Hexachlorocyclopentadiene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The capacity of the registered substance to induce gene mutation in bacteria and mammalian cells in vitro was investigated using methods similar to the OECD Testing Guidelines 471 and 476 respectively. The registered substance did not induce gene mutation in bacteria and mammalian cells under the conditions of these tests.

 

In regard to the capacity of the registered substance to induce structural chromosomal aberration in mammalian cells in vitro, it was not relevant to perform a test as in vivo studies investigating this endpoint were available.

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

his

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.

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).

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

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

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

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

See attached background material for full results table.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

Low doses were used due to the substance being cytotoxic

GLP compliance:

no

Type of assay:

other: Mouse Lymphoma Forward Mutation Assay

Target gene:

thymidine kinase (tk)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Fischer mouse

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Fischer's medium for leukemic cells of mice with 10% horse serum and sodium pyruvate.

Additional strain / cell type characteristics:

other: heterozygous for a specific autosomal mutation at the TK locus and sensitive to BUdR

Metabolic activation:

with and without

Metabolic activation system:

S9 liver

Test concentrations with justification for top dose:

Ranging from 2.5 pl/ml to 1.25 μl/ml. A level that showed at least 50% reduction in growth potential was used as the highest dose, with at least 3 lower doses including levels that were below the toxic range.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

growth medium without the addition of solvent

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (1%)

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): Bromodeoxyuridine (BUdR)

OTHER: Cloning medium consisted of Fischer's medium with 20% horse serum, sodium pyruvate, and 0.37% agar. Selection medium was made by the addition of 7.5 mg BUdR to 100 ml cloning medium.

Rationale for test conditions:

Low doses were used due to the substance being cytotoxic.

Evaluation criteria:

A compound is considered mutagenic if:
- A dose-response relationship is observed over 3 of the 4 dose levels
- The minimum increase at the high level of the dose-response curve is at least 2.5 times greater than the solvent and/or negative control values
- The solvent and negative control data are within the normal range of the spontaneous background for the TK locus

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: cytoxicity was evaluated in order to select non cytotoxic concentrations for the assay

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

See attached background material for full results table.

Conclusions:

Hexachlorocyclopentadiene did not induce forward mutation in L5178Y cells. No dose related trends in either the absolute number of mutants or the mutant frequencies were observed and at no level did these parameters increase to 2.5 times the spontaneous level. Therefore, the compound was considered to be non-mutagenic under the conditions of this assay.

Executive summary:

The capacity of Hexachlorocyclopentadiene to induce gene mutation in mammalian cells was determined according to the method similar to the OECD Testing Guideline 476 (non GLP) with deviations.

The test substance was evaluated in L5178Y cells with and without metabolic activation over the concentration range of 2.5 pl/ml to 1.25 µl/ml. These low concentrations were selected due to the cytotoxicity occurring at higher concentrations.

DMSO was used a the vehicle and negative control. Ethylmethanesulfonate and N-Nitrosodimethylamine were used as positive controls. Results were within the expected range and therefore considered as valid.

Hexachlorocyclopentadiene did not induce forward mutation in L5178Y cells. No dose related trends in either the absolute number of mutants or the mutant frequencies were observed and at no level did these parameters increase to 2.5 times the spontaneous level. Therefore, the compound was considered to be non-mutagenic under the conditions of this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The capacity of the registered substance to induce structural chromosomal aberration in vivo was investigated using methods similar to the OECD Testing Guidelines 474 and 478. The registered substance did not induce structural chromosomal aberration under the tests conditions.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

Deviations:

yes

Remarks:

Information on bodyweight and toxicity signs not recorded

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Random bred male and female mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Protage, Michigan
- Age at study initiation: at least 8 weeks
- Assigned to test groups randomly: yes
- Housing: Males housed individually and females housed in pairs (except when mating) in shoe box cages on AB-SORB-DRI bedding.
- Diet: Purina Lab Chow ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Photoperiod: 12 hour light/dark cycle

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO

Duration of treatment / exposure:

Male mice exposed to several dose levels of test compound over 5 days

Frequency of treatment:

Daily

Post exposure period:

Rested for 2 days following treatment, then mated weekly for 7 weeks.

