Benzoic acid, 2,3,4,5-tetrachloro-6-cyano-, methyl ester, reaction products with p-phenylenediamine and sodium methoxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD guideline study, not in triplicate (duplicates only)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997

Deviations:

yes

Remarks:

only plate duplicates per treatment were used

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Mixture of octachloro, monomethoxy-heptachloro and bismethoxy-hexachloro derivatives of 3,3'-(1,4-phenylenedinitrilo)bis[2,3-dihydro-1H-isoindol-1-one]

EC Number:

600-736-8

Cas Number:

106276-80-6

Molecular formula:

C9 H3 Cl4 N O2 .C6 H8 N2 .C H40 .Na

IUPAC Name:

Mixture of octachloro, monomethoxy-heptachloro and bismethoxy-hexachloro derivatives of 3,3'-(1,4-phenylenedinitrilo)bis[2,3-dihydro-1H-isoindol-1-one]

Method

Target gene:

Salmonella: his-
Escherichia: trp-

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix of liver from rats induced with Phenobarbital/ 5,6-benzoflavone

Test concentrations with justification for top dose:

Dose-finding test: 5, 20, 78, 313, 1250 and 5000 µg/ plate
Main test: 313, 625, 1250, 2500 and 5000 µg/ plate
Confirmation test (only with TA 98 and TA100 with metabolic activation): 1000, 2000, 3000, 4000 and 5000 µg/ plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see "Details on test system"

Details on test system and experimental conditions:

Dose finding test, main test & confirmation test: Preincubation assay
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S-9 mix (10% S9 supernatant plus 90% cofactor solution) are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 mL of top agar is added and, after mixing, the samples are poured onto the minimum glucose agar plates.

In each experiment 2 test plates per dose or per control used; after incubation at 37°C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

POSITIVE CONTROL:
with metabolic activation: 2-aminoanthracene for TA 1535 (2.0 μg/plate) and WP2 uvr A (10 µg/plate); benzo[a]pyrene (5.0 μg/plate) for TA 98, TA 100 and TA 1537
without metabolic activation: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide for TA 98 (0.1 μg/plate), TA100 and WP2 uvr A(both 0.01 μg/plate); sodium azide (0.5 μg/plate) for TA 1535; 9-aminoacridine (80 μg/plate) for TA 1537.
All substances were dissolved in DMSO.

The titer was determined and the strain characteristics were checked in regular measurements. Sterility control was performed.

Evaluation criteria:

According to the report, the test substance was considered to have a reverse mutagenic potential when the number of revertant colonies per plate treated with the test substance was more than twice that of the negative (vehicle) control and there was reproducibility in test results, and when negative (vehicle) control values, positive control values and test results satisfied the acceptable standards.
Generally, a dose dependency should be observed in addition.

Statistics:

The arithmetic mean was calculated.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

in precipitating doses

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

other: S. typhimurium TA 1535, TA 1537 and TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

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:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: TA 98

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Dose-finding test (Preincubation test; 5 - 5000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor*	dose dependency	Assessment
TA 98	no	19	1.1	no	negative
	yes	33	1.1	no	negative
TA 100	no	117	1.1	no	negative
	yes	138	1.1	no	negative
TA 1535	no	8	1.0	no	negative
	yes	9	0.9	no	negative
TA 1537	no	7	0.9	no	negative
	yes	12	0.9	no	negative
WP2 uvr A	no	19	1.1	no	negative
	yes	25	0.9	no	negative
Main test (Preincubation test; 313 - 5000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor*	dose dependency	Assessment
TA 98	no	25	n.a.	no	n.a.
	yes	36	n.a.	no	n.a.
TA 100	no	119	n.a.	no	n.a.
	yes	123	n.a.	no	n.a.
TA 1535	no	11	n.a.	no	n.a.
	yes	11	n.a.	no	n.a.
TA 1537	no	7	n.a.	no	n.a.
	yes	12	n.a.	no	n.a.
WP2 uvr A	no	27	n.a.	no	n.a.
	yes	26	n.a.	no	n.a.
Confirmation test (Preincubation test; 1000 - 5000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor*	dose dependency	Assessment
TA 98	no	only neg. ctrl. performed
	yes	34	n.a.	no	n.a.
TA 100	no	only neg. ctrl. performed
	yes	134	n.a.	no	n.a.
* in non-cytotoxic, non-precipitating doses (>= 313 μg/plate without metabolic activation and >= 78 μg/plate with metabolic activation)
n.a. = not applicable

In TA98 with metabolic activation in precipitating doses, the revertant factor was maximum increased by a factor of 5.2, 5.3 and 4.8 in the highest dose in the dose finding, main and confirmation test, respectively; the increase of revertants showed a dose dependence in all three tests. In all other treatments with precipitating doses, no relevant increase of mean revertant number by a factor >= 2.0 was observed.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation in precipitating doses (TA 98)