4-[bis(phenylsubstituted)substituted]phthalic 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:

Experiment Starting Date: July 22, 2015 Experiment Completion Date: July 30, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Type:

impurity

Test material form:

solid: particulate/powder

Method

Target gene:

S. typhimurium - histidine locus
E. coli - tryptophan locus

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 mix (prepared from from induced rat liver)

Test concentrations with justification for top dose:

Main test 1:
+/- S9 mix
All strains: 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate

Main test 2:
- S9 mix
TA100: 39.1, 78.1, 156, 313, 625, 1250 and 2500 µg/plate
TA1535: 19.5, 39.1, 78.1, 156, 313, 625, 1250 and 2500 µg/plate
WP2uvrA: 156, 313, 625, 1250, 2500 and 5000 µg/plate
TA98 and TA1537: 39.1, 78.1, 156, 313, 625 and 1250 µg/plate

+ S9 mix
All strains: 313, 625, 1250, 2500 and 5000 µg/plate

Dose selection:

Main test 1:
5000 µg/plate was the highest dose and five lower doses were diluted with a geometric progression of 4.

Main test 2:
The result of the main test 1 showed that the number of revertant colonies in the test substance treatment groups was increased to twice or more than that in the negative control for TA100 and TA1535 without S9 and for TA100 with S9 mix. The bacterial growth inhibition was observed at 1250 µg/plate or more for TA100, TA1535, TA98 and TA1537 and at 5000 µg/plate for WP2uvrA without S9 mix. The precipitation of the test substance was not observed in either the presence or absence of S9 mix.
Therefore, in consideration of the increase of the number of revertant colonies and the bacterial growth inhibition, the dose levels described above for main test 2 were selected.

Vehicle / solvent:

Vehicle- DMSO
The test substance was insoluble in distilled water at 50.0 mg/mL and soluble in DMSO at 50.0 mg/ml. The test substance solution of 50,0 mg/mL prepared with DMSO was considercd to be stable from the facts that there were no change in color, exothermic reaction nor gas generation at room temperature within 2 hours after preparation. Therefore, DMSO was selected as a vehicle.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

0.01 µg/plate for strain TA100 and WP2uvrA, 0.1 µg/plate for strain TA98

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

- S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/plate for strain TA1535

Positive control substance:

sodium azide

Remarks:

- S9 mix

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/plate for strain TA1537

Positive control substance:

other: ICR-191

Remarks:

- S9 mix

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/plate for strain TA98, 1 µg/plate for strain TA100, 2 µg/plate for strains TA1535 and TA1537, 10 µg/plate for strain WP2uvrA

Positive control substance:

other: 2-Aminoanthracene

Remarks:

+ S9 mix

Details on test system and experimental conditions:

Medium and S9 Mix:
Medium:
- Minimal glucose agar plate
- Soft agar: A solution containing 0.5 mM histidine and 0.5 mM biotin for S.typhimurium strains or 0.5 mM tryptophan for E.coli strain was added to a soft agar solution containing 0.6 w/v% agar and 0.5 w/v% NaCl at a volume ratio of 1 to 10.

S9 mix:
Rat liver S9: S9 was prepared from the liver of 7-week old rats administered with phenobarbital and 5,6-benzoflavone.
Composition of S9 mix: S9 mix was prepared just before use. 1 ml of S9 mix consisted of 8 µmol MgCl2, 33 µmol KCl, 5 µmol Glucose-6-phosphate, 4 µmol NADPH, 4 µmol NADH, 100 µmol sodium-phosphate buffer (pH 7.4) and 0.1 mL of S9.

Pre-cultures of the tester strains:
Frozen stock culture of bacterial strains, 20 µl for TA100, TA98, TA1535 and TA1537, 5 µl for WP2uvrA, were respectively inoculated to 11 ml of Nutrient broth No. 2 in L- tube (volume: 27 ml). The culture was incubated at 37 ± 0.5 °C for 9 hours with shaking at about 50 times/minute by a seesaw type of shaker.
The number of viable cells was calculated from O.D value at 660 nm measured by a photometer (miniphoto 518R, TAITEC) at the end of incubation. It was confirmed that the numbers of viable cells were more than 1.0 x 10^9 cells/ml.
The final numbers of viable cells are shown below:
TA100: 2.1 x 10^9 cells/ml
TA1535: 2.0 x 10^9 cells/ml
WP2uvrA: 4.7 - 4.8 x 10^9 cells/ml
TA98: 2.2 x 10^9 cells/ml
TA1537: 1.8 - 1.9 x 10^9 cells/ml

Preparation of test substance solution and positive control substance solutions:
a) Preparation of test substance solution
1) Preparation method
The test substance was weighed, added into DMSO and mixed by laboratory mixer to make a 50.0 mg/ml as original solution. The test substance solutions of each required concentration were prepared by diluting with the same vehicle.
2) Timing of preparation
The test substance solutions were prepared just before use, kept under yellow light at room temperature and used within 2 hours.
b) Preparation of positive control substance solutions
1) Preparation method and storage conditions
NaN3 was dissolved in distilled water, AF-2, ICR-191 and 2AA were dissolved in DMSO. Positive control solutions were stored below -80°C. The positive control substances were thawed just before use.

