[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay (AMES Test, OECD TG 471): negative

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:

September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator
- Solubility and stability of the test substance in the solvent/vehicle: According to ICH Draft Consensus Guideline M7, formulations were prepared freshly prior to start of treatment. Thus, no stability test in the solvent was performed.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Final preparation: clear colorless solution (only used at the same day) - The purity of the test item was not taken into account for the calculation of the dosages.

Target gene:

Histidine gene locus

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male Sprague Dawley rat liver S9 mix

Test concentrations with justification for top dose:

0, 16, 50, 160, 500, 1600, 5000 µg/plate (+/-S9 mix, all strains)

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA 1535, TA 100), 4-nitro-1,2-phenylene diamine (TA 1537), 2-Nitrofluoren (TA 98), Cumene hydroperoxide (TA 102), 2-aminoanthracene (all strains).

Details on test system and experimental conditions:

METHOD: each concentration including the controls was tested in triplicate.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA1535, TA100, TA1537 and TA98 this increase should be about twofold as compared to the solvent controls. For TA102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Key result

Species / strain:

S. typhimurium, other: TA1535, TA100, TA1537, TA98 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

test item precipitation was not observed.

Table 1: Summary of results from the Salmonella mutagenicity assay with FMA (mean values of revertants per plate)

 

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
solvent control	8	109	6	14	233
16	6	107	6	17	233
50	7	112	7	16	200
160	7	116	7	14	215
500	6	101	6	15	197
1600	7	118	8	13	205
5000	7	122	7	16	209
Positive control	643	780	43	1250	638
Dose ( µg per plate )	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
solvent control	11	133	8	18	336
16	10	139	5	20	332
50	11	134	6	17	302
160	9	144	7	22	340
500	9	121	7	19	322
1600	9	129	7	20	311
5000	10	130	7	14	300
Positive control	127	2293	129	2474	2133

 

 

 

Historical negative control data demonstrated that no deleterious or mutagenic effects were induced by the chosen solvent. The positive controls sodium azide, 4-nitro-1,2 -phenylene diamine, 2-nitrofluoren, cumene hydroperoxide and 2-aminoanthracene increased mutant counts to well over those of the solvent controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

None of the five strains used showed a dose-related and biologically relevant increase in mutant counts over those of the solvent controls in the preincubation test. This applied both to the tests with and without S9 mix.

 

 

Conclusions:

No evidence of mutagenic activity.

Executive summary:

The test item, dissolved in DMSO, was administered in doses of up to and including 5000 μg per plate without and with S9 mix on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA1535, TA100, TA1537, TA98 and TA102.
Doses up to and including 5000 μg per plate did not cause any bacteriotoxic effects. Test item precipitation occurred at the dose of 1600 μg per plate and above.
Evidence of mutagenic activity of the test item was not seen. In the preincubation modification, no biologically relevant increase in the mutant count, in comparison to the solvent controls, was observed in any of the strains tested, without and with S9 mix, under the experimental conditions applied.
The employed positive controls induced a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding solvent controls.
Therefore, the test item is considered to be non-mutagenic in the preincubation modification of the Salmonella/microsome test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In silico assessments on the mutagenic potential using (Q)SAR methologies according to ICH M7 Guideline are accepted in the pharmaceutical context by regulators as valid tools for detecting DNA reactive substances (ICH=International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use; M7 Guideline is on "Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk").

With regard to the prediction models used (Leadscope, DEREK, VITIC) the substance raised a structural alert for mutagenicity based on the general mutagenic potential of aromatic nitro compounds in DEREK (329-Aromatic nitro compound; no findings in VITIC) [Result-Collected, March 2019]. However, since experimental data for this substance are available (Ames Test, OECD TG 471) no mutagenic potential will be concluded for the substance.

Justification for classification or non-classification

Based on the study results a classification according to Regulation (EC) No. 1272/2008 (CLP) is not required.