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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Qualifier:
according to
Guideline:
EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Peripheral blood lymphocytes were obtained from a healthy 40 year old adult female with no recent history of either radiotherapy, viral infection or the administration of drugs.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S-9
Test concentrations with justification for top dose:
First assay: 0.25, 0.75, 2.5, 7.5, 25, 75, 250, 750 and 2500 µg/ml; concentrations evaluated for CA: 25, 75, 250 and 750 µg/ml (without S9 mix) and 7.5, 25, 75 and 250 µg/ml (with S9 mix). Second assay: 10, 19, 38, 75, 150, 300 and 600 µg/ml; concentrations evaluated for CA: 75, 150, 300 and 600 µg/ml (without S9 mix - 20 hrs harvest); 38, 75, 150 and 300 µg/ml (without S9 mix - 44 hrs harvest); 38, 75, 150 and 300 µg/ml (with S9 mix - 20 hrs harvest); 75, 150, 300 and 600 µg/ml (with S9 mix - 44 hrs harvest)
Vehicle / solvent:
A solubility test was conducted using dimethylsulfoxide (DMSO) which was selected by the Sponsor as the solvent of choice. The test was performed to determine the level of solubility of the test article in DMSO, which permitted preparation of the highest soluble or workable stock concentration, up to 500 mg/ml.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: Mitomycin C in absence of S9 and Cyclophosphamide in the presence of S9.
Details on test system and experimental conditions:
METHOD OF APPLICATION:
0.6 ml heparinized blood was inoculated into centrifuge tubes containing 9.4 ml complete medium supplemented with 1% PHA. The tubes were incubated at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO, in air for approximately 44-48 hours. Treatment was carried out by refeeding with approximately 10 ml fresh complete medium or S9 reaction mixture to which was added 100 ml of dosing solution of test or control article in solvent or solvent alone.

DURATION
- Preincubation period:
44-48 hours

- Exposure duration:
In the absense of metabolic activation:20 harvest (first harvest) or 44 hours (delayed harvest)
In the presence of metabolic activation: 4 hours

- Expression time (cells in growth medium):
16 hours (first harvest) or 40 hours (delayed harvest)

- Selection time (if incubation with a selection agent):
2 hours

- Fixation time (start of exposure up to fixation or harvest of cells):
20 hours (first harvest) or 44 hours (delayed harvest)

SPINDLE INHIBITOR (cytogenetic assays):
Colcemid®

STAIN (for cytogenetic assays):
5% Giemsa

NUMBER OF REPLICATIONS:
2

NUMBER OF CELLS EVALUATED:
100 per replicate

DETERMINATION OF CYTOTOXICITY
- Method: The toxic effects of treatment are based upon mitotic inhibition relative to the solvent-treated control and are presented for both the initial and the independent repeat chromosome aberration assays.

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
Evaluation criteria:
The number and types of aberrations, the percentage of structurally damaged cells (percent aberrant cells) in the total population of cells examined, the percentage of numerically damaged cells in the total population of cells examined, and the frequency of structural aberrations per cell (mean aberrations per cell) was reported for each treatment group. Chromatid and isochromatid gaps were presented in the data but not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell.
Statistics:
Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test at any test article dose level, the Cochran-Armitage trend test was used to measure dose-responsiveness.

Results and discussion

Test results
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
no clastogenic effects
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
none reported

RANGE-FINDING/SCREENING STUDIES:
the initial chromosome aberration assay, was conducted to establish the dose range for testing and to evaluate the clastogenic potential of the test article.

COMPARISON WITH HISTORICAL CONTROL DATA:
Not reported.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Initial Assay

In the initial chromosome aberration assay HPBL were exposed to nine concentrations of test article ranging from 0.25 µg/ml to 2500 µg/ml. Test article concentrations greater than 2500 µg/ml were insoluble in treatment medium and not tested in the assay. The test article was soluble but cloudy in treatment medium at dose levels 75 and 250 µg/ml and workable in treatment medium at dose levels 750 and 2500 µg/ml. The test article was soluble in treatment medium at all other dose levels tested. The osmolality and pH of the highest concentration tested, 2500 µg/ml, were 419 mmol/kg and approximately 8, respectively. The osmolality of the solvent, DMSO, was 447 mmol/kg. Metaphase cells were collected for microscopic evaluation at 20 hours after the initiation of treatment.

