4-[(6-chloro-2-[(4-cyanophenyl)amino]-4-pyrimidinyl]oxy]-3,5-dimethylbenzonitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-02-20 to 2003-03-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4D, dated May 19, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-({6-chloro-2-[(4-cyanophenyl)amino]pyrimidin-4-yl}oxy)-3,5-dimethylbenzonitrile

EC Number:

928-806-6

Cas Number:

1070377-34-2

Molecular formula:

C20H14ClN5O

IUPAC Name:

4-({6-chloro-2-[(4-cyanophenyl)amino]pyrimidin-4-yl}oxy)-3,5-dimethylbenzonitrile

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): T002615
- Physical state: solid
- Appearance white solid

Specific details on test material used for the study:

Batch No.: 00403487
Aggregate State at Room Temperature: solid
Colour: white
Purity: > 98 %
Stability in Solvent: not indicated by the sponsor
Storage: room temperature
Expiration Date: retest date - 2003-12

Method

Target gene:

histidine locus ( S. Typhimurium strains) ; tryptophan locus (E. coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: see "Any other info. on material and methods incl. tables" section

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: see "Any other info. on material and methods incl. tables" section

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/B-naphtoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-experiment on toxicity: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Both plate incorporation test (experiment I) and the pre-incubation test (experiment II) (with and without S9 mix): 33, 100, 333, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation; TA 1535 and TA 100 at 10 µg/plate

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

Without metabolic activation; TA 1537 at 50 µg/plate and TA 98 at 10 µg/plate

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation; WP2 uvrA at 4 µL/plate

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation; 2.5 µg/plate( TA 1535, TA 1537, TA 98, TA 100), and 10 µg/plate (WP2 uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: experiment I: in agar (plate incorporation test); experiment II: preincubation test

EXPERIMENTAL PERFORMANCE:
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL: Test solution at each dose level, solvent (negative control) or 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: Bacteria suspension (cf. test system, pre-culture of the strains),
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 suspension were mixed in a test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

DURATION
- Preincubation period: 60 minutes (experiment II)
- Exposure duration: 48 hours (experiment I and II)
- Selection time (if incubation with a selection agent): 48 hours ( simultaneous with exposure)

SELECTION AGENT (mutation assays): histidine and tryptophan locus

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Evaluation criteria:

- The test substance was 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 WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control was observed.
- A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent control, such an increase iss not considered biologically relevant.

Statistics:

No statistical evaluation of the data is required.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: no data
- Precipitation: see detailed table in section "any other information on results"

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment the concentration range of the test item was 3-5000 µg/plate. The pre-experiment is reported as part of experiment I since no toxic effect were observed and 5000 µg/plate were chosen as maximal concentration.

COMPARISON WITH HISTORICAL CONTROL DATA:
The historical range of positive controls was exceeded in strains TA 1535 (exp.I) without metabolic activation and in strain 98 (exp.II) with metabolic activation. This effect indicates the sensitivity of the strains rather than compromising the assay.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

The assay was performed in two independent experiments both with and without liver microsomal activation. Due to a technical failure, no bacterial growth was observed in strain WP2 uvrA in experiment I (plate incorporation test). An additional experiment was performed under identical conditions using strain WP2 uvrA. The results of this additional experiment are included in this report as experiment I.

Precipitation of the test item

strain	Experiment I		Experiment II
	without S9 mix	With S9 mix	without S9 mix	With S9 mix
TA1535	1000-5000	1000-5000	2500, 5000	333, 2500, 5000
TA1537	1000-5000	1000-5000	1000-5000	1000-5000
TA98	1000-5000	1000-5000	2500, 5000	2500, 5000
TA100	5000	2500, 5000	2500, 5000	2500, 5000
WP2uvrA	1000-5000	1000-5000	2500, 5000	5000

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshfits in the genome of the strains used.