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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation (OECD 471): negative

Gene mutation in mammalian cells (OECD 476): negative

Cytogenicity/chromosome aberration in mammalian cells (no guideline followed but general compliant with OECD 473): negative

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: peer reviewed data
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 1997
Deviations:
yes
Remarks:
no 5th strain (TA 102 or E.coli) was tested
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix) from livers of Aroclor-1254-treated male Sprague-Dawley rat or Syrian hamster liver
Test concentrations with justification for top dose:
0.1, 0.3, 1.0, 3.0, 10.0, 33.0, 66.0, 100.0 µg/plate with and without metabolic activation
Vehicle / solvent:
not specified
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-aminoanthracene (2-AA), 4-nitro-1,2-phenylene diamine (4-NOPD)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, other: thinning, absence of bacterial lawn, appearance of his(-) pinpoint colonies
Evaluation criteria:
The criteria used for data evaluation are summarized as follows: 1) mutagenic response: a dose-delated, reproducible increase in the number of revertants over background, even if the increase was less than twofold; 2) nonmutagenic response: when no increase in the number of revertants was elicited by the chemical; 3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity. The initial determination of mutagenic, nonmutagenic, or equivocal was made by the testing laboratory; the final determination was made by the project officer.
Statistics:
Mean values and standard deviation were calculated.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slight toxicity at 100 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slight toxicity at 100 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slight toxicity at 33 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(slight) toxicity at 33 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The test substance was initially tested with strain TA 100 in the presence and the absence of the metabolic activation systems, over a wide dose range with an upper limit of 10 mg/plate, or less when solubility problems were encountered. Toxicity was evidenced by one or more of the following phenomena: appearance of his - pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bactenal lawn. As a rule, at least one toxic dose was incorporated into the first mutagenicity test; the repeat test occasionally had the doses adjusted so that an apparent toxic dose was not reached.

Please refer to the results provided under "Attached background material".

Genetic toxicity in vivo

Description of key information

Erythrocyte micronucleus study in mice (OECD 474): negative

Additional information

Reliable studies regarding genetic toxicity are available for the test substance.

 

In vitro:

 

- Gene mutation in bacteria:

The studies were coded and performed onS. typhimuriumTA 1535, PA 1537, TA 100 and TA 98. The metabolic activation was carried out with S9 mix containing 10 % of an S9 fraction obtained from male Sprague-Dawley rats or Syrian hamsters induced with Aroclor 1254. A pre­incubation method or which the bacterium strain concerned was incubated for 20 minutes at 37°C together with the test substance and S-9 mix or buffer was used. Soft agar was then added and the mixture plated on minimal medium. The plates were then incubated and inspected. Each test was performed at 5 concentrations, plus negative and positive control, on 3 plates each. The tests were not repeated. As, however, the tests were performed separately by two laboratories, those of the second laboratory may be looked upon as a repetition. The doses were chosen on the basis of the toxicity of the test substance and ranged from 100 µg to 0.1 µg per plate The tests were performed by EG&G Mason Research Institute and SRI International. The tests gave no indication for mutagenicity of the test substance in the tester strains. Thus, under the conditions of this study, the test substance does not induce mutations in the tested strains of S. typhimurium.

 

- Gene mutation in mammalian cells

The test substance was evaluated for mutagenic activity at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in L5178Y mouse lymphoma cells (forward mutation assay) according to OECD Guideline 476. In the mutation experiments, cultures were treated at concentrations up to and including 35 µg/mL in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9). The maximum concentration was based on the results of a cytotoxicity range-finding experiment. In the absence and presence of S-9, the test substance induced significant concentration-related cytotoxic effects. Due to extreme toxicity (<4% relative survival) cultures treated at 30 µg/mL and above in the absence of S-9 (both experiments) and at 35 µg/mL in its presence (Experiment 2) were not considered for assessment of mutagenicity. No precipitation was observed in the mutation experiments.

No biologically relevant increases in mutant frequency, compared to control values, were observed in the absence and presence of S-9 in Experiments 1 and 2.

The positive control chemicals 4-Nitro­quinoline 1-oxide and Benzo[a]pyrene induced clear mutagenic effects in the absence and presence of S-9 respectively, thereby demonstrating the sensitivity of the test system and the activity of the S-9 mix.

