Reaction mass of sodium amino-bis{[4-(ethenylsubstituted)phenyl]diazenyl}-hydroxynaphthalenesulfonate and polysodium amino-{[4-(ethenylsubstituted)phenyl]diazenyl}-{[4-(ethenylsubstituted)-2-sulfonatophenyl]diazenyl}-hydroxynaphthalenesulfonate and polysodium amino-bis{[4-(ethenylsubstituted)-2-sulfonatophenyl]diazenyl}-hydroxynaphthalenesulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Test item FAT 40865/A TE did not induce gene mutations either by base pair substitution or by frame shifts in the genome of the strains used under the conditions of the assay with and without metabolic activation.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

None

Target gene:

Histidine auxotrophic genes of S. typhimurium

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

None

Additional strain / cell type characteristics:

nitroreductase deficient

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0.050, 0.158, 0.501, 1.582 and 5 mg/plate, both in absence (-S9) and in the presence (+S9) of metabolic activation.

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine, 2-Aminoanthracene

Remarks:

None

Details on test system and experimental conditions:

Test System
The bacterial strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 were obtained from Xenometrix AG, Gewerbestrasse 25, CH-4123 Allschwil (Switzerland)
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation

Conditions of the experiment :
For each strain and dose level, including the controls, three plates (triplicate) were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level, negative control and 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 Bacterial suspension,
2000 µL Overlay agar
In the pre-incubation assay 100 µL test solution, 500 µL S9 mix/S9 mix substitution buffer and 100 µL bacterial suspensions were mixed in a test tube and incubated at 37 ± 2°C for 60 mi-nutes. After pre-incubation 2.0 ml overlay agar (47 ± 2°C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated in inverted position for 48 hours at 37 ± 2°C

DURATION
- Preincubation period: 1 hour

NUMBER OF REPLICATIONS: Three replicates per concentration

DETERMINATION OF CYTOTOXICITY
- other:Bacterial background lawn and Revertant colony count

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 TA 102] or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding negative 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 control such an increase is not considered biologically relevant.

Statistics:

Descriptive

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Water solubility: Soluble
Solvent used RO (reverse osmosis) Water
Quantity of test item : 50 mg
Volume of Vehicle added : 1000 µL
Final Concentration : 50 mg /mL
Solubility status : soluble
As mentioned in the above table, solubility of test item was checked in RO water and found soluble. The test item was found soluble in RO water at 50 mg/ml to give final treatment concentration of 5 mg/plate (recommended maximum test concentration for soluble non-cytotoxic substances). Therefore, RO water was chosen as solvent for the study.

- Precipitation:
Precipitation was checked as insolubility to assess precipitation in the final mixture under the actual test conditions an evident to the unaided eye. Test item dissolved in RO water at 50 mg/mL concentration was checked for precipitation. Details given in table below:
Precipitation Record
Overlay agar volume Test item preparation volume Concentration/Plate Result
2 mL 100 µL 5 mg No Precipitation
An amount of test item preparation (50 mg/mL) was added to 2 ml of overlay agar (Top agar) in test tubes to give test item concentration of 5 mg/plate and plated on minimal glucose agar (MGA) plates. No precipitation was observed which was assumed non-interfering with the scoring. Therefore 5 mg/plate was selected as the highest concentration for pre-experiment.

RANGE-FINDING/SCREENING STUDIES:
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate) were tested for toxicity and mutation induction with each 3 plates (triplicates). The experimental conditions in this pre-experiment were the same as described for the Trial-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.
No distinct reduction in the number of revertant colony count as well as background lawn was observed in any of the test item concentrations tested 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate both in absence and in the presence of metabolic activation, when compared to that of the negative control group.
Based on the results of pre-experiment following doses were selected for the main study trials: 0.050, 0.158, 0.501, 1.582 and 5 mg/plate, both in absence (-S9) and in the presence (+S9) of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous reversion rates in the negative control are in the range of in house historical data.

None

Conclusions:

FAT 40865/A TE did not induce gene mutations either by base pair substitution or by frame shifts in the genome of the strains used under the conditions of the assay with and without metabolic activation.

Executive summary:

A study was performed as per OECD Guideline 471 to assess the mutagenic potential of FAT 40865/A TE to induce gene mutations in comparison to negative control according to the plate incorporation method (Trial-I) and the pre-incubation method (Trial-II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative and positive controls was tested in triplicates. The test item was tested at the following concentrations i.e., 0.050, 0.158, 0.501, 1.582 and 5 mg/plate, both in presence (+S9) and in absence (-S9) of metabolic activation.

No significant increase in revertant colony numbers in any of the tester strains were observed following treatment with FAT 40865/A TE at any dose level in both the confirmatory trials, neither in the presence nor in the 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. The spontaneous reversion rates in the negative and positive controls are within the range of in house historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. It can be concluded that the test item FAT 40865/A TE did not induce gene mutations either by base pair substitution or by frame shifts in the genome of the strains used under the conditions of the assay with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A study was performed as per OECD Guideline 471 to assess the mutagenic potential of FAT 40865/A TE to induce gene mutations in comparison to negative control according to the plate incorporation method (Trial-I) and the pre-incubation method (Trial-II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative and positive controls was tested in triplicates. The test item was tested at the following concentrations i.e., 0.050, 0.158, 0.501, 1.582 and 5 mg/plate, both in presence (+S9) and in absence (-S9) of metabolic activation.

No significant increase in revertant colony numbers in any of the tester strains were observed following treatment with FAT 40865/A TE at any dose level in both the confirmatory trials, neither in the presence nor in the 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. The spontaneous reversion rates in the negative and positive controls are within the range of in house historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. It can be concluded that the test item FAT 40865/A TE did not induce gene mutations either by base pair substitution or by frame shifts in the genome of the strains used under the conditions of the assay with and without metabolic activation.

Justification for classification or non-classification

Test item FAT 40865/A TE did not induce gene mutations either by base pair substitution or by frame shifts in the genome of the strains used under the conditions of the assay with and without metabolic activation. Hence, it does not warrant the classification for genetic toxicity as per the CLP (Regulation 1272/2008) criteria.