2-amino-5-(2-hydroxyethyl)-6-methyl-4(3H)-pyrimidinone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The study was designed to evaluate the in vitro mutagenicity of a solution of the test item in a bacterial reverse mutation assay, with a polar solvent (NaCL 0.9%) and another solvent appropriate to he nature and use of the test item (DMSO). The following concentrations were prepared: 7.0, 2.3, 0.78, 0.26, 0.086 and 0.028 mM in NaCl 0.9%, and 65, 21.7, 7.22, 2.41, 0.80 and 0.27 mM in DMSO. As the assay requires a 1:25 dilution step, final concentrations tested are divided 1 : 25. Positive and negative controls complied with the acceptance criteria, hence the assay was valid with all strains tested, with and without enzymatic activation S9. Based on the results of the performed Ames test, SP02 is concluded not to be genotoxic.

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:

July - August 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Also according to EN ISO 10993-3:2009

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 103901

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: temperature = 20 +/- 5 Celsius
- Stability under test conditions: stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dissolution
- Final dilution of a dissolved solid, stock liquid or gel: In NaCl 0.9%: 0.28, 0.092, 0.0308, 0.0867, 0.0287, 0.0097 mM. In DMSO: 2.6, 0.08668, 0.2888, 0.0964, 0.032, 0.0108 mM

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

In NaCl 0.9%: 0.28, 0.092, 0.0308, 0.0867, 0.0287, 0.0097 mM
In DMSO: 2.6, 0.08668, 0.2888, 0.0964, 0.032, 0.0108 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [DMSO; physiol. saline (NaCl 0.9%)]
- Justification for choice of solvent/vehicle: compatible with the test system.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

other: N4-aminocytidine; 2-aminoanthracene

Evaluation criteria:

Fold increase greater than 2 times the baseline level was classified as possibly positive if multiple increases >=2 were observed or if single increase >=2 occurred at highest concentration in the assay.
Multiple responses of greater than 2-fold the baseline level with a dose-reponse lead to the test compound being classified as a clear positive. A test compound was classified negative if no response greater than 2 times the baseline and no statistical significance was recorded; and if single 2-fold increase occurred at lower concentration.
Although statistical analysis was be apllied to all data collected, increases in revertant yields were not classified as positive if less than 2-fold over the baseline vule and if no dose-response was observed.

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

in DMSO

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

in DMSO

Metabolic activation:

with

Genotoxicity:

other: possibly positive

Remarks:

However, no dose-dependent response observed

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

in DMSO

Metabolic activation:

without

Genotoxicity:

other: possibly positive

Remarks:

However, no dose-dependent response observed.

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

in DMSO

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli, other: mix

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

in NaCl 0.9%

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

in NaCl 0.9%

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

The conclusion of the authors is as follows:
- AMES performed with NaCl 0.9% negative (with and without S9)
- Ames performed with DMSO possibly genotoxic (TA 98 with S9 and TA1535 without S9)

However:

- The observed possible genotoxicity is a statistical artefact and has no biological relevance.
- There is no observation of a dose-dependent response. Normally, this is 1 of the most important evaluation criteria. The authors did not include these criteria in the analysis.
- In addition, it is noticeable that strain TA-1535 is indicated as positive (-S9) but TA-100 is not. TA-100 and TA-1535 are derived from the same G-46 strain and therefore typically give the same result. The fact that this is not the case here also indicates the lack of biological relevance.
- Generally, when a strain responds positively without S9, it also does so with S9. In the case of TA 1535, this is not the case either.

We therefore conclude that SP02 has no mutagenic potential.

Conclusions:

The authors of this key study conclude that the test item SP02 was identified as not mutagenic in the Ames MPF assay with Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E. coli uvrA and pKM101 strains; with and without metabolic activator S9 with solution in NaCl 0.9%. Test item was identified as possible mutagenic in the Ames MPF assay with Salmonella typhimurium TA98 with S9 and with TA1535 without S9 with solution on DMSO.

The conclusion above on the possible mutagenicity in DMSO is in the opinion of this registrant incorrect as based on the supporting OECD 476 study entered also in this dossier and the following observations:

- The observed possible genotoxicity is a statistical artefact and has no biological relevance.

- There is no observation of a dose-dependent response. Normally, this is one of the most important evaluation criteria. The key study authors did not include these criteria in the analysis.

- In addition, it is noticeable that strain TA-1535 is indicated as positive (-S9) but TA-100 is not. TA-100 and TA-1535 are derived from the same G-46 strain and therefore typically give the same result. The fact that this is not the case here also indicates the lack of biological relevance.

- Generally, when a strain responds positively without S9, it also does so with S9. In the case of TA 1535, this is not the case either.

Therefore, the mutagenicity of test item SP02 in DMSO is negative.

Executive summary:

The study was designed to evaluate the in vitro mutagenicity of a solution of the test item in a bacterial reverse mutation assay, with a polar solvent (NaCL 0.9%) and another solvent appropriate to he nature and use of the test item (DMSO).

The following concentrations were prepared: 7.0, 2.3, 0.78, 0.26, 0.086 and 0.028 mM in NaCl 0.9%, and 65, 21.7, 7.22, 2.41, 0.80 and 0.27 mM in DMSO. As the assay requires a 1:25 dilution step, final concentrations tested are divided 1 : 25.

Positive and negative controls complied with the acceptance criteria, hence the assay was valid with all strains tested, with and without enzymatic activation S9. Based on the results of the performed Ames test, SP02 is concluded not to be genotoxic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The GHS criteria for classification were not met.