Dose / conc.:

1 mg/kg bw/day (nominal)

Dose / conc.:

0.3 mg/kg bw/day (nominal)

Dose / conc.:

0.1 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 random bred mice assigned to 1 of 5 groups. Three groups received different dose levels of hexachlorocyclopentadiene; a fourth group received only the solvent; a fifth group received a known mutagen as the positive control. Following treatment, each male was mated with 2 virgin females which were replaced weekly for 7 weeks.

Control animals:

yes, concurrent vehicle

yes, sham-exposed

yes, historical

Positive control(s):

Triethylenemelamine (TEM)
- Justification for choice of positive control(s):
- Route of administration: single intraperitoneal injection

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Range finding studies were conducted to calculate dose information. LD50, LD5 and LD1 concentrations were computer generated based on the preliminary study. The high dose level was selected from these data; 1/3rd and 1/10th of the high dose were used as the intermediate and low dose levels respectively.

TREATMENT AND SAMPLING TIMES: Male mice were exposed to the test compound for 5 days then mated over the entire period of spermatogenesis to unexposed virgin females. At mid-pregnancy the females were killed and scored for number of living and dead implants, as well as fertility level.

METHOD OF ANALYSIS: The number of dead and living fetuses, resorption sites, and total implantation sites were recorded. Data were analysed for statistical significance. Results were compared to data from control animals and used to determine the degree of induced dominant lethality.

Evaluation criteria:

Dominant lethality is determined from:
a) a mutation index derived from dead to total implants; or
b) the number of dead implants per pregnant female

Statistics:

See attached background material.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

A preliminary test was performed in order to determine the toxicity of the substance and adapt the final doses selected

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 50 mg/kg to 5000 mg/kg (first trial). 7.6 mg/kg to 76 mg/kg (second trial).
- Clinical signs of toxicity in test animals: Mortality occurred at all the doses investigated. All animals died at 76 mg/kg and above.

See attached background material for full results table.

Conclusions:

Hexachlorocyclopentadiene was not active in this Mouse Dominant Lethal Assay.

Executive summary:

A mouse dominant lethal assay was performed to determine the potential of the assay to induce structural and numerical chromosome aberrations according to a method similar to the OECD Tetsing Guideline 478 (non-GLP) with deviations.

Hexachlorocyclopentadiene was tested for toxicity over a range of 50 mg/kg to 5,000 mg/kg. Based on this toxicity and acute LD50 level of 76 mg/kg was calculated. The high dose was selected as the LD50 and concentrations of 25.3 mg/kg and 7.6 mg/kg were employed as the two lower doses levels. These concentrations proved to be too toxic for a five-day exposure since all ten animals died before five days. A second LD50 was calculated and identified 1 mg/kg as the high dose and 1/3 and 1/10 of the LD50 levels for the study.

The test material was examined for its ability to induce dominant lethality in mice treated with 0.1, 0.3 and 1.0 mg/kg bw/d for five days.

There was no evidence for significant dominant lethal activity by Hexachlorocyclopentadiene in mice. All data were within the concurrent and historical control levels. The positive control compound produced the expected dominant lethal effects in the first three mating weeks. Hexachlorocyclopentadiene was not active in the Mouse Dominant Lethal Assay conducted as part of this evaluation.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

No data on animal toxicity.

GLP compliance:

no

Type of assay:

other: mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Frederick Cancer Research Facility
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation: Females: 101 to 108g / Males: 118 to 127g
- Housing: individually in stainless steel cages (Hazleton Systems)
- Diet (e.g. ad libitum): NIH-07 pelleted rodent diet ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C-21°C
- Humidity (%): 35%-65%
- Air changes (per hr): 20 changes/h
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

inhalation: vapour

Vehicle:

- Vehicle(s)/solvent(s) used: air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel whole-body inhalation chamber (Hazleton Systems)
- Source and rate of air: fresh air
- System of generating particulates/aerosols: Vaporizer. Gardner Type CN condensation nuclei detector used to ensure the generation of vapour and not of aerosol.

TEST ATMOSPHERE
- Brief description of analytical method used: On-line gas chromatograph with electron capture detector.
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours per day, 5 days per week

Dose / conc.:

0 ppm (nominal)

Dose / conc.:

0.01 ppm (nominal)

Remarks:

Equivalent to 0.08 mg/m3.

