4-[(3,5-Dichlorobenzoyl)amino]-3-hydroxybenzoic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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:

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

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-(3,5-dichlorobenzamido)-3-hydroxybenzoic acid

EC Number:

832-365-4

Cas Number:

1184581-58-5

Molecular formula:

C14H9Cl2NO4

IUPAC Name:

4-(3,5-dichlorobenzamido)-3-hydroxybenzoic acid

Test material form:

solid: particulate/powder

Details on test material:

Batch E010014831, re-test date: 30 June 2015

Specific details on test material used for the study:

Description White to off white powder
Batch E010014831
Purity/Composition 99.5%
Test substance storage At room temperature in the dark
Stable under storage conditions until 30 June 2015 (Retest date)
Purity/composition correction factor Not required
Stability at higher temperatures Not indicated
Stability in vehicle Dimethyl sulfoxide: Unknown
Solubility in vehicle Dimethyl sulfoxide: Not indicated

Method

Target gene:

S. typhimurium- histidine
E. coli - Tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa, gaol, chl, bio, uvrB

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate); male Sprague Dawley; Aroclor

Test concentrations with justification for top dose:

Dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main test: 3, 10, 33, 100, 333, 1000, 3330 µg/plate- Due to precipitate of the test substance on the
plates, the highest concentration of PF-06410251 used in the subsequent mutation assay was 3330
µg/plate.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191: TA 1537 2.5µg/plate; 2 aminoanthracene- all strains +S9 1-15µg/plate

Details on test system and experimental conditions:

Test system Salmonella typhimurium bacteria and Escherichia coli bacteria
Rationale Recommended test system in international guidelines (e.g. OECD, EC).
Source Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames)
(TA1535: 2006, TA1537: 2009, TA98: 2006, TA100: 2006) and (Master culture
from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK)
(WP2uvrA, 2008)
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 his C3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 his G46 Base-pair substitutions
TA100 his G46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)
Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)
The Salmonella typhimurium strains are regularly checked to confirm their histidine-requirement,
crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of
spontaneous revertants.
The Escherichia coli WP2
uvrA strain detects base-pair substitutions. The strain lacks an excision repair
system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of
mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment (Ref.1).
The strain is regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number
of spontaneous revertants.
Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

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 negative control data (number of spontaneous revertants per plate) should be within the
laboratory historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains, which are within the
laboratory historical range documented for each positive control substance. Furthermore, the mean
plate count should be at least three times the concurrent vehicle control group mean.
c) 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.

Statistics:

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the
concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or
WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent
control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is
greater than three (3) times the concurrent vehicle 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 independently repeated
experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final
evaluation decision

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Dose range finding test/first mutation experiment  

PF-06410251 was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33,

100, 333, 1000, 3330 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: 33, 100, 333, 1000

and 3330 µg/plate. The results are shown in Table 1 and Table 2.

Precipitate  

Precipitation of  PF-06410251 on the plates was observed at the start and at the end of the incubation

period at concentrations of 3330 µg/plate and upwards.

Toxicity

To determine the toxicity of  PF-06410251, the reduction of the bacterial background lawn, the increase

in the size of the microcolonies and the reduction of the revertant colonies were examined.

The bacterial background lawn was not reduced at any of the concentrations tested and no biologically

relevant decrease in the number of revertants was observed up to the dose level of

3330 µg/plate. Since  PF-06410251 precipitated heavily on the plates at the test substance

concentration of 5000 µg/plate the number of revertant colonies of this dose level could not be

determined.

Mutagenicity

No increase in the number of revertants was observed upon treatment with  PF-06410251 under all

conditions tested.

Experiment 2

To obtain more information about the possible mutagenicity of  PF-06410251, a second mutation

experiment was performed in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based

on the results of the first mutation assay,  PF-06410251 was tested up to the dose level of

3330 µg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Precipitate

Precipitation of  PF-06410251 on the plates was observed at the start and at the end of the incubation

period at the concentration of 3330 µg/plate.

Toxicity

In the second mutation assay, there was no reduction of the bacterial background lawn and no

biologically relevant decrease in the number of revertants at any of the concentrations tested in all

tester strains in the absence and presence of S9-mix.

Mutagenicity

In the second mutation assay, no increase in the number of revertants was observed upon treatment

with  PF-06410251 under all conditions tested.

Applicant's summary and conclusion

Conclusions:

All bacterial strains showed negative responses over the entire dose range, i.e. no significant doserelated
increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within the laboratory historical control
data ranges indicating that the test conditions were adequate and that the metabolic activation system
functioned properly.
Based on the results of this study it is concluded that PF-06410251 is not mutagenic in the Salmonella
typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of  PF-06410251 in the Salmonella typhimurium reverse mutation

assay and the Escherichia  coli  reverse mutation assay (with independent repeat).  

PF-06410251 was tested in the Salmonella typhimurium reverse mutation assay with four  histidinerequiring

strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia

coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA).

The test was performed in two independent experiments in the presence and absence of

S9-mix (rat liver S9-mix induced by Aroclor).

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

guidelines.

Batch E010014831 of  PF-06410251 was a white to off white powder with a purity of 99.5%. The test

substance was dissolved in dimethyl sulfoxide.

In the dose range finding test,  PF-06410251 was tested up to concentrations of 5000 µg/plate in the

absence and presence of S9-mix in the strains TA100 and WP2uvrA.  PF-06410251 precipitated on the

plates at dose levels of 3330 and 5000 µg/plate. The bacterial background lawn was not reduced at

any of the concentrations tested and no biologically relevant decrease in the number of revertants was

observed up to the dose level of 3330 µg/plate. Since  PF-06410251 precipitated heavily on the plates

at the test substance concentration of 5000 µg/plate, the number of revertant colonies of this dose

level could not be determined. Results of this dose range finding test were reported as part of the first

mutation assay.

Based on the results of the dose range finding test,  PF-06410251 was tested in the first mutation

assay at a concentration range of 33 to 3330 µg/plate in the absence and presence of 5% (v/v) S9-mix

in tester strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional

parameters,  PF-06410251 was tested at the same concentration range as the first assay in the

absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and

WP2uvrA.  PF-06410251 precipitated on the plates at the top dose of 3330 µg/plate. The bacterial

background lawn was not reduced at any of the concentrations tested and no biologically relevant

decrease in the number of revertants was observed.

PF-06410251 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 an independently repeated experiment.

In this study, the negative and strain-specific positive control values were within the laboratory

historical control data ranges indicating that the test conditions were adequate and that the metabolic

activation system functioned properly.

Based on the results of this study it is concluded that  PF-06410251 is not mutagenic in the Salmonella

typhimurium reverse mutation assay and in the Escherichia  coli  reverse mutation assay.