Methods:
The pre-incubation method was used and the test was carried out in triplicate (for negative control, positive control and test substance treatment groups).

Procedures:
After 0.1 ml of the test substance solution, vehicle or positive control solutions, 0.5 ml of 0.1 M sodium phosphate buffer (pH 7.4) or S9 mix, and 0.1 ml of the bacterial culture were added to a test tube, the mixture was shaken at 37 ± 0.5 ºC for 20 minutes. Two millilitres of the soft agar were then added to each tube and the mixture was then poured onto a minimal agar plate. The number of revertant colonies was counted after incubation at 37 ± 0.5 ºC for 48 hours.

Confirmation of Sterility:
The highest concentration of the test substance solution (0.1 mL) and S9 mix (0.5 mL) were individually mixed with 2 mL of the soft agar and were poured onto each minimal glucose agar plate in order to examine bacterial contamination. Bacterial contamination was judged with those plates after incubation at 37 ± 0.5 ºC for 48 hours.

Observation:
The precipitation of the test substance was observed macroscopically and the bacterial growth inhibition was observed by using a stereomicroscope at the end of the incubation

Colony counting:
For the plates in which the bacterial growth inhibition was observed, the number of colonies was counted manually, and for the other plates with a colony analyser. Square correction and miscounting correction were conducted when counting with the colony analyser.

Rationale for test conditions:

The ability of the test substance to induce mutations was investigated by using S.typhimurium and E.coli.

Evaluation criteria:

The test substance was judged to be positive when the number of revertant colonies increased twice or more than that in the negative control and when the responses were dose-related and/or reproducible. The other cases were judged to be negative. No statistical methods were used.
In the case of a positive result, the specific activity (number of revertant colonies/mg) was calculated by the following formula for the dose levels at which the number of revertant colonies was shown twice or more than that in the negative control, and the highest value in the specific activity wa designated as the specific activity of the test substance.
Specific activity = (N – N0) / D
N: mean number of revertant colonies at the corresponding dose
N0: mean number of revertant colonies in the negative control
D: the corresponding dose (mg/plate)

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

(with and without S9-mix)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(without S9-mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9 mix only

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9 mix only

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Main Test 1:
The number of revertant colonecs in the test substance treatment groups was increased to twice or more than that in the negative control for TA100 and TA1535 without S9 mix and for TA100 with S9 mix. The bacterial growth inhibition was observed at 1250 µg/Plate or more for TA100, TA1535, TA98 and TA1537, and at 5000 µg/Plate for WP2uvrA without S9 mix.
The precipitation of the test substance was not observed in either the presence or absence of S9 mix.

Main Test 2:
The number of revertant colonies in the test substance treatment groups was increased to twice or more than that in the negative control for TA100 and TA1535 without S9 mix and for TA100 with S9 mix. The bacterial growth inhibition was observed at 1250 µg/plate or more for TA100, at 625µg/plate or more for TA1535, at 5000 µg/plate for WP2uvrA, and at 625 µg/plate or more for TA98 and TA1537 without S9 mix. The precipitation of the test substance was not observed in either the presence or absence of S9 mix.

Any other information on results incl. tables

The results of tests 1 and 2 are shown in attached background material (result tables and dose-response curves).

Applicant's summary and conclusion

Conclusions:

It was concluded that CIM-43 had ability to induce mutations under the test conditions

Executive summary:

The ability of CIM-43 to induce mutations was investigated using Salmonella typhurium strains TA100, TA1535, TA98 and TA1537 and Escherichia coli strain WP2uvrA with a pre-incubation method in the presence and absence of a metabolic activation system (S9 mix).

The mutagenicity of the test substance was judged to be positive because the number of revertant colonies in the test substance treatment groups increased to twice or more than that in the negative control for TA100 and TA1535 without S9 mix and for TA100 with S9 mix, and the reproducibility was confirmed. In the other test conditions, the number of revertant colonies in the test substance treatment groups was less than twice that in each negative control.

Consequently, it was concluded that the CIM-43 had ability to induce mutations under the present test conditions.