The activity of HBCDD in the induction of chromosome aberrations in HPBL was tested following a 20 hour exposure period in the absence of an exogenous source of metabolic activation. The findings of the cytogenetic analysis are presented by treatment culture in Table 1 and summarized by group in Table 3. At the highest test concentration evaluated (750 µg/ml) microscopically for chromosome aberrations, the mitotic index was reduced 56% relative to the solvent control. Dose level 2500 µg/ml was not selected for analysis due to an insufficient number of scorable cells. Dose levels 0.25, 0.75, 2.5 and 7.5 µg/ml were tested but not required for analysis. The percentage of cells with structural aberrations in the test article-treated groups was not significantly increased above that of the solvent control at any dose level (p >0.05, Fisher's exact test). The percentage of aberrant cells in the MMC group was 8.5% (p ≤0.01, Fisher's exact test).

The activity of HBCDD in the induction of chromosome aberrations in HPBL cells was tested using a 4 hour exposure and a 16 hour recovery period in the presence of an exogenous source of metabolic activation. The findings of the cytogenetic analysis are presented by treatment flask in Table 2 and summarized by group in Table 3. At the highest test concentration evaluated (250 µg/ml) for chromosome aberrations, the mitotic index was reduced 13% relative to the solvent control. Dose levels 750 and 2500 µg/ml were not selected for analysis due to an insufficient number of scorable cells. Dose levels 0.25, 0.75, and 2.5 µg/ml were tested but not required for analysis. The percentage of cells with structural aberrations in the test article-treated groups was not significantly increased above that of the solvent control at any dose level (p >0.05, Fisher's exact test). The percentage of aberrant cells in the CP group was 6.5% (p ≤0.01, Fisher's exact test).

Independent Repeat Assay

Based on the results of the initial assay, dose levels of 10, 19, 38, 75, 150, 300 and 600 µg/ml were selected for further study in both the non-activated and S9-activated portions of the independent repeat assay with 20 and 44 hour cell harvest times. The test article was soluble but cloudy in treatment medium at dose level 75 pg/ml, and was workable in treatment medium at dose levels 150 µg/ml and higher. The test article was soluble in treatment medium at all other concentrations tested. The osmolality of the highest concentration (600 µg/ml) tested was 381 mmol/kg. The osmolality of the solvent, DMSO, was 393 mmol/kg. The pH of the highest concentrations tested was approximately 8. At the time of test article administration, due to technical oversight, the cultures were treated approximately 43 minutes to 1 hour 13 minutes beyond the 44-48 hour time limit; since the result of the study was negative, the Study Director determined this deviation to have had no significant effect upon the test system.

The findings of the cytogenetic analysis for groups tested in the absence of an exogenous source of metabolic activation are presented by treatment culture in Tables 4 and 5 and summarized by group in Table 8. At the highest test concentrations evaluated for chromosome aberrations, 600 and 300 µg/ml, the mitotic indices were reduced 55% and 94% relative to the solvent control at the 20 hour and 44 hour harvests, respectively. Dose level 600 µg/ml in the 44 hour harvest was not analyzed due to complete mitotic inhibition. Dose levels 10, 19, and 38 µg/ml in the 20 hour harvest were tested but not required for analysis. In the 44 hour harvest, dose levels 10 and 19 µg/ml were not required for analysis. The percentage of cells with structural aberrations in the test article­treated groups was not significantly increased above that of the solvent control at any dose level or harvest time (p >0.05, Fisher's exact test). The percentage of cells with numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control at the 44 hour harvest time, regardless of dose level (p >0.05, Fisher's exact test). The percentages of aberrant cells in the MMC groups were 7.5% and 35% (p ≤0.01, Fisher's exact test) for the 20 and 44 hour harvest times, respectively.

The findings of the cytogenetic analysis for groups tested in the presence of an exogenous source of metabolic activation are presented by treatment flask in Tables 6 and 7 and summarized by group in Table 8. At the highest test concentrations evaluated for chromosome aberrations, 300 and 600 µg/ml, in the 20 and 44 hour harvests, respectively, the mitotic indices were reduced 71% and 69% relative to the solvent control. Dose level 600 µg/ml in the 20 hour harvest was not analyzed due to complete mitotic inhibition. Dose levels 10 and 19 µg/ml in the 20 hour harvest and 10, 19, and 38 µg/ml in the 44 hour harvest were tested but not required for analysis. The percentage of cells with structural aberrations in the test article-treated groups was not statistically increased above that of the solvent control at any dose level or harvest time (p >0.05, Fisher's exact test). The percentage of cells with numerical aberrations in the test article-treated groups was not statistically increased above that of the solvent control at the 44 hour harvest time, regardless of dose level (p >0.05, Fisher's exact test). The percentages of aberrant cells in the CP groups were 6% and 8.5% (p ≤0.01, Fisher's exact test) for the 20 and 44 hour harvests, respectively.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The positive and negative controls fulfilled the requirements for a valid test.

