2-Chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

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:

19 May - 12 Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

adopted in 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

Cas Number:

1211443-61-6

Molecular formula:

C14 H17 Cl N4 O

IUPAC Name:

2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA1535, TA97, TA98, TA100 and TA102

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats treated with Aroclor 1254 (500 mg/kg bw); S9 mix was tested in two concentrations of 5% and 10% (v/v)

Test concentrations with justification for top dose:

Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate for TA100 with (5% v/v) and without metabolic activation

The dose-range finding test is further reported as a part of Experiment 1.

Experiment 1: 52, 164, 512, 1600 and 5000 μg/plate for remaining strains with (5% v/v) and without metabolic activation
Experiment 2: 492, 878, 1568, 2800 and 5000 µg/plate for all strains with (10% v/v) and without metabolic activation

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191: -S9, 0.75 µg/plate in DMSO for TA97; tert-butyl hydroperoxide (TBH): -S9, 250 µg/plate in DMSO for TA102; 2-aminoanthracene (2AA): +S9, 2.5, 5, 1, 1, 2 and 10 µg/plate in DMSO for strains TA1535, TA97, TA98, TA100, TA 100 and TA102, respectively

Remarks:

Each S9 batch is characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation

DURATION : 48 ± 4 h

NUMBER OF REPLICATIONS: Triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn, increase in the size of the microcolonies and the reduction of revertant colonies

OTHER:
Revertant colonies were counted with the Sorcerer Colony Counter.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100, TA97 and TA102 is not greater than 2 times the concurrent control, and the total number of revertants in tester strains TA1535, and TA98 is not greater than 3 times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100, TA97 and TA102 is greater than 2 times the concurrent control, or the total number of revertants in tester strains TA1535 and, TA98 is greater than 3 times the concurrent control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

Mean values and standard deviations were calculated.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA1535, TA97, TA98, TA100 and TA102

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test substance was observed in either the presence or absence of S9 mix.

HISTORICAL CONTROL DATA : The test results showed that the numbers of revertant colonies in the negative and positive controls were within the range of the historical data at the testing facility.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Remarks on result:

other:

Remarks:

In strain TA102, a fluctuation in the number of revertant colonies above the laboratory historical control data range was observed in the presence of S9-mix at 512 μg/plate. Since the increase was less than two-fold (a maximum of 1.2-fold was reached), it was not considered to be biologically relevant.

Any other information on results incl. tables

Table 1. Results of dose-range finding test (as part of Experiment 1)

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.)
	TA100
	-S9 mix			+S9 mix (5%)
Positive control	706	±	115	533	±	63
Solvent control	91	±	6	94	±	27
1.7	104	±	11	85	±	12
5.4	105	±	6	85	±	6
17	95	±	13	85	±	19
52	86	±	18	91	±	16
164	97	±	6	89	±	7
512	106	±	4	87	±	9
1600	92	±	6	95	±	23
5000	90	±	10	75	±	13

Table 2. Results of Experiment 1

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.)
	TA1535			TA98			TA102			TA97
	-S9 mix
Positive control	800	±	5	1411	±	56	876	±	136	1605	±	31
Solvent control	5	±	2	10	±	5	403	±	24	136	±	19
52	11	±	4	11	±	4	300	±	11	131	±	11
164	13	±	2	14	±	5	322	±	26	122	±	10
512	14	±	3	14	±	4	375	±	23	121	±	6
1600	7	±	1	16	±	4	388	±	15	125	±	8
5000	5	±	4	10	±	4	334	±	21	113	±	8
+S9 mix (5%)
Positive control	176	±	18	1062	±	76	732	±	152	2074	±	103
Solvent control	7	±	4	17	±	2	371	±	21	142	±	12
52	9	±	4	18	±	5	420	±	27	154	±	21
164	7	±	3	17	±	1	394	±	71	155	±	7
512	8	±	4	17	±	2	440	±	36	139	±	13
1600	9	±	3	17	±	8	408	±	27	147	±	29
5000	4	±	1	13	±	4	401	±	16	137	±	20

 

Table 3. Results of Experiment 2

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.)
	TA1535			TA98			TA100			TA102			TA97
	-S9 mix
Positive control	922	±	27	1172	±	232	1266	±	119	1331	±	296	1488	±	76
Solvent control	10	±	5	14	±	6	104	±	11	327	±	41	152	±	6
492	14	±	1	30	±	9	109	±	13	238	±	51	152	±	9
878	10	±	5	24	±	5	102	±	25	267	±	31	163	±	20
1568	10	±	3	12	±	4	105	±	8	306	±	29	155	±	7
2800	14	±	6	10	±	2	122	±	3	239	±	46	155	±	15
5000	10	±	2	18	±	9	104	±	11	247	±	18	171	±	21
	+S9 mix (10%)
Positive control	251	±	71	789	±	88	1206	±	152	718	±	53	1812	±	179
Solvent control	21	±	6	24	±	5	92	±	23	315	±	52	174	±	20
492	17	±	3	26	±	9	90	±	10	313	±	19	177	±	6
878	13	±	2	18	±	5	85	±	15	303	±	30	197	±	24
1568	12	±	4	21	±	3	80	±	6	326	±	34	173	±	7
2800	14	±	2	22	±	4	79	±	13	300	±	11	183	±	5
5000	8	±	4	21	±	4	87	±	1	308	±	29	167	±	20

Table 4. Historical solvent control data from experiments performed between May 2015 and May 2017

	TA1535		TA97		TA98		TA100		TA102
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	3 -36	3 - 32	85 - 191	93 - 199	8 - 41	9 - 55	66 - 161	63 - 160	195 - 475	264 - 428
Mean	11	11	122	151	16	23	105	105	269	336
SD	4	4	31	27	5	7	19	20	44	37
n	2057	2039	45	45	2023	2083	2027	2033	267	288

SD = standard deviation

n = number ob observations

Table 5. Historical positive control data from esperiments performed between May 2015 and May 2017

	TA1535		TA97		TA98		TA100		TA102
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	125 - 1381	78 - 1058	652 - 2026	915 – 2405	410 – 1995	250 - 1977	537 – 1848	408 - 2651	618 – 1215	628 – 1154
Mean	839	220	1131	1785	1369	929	908	1330	857	885
SD	153	112	312	446	310	345	178	324	255	153
n	2065	1967	48	45	1920	2014	2007	2020	10	26

SD = standard deviation

n = number ob observations

Applicant's summary and conclusion

Conclusions:

Under the conditions of the conducted study the substance was not mutagenic in any of the five Salmonella typhimurium strains (TA1535, TA97, TA98, TA100 and TA102) tested with and without metabolic activation.