It is concluded that, under the conditions employed in this study, the test substance was not mutagenic in this test system when tested up to its limit of toxicity in the absence and presence of metabolic activation by S-9. Thus, under the conditions of this study, the test substance is negative in the HGPRT forward mutation assay in L5178Y cells.

 

- Cytogenicity / chromosome aberration in mammalian cells:

The test substance was tested in the chromosome aberration assay using Chinese hamster ovary cells. The assay was conducted in the absence of a metabolic activation system at dose levels of 4, 8, 15, 30, and 60 µg/mL and in the presence of an Aroclor-induced S-9 activation system at dose levels of 0.33, 0.65, 1.3, 2.5, 5, 10, and 20 µg/mL. Due to an observed delay in cell cycle kinetics in the toxicity study, metaphase cells were collected at 20 hours after initiation of treatment in order to assure microscopic evaluation of first-division metaphase cells. The test article was soluble in solvent and treatment medium at all dose levels tested.

Toxicity was a limiting factor in the analysis of test concentrations in the non-activated and S-9 activated studies. Excessive toxicity, (i.e., complete mitotic inhibition) precluded evaluation of dose level 60 µg/mL in the non-activated study and dose level 20 µg/mL in the S-9 activated study. To assure four scorable dose levels, dose levels 0.65, and 0.33 µg/mL were also tested but was not required for microscopic analysis in the S-9 activated study.

No increase in chromosome aberrations was observed in the non-activated or S-9 activated test systems relative to the solvent control group.Thus, under the conditions of this study, the test substanceis negative in the in-vitro chromosome aberration assay in Chinese hamster ovary cells.

 

In vivo:

- Cytogenicity / micronucleus test in mice:

The effect of the test substance on chromosome structure in bone marrow cells was investigated using an in vivo micronucleus test according to OECD Guideline 474.

A preliminary toxicity test was first conducted, using dosages of 60, 200, 600, 2000 and 5000 mg/kg. Subsequently male mice were given a single dose of the test substance at 500, 1000 or 2000 mg/kg and female mice were given a single dose at 250, 500 or 1000 mg/kg. In all cases the test substance was dosed by oral gavage, dissolved in maize oil. Concurrent vehicle and positive control groups of mice were similarly dosed with maize oil or chlorambucil (30 mg/kg) respectively. Five males and five females from each group were killed 24 hours after treatment; further lots of five males and five females, given the test substance at the highest dose levels or the vehicle, were killed 48 and 72 hours after treatment. Bone marrow smears on glass slides were made from each animal. These slides were then stained and prepared for examination.

A total of at least 2000 erythrocytes per animal were then examined for the presence of micronuclei, using the light microscope. Calculated values of micronuclei per 1000 polychromatic erythrocytes were analysed statistically using the Mann-Whitney U test. The ratio of polychromatic mature cells was also calculated for each animal, as an indicator of gross toxicity. No indication of bone marrow toxicity, as evidenced by depression of bone marrow proliferation, was noted in any treated group.

Males treated at 2000 mg/kg bw and females at 1000 mg/kg bw showed clinical signs including prone or hunched posture, piloerection, closed eyes, and reduced motor activity. Two high-dose males and one high dose female were sacrificed in extremis approx 4 h after dosing. No adverse reactions were noted in the lower dose groups.

Frequencies of micronucleated polychromatic erythrocytes in animals killed 24, 48 or 72 hours after administration of the test substance were generally similar to those in concurrent vehicle controls. A small but statistically significant (p<0.05) increase was observed in the group of female mice treated at 250 mg/kg and sacrificed 24 hours later. However, this is not thought to be of biological significance as the values for individual mice in this group all fell within the range observed for the vehicle control mice throughout the study (0.0 - 3.0) and no such effect was seen in mice treated with higher dosages of the test substance. Biologically and statistically significant increases over control values were, however, seen in positive control group animals given chlorambucil at 30 mg/kg (p < 0.01).Thus, under the conditions of this study, the test substanceis negative in the in vivo micronucleus test in mice.

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.

In fact, this is further in line with the conclusion of the ECHA Committee for Risk Assessment (RAC) Opinion for harmonised EU classification and labelling of Chlorophene based on the same reliable data, which was adopted in 2015 ( RAC CLH-O-0000001412-86-58/F, 12 March 2015). As a result, and referring to the Adaptation to Technical Progress (ATP) to CLP Regulation, Chlorophene was inserted in ATP10 with no classification for genetic toxicity.