Dose / conc.:

0.05 ppm (nominal)

Remarks:

Equivalent to 0.39 mg/m3.

Dose / conc.:

0.2 mg/kg bw/day (nominal)

Remarks:

Equivalent to 1.56 mg/m3.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control(s):

no data

Tissues and cell types examined:

Micronuclei in polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs).

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: Peripheral blood samples were obtained from mice after 13-week inhalation toxicity study. Smears were immediately prepared and fixed in absolute methanol, and later stained with a chromatin-specific fluorescent dye mixture of Hoechst 33258/pyronin Y.

METHOD OF ANALYSIS: Slides scanned to determine the frequency of micronuclei in 2,000 polychromatic erythrocytes (PCEs) and 10,000 normochromatic erythrocytes (NCEs). Percentage of PCEs amoung the total erythrocyte population was also determined.

Evaluation criteria:

The criteria of Schmid (1976) were used to define micronuclei. It was also required that the micronuclei exhibit the characteristic fluorescent emissions of DNA; the minimum size limit was approximately one-twentieth the diameter of the NCE cell.

Statistics:

Before statistical analysis, log transformation of the NCE data, testing for normality by Shapiro-Wilk test, and heterogeneity of variance by Cochran's test were performed. Frequency of micronucleated cells amoung NCEs was analysed by analysis of variance using the SAS GLM procedure. The Student's t-test was used to compare the NCE data for each dose group with the concurrent solvent control. The Cochran-Armitage trend test was used to analyse the frequency of micronucleated cells amoug PCEs, and individual dose groups were compared to the concurrent solvent control by Kastenbaum-Bowman's bionomial test. The percentage of PCEs among total erythrocytes was analysed by an analysis of variance on ranks (classed by sex), and individual dose groups were compared with the concurrent solvent control using a t-test on ranks.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not examined

Remarks:

Doses were selected based on previous toxicological testing

Vehicle controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No increase in the frequency of micronucleated erythrocytes was observed in peripheral blood samples obtained from male and female B6C3Fl mice exposed to hexachlorocyclopentadiene by inhalation for 13 weeks
- Ratio of PCE/NCE (for Micronucleus assay): See attached background information.
- Statistical evaluation: Exposed groups do not differ from the control (by Student's t-test, Kastenbaum-Bowman's binomial test, or t-test on ranks).

See attached background material.

Conclusions:

No increase in the frequency of micronucleated erythrocytes was observed in peripheral blood samples obtained from male and female B6C3F1 mice exposed to hexachlorocyclopentadiene by inhalation for 13 weeks. It was concluded that the substance did not induce structural chromosomal aberrations under the conditions of the study.

Executive summary:

The potential of Hexachlorocyclopentadiene to induce structural chromosomal aberration in mice was determined by the US NTP, using a Mouse Peripheral Blood Micronucleus Test protocol. The study is not GLP compliant however the procedure is comparable to the OECD Testing Guideline 474. Male and female B6C3F1 mice were exposed to hexachlorocyclopentadiene by inhalation for 13 weeks, at doses of 0.00, 0.01, 0.05, and 0.20 ppm. After treatment peripheral blood samples were taken. Smears were prepared and fixed in absolute methanol; later they were stained with a chromatin-specific fluorescent dye. Slides were scanned to determine micronuclei frequency in 2,000 polychromatic erythrocytes (PCEs) and 10,000 normochromatic erythrocytes (NCEs) in 10 animals per dose group. Percentage of PCEs among the total erythrocyte population was determined. The results were analysed using a variety of statistical tests, and there was found to be no increase in the frequency of micronucleated erythrocytes observed in the peripheral blood samples. It was concluded that the substance did not induce structural chromosomal aberrations under the conditions of the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The genotoxicity studies examined were non GLP compliant however they followed procedures similar to the OECD Guidelines for Testing of Chemicals, with acceptable restrictions. Results of these studies performed on Hexachlorocyclopentadiene were negative. Therefore the substance did not meet the criteria for classification as mutagenic according to Regulation (EC) N° 1272/2008.