2,4-Dichloro-pyrimidin-5-ylamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type fo genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07Nov 2016 - 26 Jan 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 471

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,4-Dichlor-pyrimidin-5-ylamine

EC Number:

664-402-3

Cas Number:

5177-27-5

Molecular formula:

C4H3Cl2N3

IUPAC Name:

2,4-Dichlor-pyrimidin-5-ylamine

Test material form:

solid: particulate/powder

Method

Target gene:

Four strains of Salmonella typhimurium bacteria (TA98, TA100, TA1535 and TA1537) and
one strain of Escherichia coli bacteria (WP2 uvrA pKM101) were used in this study. Strains
TA98, TA1535 and TA1537 were originally obtained from the UK NCTC. Strain TA100 was
derived from cultures originally obtained from Covance Laboratories Inc., USA. Strain WP2
uvrA pKM101 was obtained from MolTox Inc., USA.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9Mix

Test concentrations with justification for top dose:

Experiment 1 treatments of all the tester strains were performed in the absence and in the
presence of S-9, using final concentrations of CD 314 at 5, 16, 50, 160, 500, 1600 and
5000 μg/plate, plus vehicle and positive controls. Following these treatments evidence of
toxicity in the form of a thinning of the background bacterial lawn and a marked reduction in
revertant numbers was observed but only at 5000 μg/plate in strain TA1535 in the absence of
S-9 and in strain TA1537 in the presence of S-9.
Experiment 2 treatments of all the tester strains were performed in the absence and in the
presence of S-9. The maximum test concentration of 5000 μg/plate was retained for all
strains. Narrowed concentration intervals were employed covering the range
333-5000 μg/plate, in order to examine more closely those concentrations of CD 314
approaching the maximum test concentration and considered therefore most likely to provide
evidence of any mutagenic activity. Following these treatments, no clear evidence of toxicity
was observed.
The test article was completely soluble in the aqueous assay system at all concentrations
treated, in each of the experiments performed.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene (AAN)

Details on test system and experimental conditions:

The test system was suitably labelled to clearly identify the study number, bacterial strain,
test article concentration (where appropriate), positive and vehicle controls, absence or
presence of S-9 mix.

Rationale for test conditions:

1. The vehicle control counts fell within the laboratory’s historical control ranges.
2. The positive control chemicals induced increases in revertant numbers of ≥2-fold (in
strains TA98, TA100 and WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 and
TA1537) the concurrent vehicle control confirming discrimination between different
strains, and an active S-9 preparation
3. At least five analysable concentrations of the test article were available.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥2-fold (in strains TA98,
TA100 or, WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 or TA1537) the concurrent
vehicle control values
2. Any observed response is reproducible.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case
basis. Biological relevance was taken into account, for example consistency of response
within and between concentrations and (where applicable) between experiments.

Statistics:

Individual plate counts were recorded separately and the mean and standard deviation of the
plate counts for each treatment were determined. Control counts were compared with the
laboratory’s historical control ranges (Section 6.2 and Section 6.3). Data were considered
acceptable if the vehicle control counts fell within the calculated historical control ranges and
the positive control plate counts were comparable with the historical control ranges.
The presence or otherwise of a concentration response was checked by non-statistical
analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate).
However, adequate interpretation of biological relevance was of critical importance.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

It was concluded that CD 314 induced concentration-related and reproducible mutation in
histidine-requiring strain TA100 of Salmonella typhimurium in the presence of a rat liver
metabolic activation system (S-9) when tested using a plate incorporation methodology up to
the maximum recommended concentration of 5000 μg/plate. Increases in revertant numbers
in strains TA100 in the absence of S-9 and TA98 in the presence of S-9 were considered to
provide evidence for an equivocal mutagenic response.