Under the conditions of the assay described in this report, Hexabromocyclododecane was concluded to be negative in the non-activated and S9-activated test systems in the in vitro mammalian cytogenetics test using human peripheral blood lymphocytes.
Executive summary:

HBCDD was tested in the in vitro mammalian cytogenetic test using human peripheral blood lymphocytes (HPBL) both in the absence and presence of metabolic activation. The assay was performed in two phases. The first phase, the initial chromosome aberration assay, was conducted to establish the dose range for testing and to evaluate the clastogenic potential of the test article. The second phase, the independent repeat chromosome aberration assay, was performed to confirm the test system response to the test article seen in the initial assay.

Dimethylsulfoxide (DMSO) was the solvent of choice based on information provided by the Sponsor, the solubility of the test article and compatibility with the target cells. The test article was soluble in DMSO at approximately 500 mg/ml, the maximum concentration tested.

In the initial chromosome aberration assay, the maximum dose tested was 2500 µg/ml. This dose was achieved using a stock concentration of 250 mg/ml, and a 100 µl dosing aliquot added to 10 ml fresh complete medium or S9 reaction mix. Dose levels greater than 2500 µg/ml were insoluble in treatment medium and not tested in the assay. Visible precipitate was observed in treatment medium at dose levels 750 and 2500 µg/m1 and was soluble but cloudy (no visible precipitate) at dose levels 75 and 250 µg/ml. The test article was soluble in treatment medium at all other dose levels tested. In the non­activated portion of the initial assay HPBL cells were exposed to the test article continuously for 20 hours; in the S9-activated portion of the initial chromosome aberration assay, HPBL cells were exposed to the test article for 4 hours. Metaphase cells were collected for microscopic evaluation at 20 hours after the initiation of treatment. Dose levels of 2500 µg/ml in the non-activated study and 750 and 2500 µg/ml in the S9­activated study were not analysed for chromosome aberrations due to complete mitotic inhibition. Toxicity (mitotic inhibition) of approximately 56% was observed at the highest dose level (750 µg/ml) evaluated for chromosome aberrations, in the non-activated study. In the S9-activated study, 13% toxicity was observed at the highest dose level (250 µg/ml) evaluated for chromosome aberrations. No statistically significant increases in chromosome aberrations were observed in either the non-activated or S9-activated test systems relative to the solvent control group regardless of dose level (p >0.05, Fisher's exact test).

Based on the results of the initial assay, an independent repeat chromosome aberration assay was conducted in the absence and presence of an Aroclor-induced S9 metabolic activation system at dose levels of 10, 19, 38, 75, 150, 300, and 600 µg/ml. The test article was soluble but cloudy at dose level 75 µg/ml and was workable in treatment medium at dose levels 150 µg/ml and higher. The test article was soluble in treatment medium at all other concentrations tested. In the independent repeat assay, HPBL cells were exposed to the test article continuously for 20 or 44 hours in the non-activated test system and for 4 hours in the S9-activated test system. Metaphase cells were collected for microscopic evaluation in both the non-activated and S9-activated studies at 20 and 44 hours after the initiation of treatment. Toxicity, measured by mitotic inhibition, was

approximately 55% and 94% at the 20 and 44 hour harvests, respectively, at the highest dose levels (600 and 300 µg/ml) evaluated for chromosome aberrations in the non­activated studies. In the S9-activated studies, toxicity was approximately 71% and 69% at the 20 and 44 hour harvests, respectively, at the highest dose levels (300 and 600 µg/ml) evaluated for chromosome aberrations. Dose level 600 µg/ml in the non-activated 44 hour harvest and in the S9-activated 20 hour harvest was not analysed for chromosome aberrations due to an insufficient number of scorable metaphase cells. No statistically significant increases in structural chromosome aberrations were observed in either the non­activated or S9-activated studies, regardless of dose level or harvest time (p >0.05, Fisher's exact test). No statistically significant increases in numerical chromosome aberrations were observed in either the non-activated or S9-activated studies at the 44 hour harvest time, regardless of dose level (p >0.05, Fisher's exact test).

Based on the findings of this study, HBCDD was concluded to be negative for the induction of structural and numerical chromosome aberrations in human peripheral blood lymphocytes.