5-chloro-2-(4-chlorophenoxy)phenol;[DCPP]

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:

key study

Study period:

from 1998-11-04 to 1999-02-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with the following deviation: 2-Aminoanthracene was the only positive control for the activation assays.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

yes

Remarks:

2-Aminoanthracene was the only positive control for the activation assay whereas OECD 471 recommends the use of a further positive control substance.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

429-290-0

EC Name:

-

Cas Number:

3380-30-1

Molecular formula:

C12 H8 Cl2 O2

IUPAC Name:

5-chloro-2-(4-chlorophenoxy)phenol

Details on test material:

- Name of test material (as cited in study report): FAT 80'220/A
- Physical state: solid, beige
- Analytical purity: > 99% (HPLC)
- Lot/batch No.: GRU 98
- Expiration date of the lot/batch: 2008-10-31
- Stability under test conditions: 10 day in PEG 1 %, CMC 1 %, and isopropanol 1 %
- Storage condition of test material: room temperature

Method

Target gene:

His operon (Salmonella strains), Trp operon (E. coli)

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: defective DNA repair activity ( uvrB) and defective LPS barrier (rfa) Ampicillin resistance (pKM101, TA 98 and TA 100 only)

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: uvrA DNA repair deficient.

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal S9 fraction from Wistar rats treated with phenobarbital and beta-naphtoflavone

Test concentrations with justification for top dose:

According to the dose selection criteria the test article was tested at the following concentrations:
Without S9 mix: 0.03; 0.1; 0.3; 1.0; 3.3; and 10.0 µg/plate
With S9 mix: 0.1; 0.3; 1.0; 3.3; 10.0; and 33.3 µg/plate

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: TA1535, TA100: sodium azide, NaN3 / TA1537, TA98: 4-nitro-o-phenylene-diamine, 4-NOPD / WP2 uvrA: methyl methane sulfonate, MMS / With metabolic activation: 2-aminoanthracene, 2-AA (all strains)

Details on test system and experimental conditions:

- For each strain and dose level including the controls, three plates were used. The following materials were mixed in a test tube and poured onto the minimal agar plates:
100 µL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 mL Bacteria suspension (cf test system, pre-culture ofthe strains),
2000 µL Overlay agar
- In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45° C) was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

Evaluation criteria:

A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.
A test article producing neither a reproducible and dose related increase in the number of revertants, nor a biologically relevant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
A mutagenic response is described as follows:
A test article is considered mutagenic if in the strains TA 98, TA 100, and WP2 uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 0.1 µg/plate without S9 and at 33 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 0.1 µg/plate without S9 and at 33 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No relevant increase of the number of revertant colonies occurred in any of the strains up to the maximal concentration tested, neither in the presence nor in the absence of metabolic activation. In the confirmatory experiment, an isolated increase in revertant colony numbers reaching the threshold of 3.0 was observed in strain TA 1535 in the presence of metabolic activation. This borderline effect was considered as biologically irrelevant since it was not reproduced. Most likely, this increase was caused by toxic effects of the test article resulting in lower numbers of surviving bacteria on the corresponding plates. A low number of bacteria competing for the traces of histidine introduced with the top agar leads to bacterial colony growth until the histidine is depleted. Such colonies may be mistaken for mutant colonies although they tend to be rather small.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative