Hexachlorocyclopentadiene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Salmonella mutagenicity test Results for 250 Chemicals

Author:

Steve Haworth, Timothy Lawlor, Kristien Mortelmans, William Speck and Errol Zeiger

Year:

1983

Bibliographic source:

Environ Mutagen. 1983;5 Suppl 1:1-142

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only tested on 4 out of 5 recommended bacteria strains

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hexachlorocyclopentadiene

EC Number:

201-029-3

EC Name:

Hexachlorocyclopentadiene

Cas Number:

77-47-4

Molecular formula:

C5Cl6

IUPAC Name:

hexachlorocyclopentadiene

Test material form:

liquid

Method

Target gene:

his

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Salmonella typhimurium strains obtained from Dr. Bruce Ames, University of California, Berkeley.

Metabolic activation:

with and without

Metabolic activation system:

Liver S-9 mix from Aroclor 1254-induced male Sprague-Dawley rats and male Syrian hamsters

Test concentrations with justification for top dose:

0.00, 0.03, 0.10, 0.30, 1.00, 3.30, 10.00, 33.30, 100.00 ug/plate
The highest test dose was 10 mg/plate if no toxicity was apparent in the preliminary toxicity determination, or the upper limit of solubility was used. If toxicity was observed, the doses were chosen so that the high dose exhibited some degree of toxicity.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water; dimethyl sulfoxide (DMSO); ethanol; acetone
- Justification for choice of solvent/vehicle: The first choice of solvent was distilled water. Dimethyl sulfoxide (DMSO) was used if the chemical was insoluble or not sufficiently soluble in water, and ethanol (95%) or acetone was used if the chemical was not soluble or stable in DMSO.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-aminoanthracene (Tested on all strains in presence of S-9); 4-nitro-o-phenylenediamine (Tes ted on TA98 without S-9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
- Cell density at seeding (if applicable): 0.05ml cells added to each culture tube

DURATION
- Preincubation period: The test mixture was vortexed and allowed to incubate for 20 min at 37 °C.
- Expression time (cells in growth medium): The plates were inverted and incubated at 37°C for 48h.

NUMBER OF REPLICATIONS: At least 5 doses of each test chemical were tested on each strain in the presence of S-9 mix or buffer. Three plates were used and the experiment was repeated no less than 1 week after completion of the initial test.

Rationale for test conditions:

The preincubation procedure was selected because of evidences that it was no less sensitive than the plate test while being more effective for various chemicals.

Evaluation criteria:

A 'positive' response was indicated by a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. A negative response is obtained when no increase in revertant colonies is observed. An 'equivocal' or 'questionable' response was applied to low-level responses that were not reproducible or dose-related, or to results that showed a definite trend but with which the investigator did not feel comfortable making a positive or negative decision.

Statistics:

The data were evaluated using an analysis based on the models presented by Margolin et al (1981).

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

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:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

See attached background material for full results table.

Applicant's summary and conclusion

Conclusions:

Hexachlorocyclopentadiene was not considered to be mutagenic mutagenic to Salmonella typhinurium strains TA98, TA100, TA1535 or TA 1537 in this bacterial reverse mutation assay, both with and without metabolic activation.

Executive summary:

The in vitro genotoxicity in bacteria of the test substance was determined using a preincubation procedure, which was a modification of the Salmonella/mammalian microsome test of Ames et al [1975] and similar to the OECD Testing Guideline 471 (non GLP) with deviations.

At least five doses of test chemical, in addition to the concurrent solvent and positive controls, were tested on each bacteria strain in the presence of S-9 mix or buffer. A positive response was indicated by a reproducible, dose-related increase. Controls were valid.

Hexachlorocyclopentadiene was considered to be non-mutagenic under the conditions of the test, for both methods, with and without metabolic activation.