4-chloro-7H-pyrrolo[2,3-d]pyrimidine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

03-12-2018to 04-01-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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-chloro-7H-pyrrolo[2,3-d]pyrimidine

EC Number:

628-079-2

Cas Number:

3680-69-1

Molecular formula:

C6H4ClN3

IUPAC Name:

4-chloro-7H-pyrrolo[2,3-d]pyrimidine

Test material form:

solid

Details on test material:

Batch (Lot) Number: GR13290
Expiry date: 31 January 2020 (expiry date)
Physical Description: White to off-white solid
Purity/Composition: 99.1%
Storage Conditions: At room temperature

Method

Target gene:

Salmonella Spp - Histidine
E.Coli Spp- tryptophan

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-Fraction
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem
GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been
injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and
2-aminoanthracene, which require metabolic activation, in tester strain TA100 at
concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

Preparation of S9-Mix
S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per
10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg
glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water
(Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL
0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was
filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added
(5% (v/v) S9-fraction) to complete the S9-mix.

Test concentrations with justification for top dose:

Dose-range Finding Test
Selection of an adequate range of doses was based on a dose-range finding test with the
strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations,
1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate.
The highest concentration of PF-01323624 used in the subsequent mutation assays was
5000 µg/plate. At least five different doses (increasing with approximately half-log steps) of
the test item were tested in triplicate in each strain in the absence and presence of S9-mix.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

methylmethanesulfonate

other: All strains: 2-aminoanthracene 1-15µg with S9; TA1537 and TA989: 2-nitrofluorene10-15µg without S9; TA1537: ICR-191 2.5µg without S9

Details on test system and experimental conditions:

Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth
(Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C,
150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10
Freshly grown cultures of each strain were used for a test.
Agar plates
Agar plates (ø 9 cm) contained 25 mL glucose agar medium. Glucose agar medium contained
per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose
(Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella
typhimurium strains also contained 12.5 µg/plate biotin (Merck) and 15 µg/plate histidine
(Sigma) and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate
tryptophan (Sigma).
Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v)
sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 mL top agar were
transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for
20 min at 121 ± 3°C.
Environmental conditions
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C
(actual range 34.7 - 38.7°C). The temperature was continuously monitored throughout the
experiment. Due to addition of plates (which were at room temperature) to the incubator or
due to opening and closing the incubator door, temporary deviations from the temperature
may occur. Based on on laboratory historical data these deviations are considered not to affect
the study integrity.

Experimental Design
Dose-range Finding Test
Selection of an adequate range of doses was based on a dose-range finding test with the
uvrA, both with and without S9-mix. Eight concentrations,
1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate.
strains TA100 and WP2
The highest concentration of PF-01323624 used in the subsequent mutation assays was
5000 µg/plate. At least five different doses (increasing with approximately half-log steps) of
the test item were tested in triplicate in each strain in the absence and presence of S9-mix.
The first experiment was a direct plate assay and the second experiment was a pre-incubation
assay.
The negative control (vehicle) and relevant positive controls were concurrently tested in each
strain in the presence and absence of S9-mix.

First Experiment: Direct Plate Assay
The above mentioned dose-range finding study with two tester strains is reported as a part of
the direct plate assay. In the second part of this experiment, the test item was tested both in
the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. Top
agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were
successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture
(10e9 cells/mL) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO and
either 0.5 ml S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case
of non-activation assays).
The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto
a selective agar plate. After solidification of the top agar, the plates were inverted and
incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies
(histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp
+) for Escherichia coli) were counted.

Second Experiment: Pre-Incubation Assay
The test item was tested both in the absence and presence of S9-mix in all tester strains. Top
agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were
pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1°C, either 0.5 mL S9-mix (in case of
activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays),
0.1 mL of a fresh bacterial culture (10e9 cells/mL) of one of the tester strains, 0.1 mL of a
dilution of the test item in DMSO. After the pre-incubation period the solutions were added
to 3 mL molten top agar. The ingredients were mixed on a Vortex and the content of the top
agar tube was poured onto a selective agar plate. After solidification of the top agar, the
plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period
revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and
tryptophan independent (Trp+) for Escherichia coli) were counted.

Colony Counting
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates
with sufficient test item precipitate to interfere with automated colony counting were counted
manually. Evidence of test item precipitate on the plates and the condition of the bacterial
background lawn were evaluated when considered necessary, macroscopically and/or
microscopically by using a dissecting microscope.

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation
assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without
S9-mix) must exhibit a characteristic number of revertant colonies when compared
against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit
limited solubility as demonstrated by the preliminary toxicity range-finding test or should
extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen
event. If the results are considered invalid due to contamination, the experiment will be
repeated.
All results presented in the tables of the report are calculated using values as per the raw data
rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

No formal hypothesis testing was done.
In addition to the criteria stated below, any increase in the total number of revertants should
be evaluated for its biological relevance including a comparison of the results with the
historical control data range.

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2
uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535,
TA1537 or TA98 is not greater than three 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 or WP2
uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535,
TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat 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.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

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

Positive controls validity:

valid

Any other information on results incl. tables

First Experiment: Direct Plate Assay

PF-01323624 was initially tested in the tester strains TA100 and WP2

uvrA as a dose-range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the

absence and presence of S9-mix. Based on the results of the dose-range finding test, the

following dose-range was selected for the mutation assay with the tester strains, TA1535,

TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and

5000 µg/plate. The results are shown in Table 1 and Table 2. The individual data are

presented in Appendix 3.

Precipitate

Precipitation of  PF-01323624 on the plates was not observed at the start or at the end of the

incubation period in any tester strain.

Toxicity

To determine the toxicity of the test item, the reduction of the bacterial background lawn, the

increase in the size of the microcolonies and the reduction of the revertant colonies were

observed. The definitions are stated in Appendix 2.

Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester

strains TA1537 in the absence of S9-mix and TA98 and TA100 in the absence and presence

of S9-mix.

Mutagenicity

In the direct plate test, no increase in the number of revertants was observed upon treatment

with  PF-01323624 under all conditions tested.

9.2. Second Experiment: Pre-Incubation Assay

To obtain more information about the possible mutagenicity of the test item, a pre-incubation

experiment was performed in the absence and presence of S9-mix. Based on the results of the

first mutation assay,  PF-01323624 was tested up to the dose level of 5000 µg/plate in the

tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The results are shown in Table

3, the individual data are presented in Appendix 3.

Precipitate

Precipitation of  PF-01323624 on the plates was observed at the start of the incubation period

at the concentration of 1600 and 5000 µg/plate and at the end of the incubation period at the

top dose level of 5000 µg/plate.

Toxicity

Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in all

tester strains in the absence and presence of S9-mix, except for tester strains TA1535 in the

presence of S9-mix and WP2uvrA in the absence and presence of S9-mix.

Mutagenicity

In the pre-incubation test, no increase in the number of revertants was observed upon

treatment with the test item under all conditions tested.

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this study it is concluded that PF-01323624 is not
mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli
reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of  PF-01323624 and/or its

metabolites to induce reverse mutations at the  histidine  locus in several strains of Salmonella

typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan

locus of Escherichia  coli (E. coli)  strain WP2uvrA in the presence or absence of an exogenous

mammalian metabolic activation system (S9).

The test was performed in two independent experiments, at first a direct plate assay was

performed and secondly a pre-incubation assay.

The study procedures described in this report were based on the most recent OECD and EC

guidelines.

Batch GR13290 of  PF-01323624 was a white to off-white solid with a purity of 99.1%. The

vehicle of the test item was dimethyl sulfoxide.

In the dose-range finding study, the test item was initially tested up to concentrations of

5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay.  PF-01323624 did

not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the

number of revertants, was observed in tester strain TA100 in the absence and presence of

S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested up to concentrations of

5000 µg/plate in the strains TA1535, TA1537 and TA98.  PF-01323624 did not precipitate on

the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of

revertants, was observed in tester strains TA1537 in the absence of S9-mix and TA98 in the

absence and presence of S9-mix.

In the second mutation experiment, the test item was tested up to concentrations of

5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2

uvrA in thepre-incubation assay. The test item precipitated on the plates at the top dose of 5000 µg/plate.

Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in all

tester strains in the absence and presence of S9-mix, except in tester strains TA1535 in the

presence of S9-mix and WP2uvrA in the absence and presence of S9-mix.

In this study, acceptable responses were obtained for the negative and strain-specific positive

control items indicating that the test conditions were adequate and that the metabolic

activation system functioned properly.

The test item did not induce a significant dose-related increase in the number of revertant

(His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in

the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and

presence of S9-metabolic activation. These results were confirmed in a follow-up

experiment.

In conclusion, based on the results of this study it is concluded that  PF-01323624 is not

mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia  coli

reverse mutation assay.