at concentrations of 1000 µg/plate and higher (without metabolic activation) and at a concentration of 5000 µg/plate (with metabolic activation). The threshold value of 2.0 was exceeded and a maximum mutation factor of 3.6 was reached at a concentration of 5000 µg/plate (without metabolic activation). Biologically relevant increases of revertant colony numbers were also observed in tester strain TA 1537 at a concentration of 5000 µg/plate (with metabolic activation). The threshold value of 3.0 was exceeded and a maximum mutation factor of 4.0 was reached at a concentration of 5000 µg/plate (with metabolic activation). Moreover, a dose-response relationship was found in tester strain TA 100 (with and without metabolic activation) and in tester strain TA 1537 (with metabolic activation). The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiment. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, FAT 40062/C TE caused gene mutations by base pair changes and frameshifts in the genome of the tester strain TA 100 and TA 1537. Therefore, FAT 40062/C TE is considered to be mutagenic in this bacterial reverse mutation assay.