2-(4-chlorobenzoyl)benzoic 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

Study period:

From May 24, 1988 to June 20, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-chlorobenzoyl)benzoic acid

EC Number:

201-615-9

EC Name:

2-(4-chlorobenzoyl)benzoic acid

Cas Number:

85-56-3

Molecular formula:

C14H9ClO3

IUPAC Name:

2-(4-chlorobenzoyl)benzoic acid

Test material form:

solid

Details on test material:

Purity: 99.8 %

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

Experiment 1:
Doses: 0, 20, 100, 500, 2500 and 5000 ug/plate in DMS0 - 3 test plates per dose or per control

Experiment 2:
Doses: 0, 4, 20, 100, 500 and 2500 ug/plate in DMS0 - 3 test plates per dose or per control

Vehicle / solvent:

Comlete solubility of the test substance in DMSO.

Controls

Untreated negative controls:

yes

Remarks:

vehicle: DMSO

Positive controls:

yes

Positive control substance:

other: 10 ug 2-aminoanthracene in DMSO (with S9), 5 ug N-methyl-N -nitro-N-nitrosoguanidine (MNNG) (without S-9))

Details on test system and experimental conditions:

S-9 mix:
The S-9 mix is prepared freshly prior to each experiment (1, 2). For this purpose, a sufficient amount of S-9 -fraction is thawed at room temperature and 3 volumes of S-9 fraction are mixed with 7 volumes of S-9 supplement (cofactors). This preparation, the so-called S-9 mix, is kept on ice until used. The concentrations of the cofactors in the S-9 mix are:

MgCl2 8mM
KCl 33 mM
glucose-6--phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 100 mM. The phosphate buffer is prepared by mixing an Na2HPO4 solution with an NaH2PO4 solution in a ratio of about 4:1.

Bacteria:

The indicator organisms TA 1535, TA 1537, TA 98 and TA 100 selected for this test are derivatives of Salmonella typhimurium. All strains have a defective excision repair System (uvr9), which prevents the repair of lesions which are induced in the DNA, and this deficiency results in greatly enhanced sensitivity of some mutagens. Furthermore, all strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances.

The strains TA 1535 and TA 100 are derived from histidineprototrophic Salmonella strains by the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and TA 96 are strains for the detection of frameshift mutagens. These strains carry different frameshift markers, i.e. the .1 mutant his C 3076 in the case of TA 1537 and the .2 type his D 3052 in the case of TA 96.

The strains TA 96 and 1A 100 carry an R factor plasmid pKM 101 (4) and, in addition to having genes resistant to antibiotics, they have a modified postreplication DNA repair system, which increases the mutation rate by inducing a defective repair in the DNA; this again leads to a considerable increase in sensitivity.

Preincubation test
The experimental procedure is based on the method described by Yahagi et al. and Matsushime et al. 0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37 °C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds.
Composition of the minimal glucose agar:
960 ml aqua dest.
20 ml Vogel-Bonner E medium
15 g Oifco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37 °C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Standard plate test
The experimental procedure is based on the method of Ames et al. Test tubes containing 2 ml portions of soft agar which consists of 100 mL agar (0.6 % agar + 0.6 % NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order:
0.1 ml test solution
0.1 ml bacterial suspension
0.5 ml 5-9 mix (in tests with metabolic activation)
or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates
Titer determination
Ttiter is determined only in the experiments with S-9 mix both without test substance (solvent only) and after adding the two highest amounts of substance. For this purpose, 0.1 mL of the overnight cultures is diluted to 10 –E06 in each case. Test tubes containing 2 ml portions of soft agar containing maximal amino-acid solution (5 mM histidine * 0.5 mM biotin) are kept in a water bath at 45 °C, and the remaining components are added in the following order:
0.1 ml solvent (without and with test substance)
0.1 ml bacterial Suspension (dilution: 10-E06)
0.5 ml S-9 mix
After mixing, the samples are poured onto the Vogel-8onner agar plates within approx. 30 seconds. After incubation at 37 °C for 48 hours in the dark, the bacterial colonies are counted.

Test design:
Experiment 1:
Strains: TA 1535, TA 100, TA 1537, TA 98
Doses: 0, 20, 100, 500, 2500 and 5000 ug/plate in DMS0
Type of test, Standard plate test with and without S-9 mix
Number of plates: 3 test plates per dose or per control

Experiment 2:
Strains: TA 1535, TA 100, TA 1537, TA 98
Doses: 0, 4, 20, 100, 500 and 2500 ug/plate in DMS0
Type of test, preincubation test with and without S-9 mix
Number of plates: 3 test plates per dose or per control

Evaluation criteria:

Evaluation criteria
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

doses >5000 µg/plate (SPT) or >2500 µg/plate (PIT)

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

doses >5000 µg/plate (SPT) or >2500 µg/plate (PIT)

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

doses >5000 µg/plate (SPT) or >2500 µg/plate (PIT)

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

doses >5000 µg/plate (SPT) or >2500 µg/plate (PIT)

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance is not mutagenic in the Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98 at the dose range of: 20 µg - 5000 µg/plate (SPT) and at the dose range of: 4 µg - 2500 µg/plate (PIT) in the absence and presence of metabolic activation.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 in Bacterial Reverse Mutation Test. The test substance was examined using four strains of Salmonella typhimurium (TA 1535, TA 100, TA 1537, and TA 98). The test was performed in two experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). DMSO was used as a vehicle. Toxic effects were monitored at doses: 20 µg - 5000 µg/plate in the Standard plate test (SPT) and 4 µg - 2500 µg/plate in the Preincubation test (PIT). In both experiments 3 test plates per dose or per control were investigated. Bacteriotoxic effect were observed at doses >5000 µg/plate (SPT) or >2500 µg/plate (PIT). An increase in the number of his revertants was not observed both in the SPT and in the PIT either without S-9 mix or after the addition of a metabolizing system. Under the study conditions, the test substance is not mutagenic in the Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98 at the dose range of: 20 µg - 5000 µg/plate (SPT) and at the dose range of: 4 µg - 2500 µg/plate (PIT) in the absence and presence of metabolic activation (Gelbke, 1988).