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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
Study period:
February 13, 2012 - April 20, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
(1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: "Kanpoan No. 287 -- Environment Protection Agency", "Eisei No. 127 -- Ministry of Health & Welfare", "Heisei 09/10/31 Kikyoku No. 2 -- Ministry of Economy, Trade & Industry"
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Identity: CITRATHAL
- Analytical purity: not applicable (as it is an UVCB substance)
- Batch No.: SC00004113
- Expiration date of the lot/batch: November 16, 2012

Method

Species / strain
Species / strain / cell type:
other: Cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Blood samples
Blood samples were obtained from healthy, non-smoking donors not receiving medication. Blood was collected from a male donor (28 years old) for the first experiment, from a 27 year-old female donor for Experiment IIA and from a 30 year-old male donor for Experiment IIB. All donors had a previously established low incidence of chromosomal aberrations in their peripheral blood lymphocytes.
Blood samples were drawn by venous puncture and collected in heparinized tubes by Dr. V. Theodor (64380 Rossdorf, Germany). The tubes were sent to the laboratory to initiate cell cultures within 24 hours after collection. If necessary, the blood was stored before use at 4°C.
- Culture medium
The culture medium was DMEM:F12 (Dulbecco's modified eagle medium / Ham's F12 medium; mixture 1:1; Life Technologies GmbH (Invitrogen division), 64293 Darmstadt, Germany) already supplemented with 200 mM Glutamax. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL / 100 µg/mL) (SEROMED, 12247 Berlin, Germany), the mitogen phytohemagglutinin (PHA, final concentration 3 µg/mL, SEROMED), 10% FBS (fetal bovine serum) provided by PAA Laboratories GmbH (35091 Cölbe, Germany), 10 mM HEPES, and the anticoagulant heparin (25,000 U.S.P.-U/mL, Nattermann, 50829 Köln, Germany).
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.
Test concentrations with justification for top dose:
Highest concentration was determined to be 2264 µg/ mL (10 mM)

Evaluated:

Cytogenetic test I:
Without S9-mix, 4 h exposure: 45.0, 78.8 and 137.9 µg/mL
With S9-mix, 4 h exposure: 45.0, 137.9, 241.4 and 739.3 µg/ mL
Cytogenetic test IIA:
Without S9-mix, 22 hr exposure: 17.4, 30.5 and 53.3 µg/mL
With S9-mix, 4 hr exposure: 45.0, 78.8 and 137.9 µg/mL
Cytogenetic test IIB:
Without S9-mix, 22 hr exposure: 40.0, 50.0, 60.0 and 70.0 µg/mL

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: Ethylmethane sulfonate in nutrient medium (770 µg/mL (exp.I), 660 µg/mL (exp.IIA) and 550 µg/mL (exp.IIB))
Remarks:
without S9
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: Cyclophosphamide in saline (15 µg/mL (exp.I) and 7.5 µg/mL (exp.II))
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 72 hr
- Exposure duration: 4 hr (with and without S9-mix), 22 (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hr

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no
Evaluation criteria:
A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the historical control data.
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of the historical control data and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

The assay can indicate an aneugenic potential of the test item if the number of induced numerical aberrations is not in the range of the historical control data.
Statistics:
Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). However, both biological and statistical significance should be considered together.

Results and discussion

Test results
Species / strain:
lymphocytes: Cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of osmolality: No
Solvent control: 352, 382 and 384 mOsm/kg
2264 µg/ml: 399 mOsm/kg
500 µg/ml: 374 mOsm/kg
300 µg/ml: 382 mOsm/kg
- Effects on pH: No
Solvent control: 7.7
2264 µg/ml: 7.4
500 µg/ml: 7.7
300 µg/ml: 7.8

- Precipitation: No precipitation was observed up to and including the top dose of 2264 µg/mL (= 0.01 M). Phase separation was observed in all experiments.

INFORMATION ON CYTOTOXICITY:
- In experiment I in the absence and presence of S9-mix and in experiment IIB in the absence of S9-mix, cytoxicity was observed at the highest evaluated concentration (44.4 and 42.4% of control, respectively). In experiment IIA in the absence and presence of S9-mix, concentrations showing clear cytoxicity were not evaluable for cytogenetic damage.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.

OTHER:
Either with or without metabolic activation, no clastogenicity was observed at the concentrations evaluated. However, in Experiment I in the absence of S9 mix, one statistically significant increase was observed after treatment with 78.8 µg/mL (2.0 % aberrant cells, excluding gaps). The value is in the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps) and no dose-related response was seen; therefore considered as being biologically irrelevant. In Experiment IIA in the presence of S9 mix one single value (3.5 % aberrant cells, excluding gaps) at concentration 78.8 µg/mL slightly exceeded the laboratory’s historical control range (0.0 – 3.0 % aberrant cells, excluding gaps). Since the value was not statistically significant, compared to the respective solvent control, and no dose-related response was observed, the finding was regarded as biologically irrelevant.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Applicant's summary and conclusion

Conclusions:
A chromosome aberration study with Citrathal was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that Citrathal is not clastogenic in human lymphocytes.
Executive summary:

A chromosome aberration study with Citrathal was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments up to cytotoxic concentrations with and without S9 -mix.

Either with or without metabolic activation, no clastogenicity was observed at the concentrations evaluated. However, in Experiment I in the absence of S9 mix, one statistically significant increase was observed after treatment with 78.8 µg/mL (2.0 % aberrant cells, excluding gaps). The value is in the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps) and no dose-related response was seen; therefore considered as being biologically irrelevant. In Experiment IIA in the presence of S9 mix one single value (3.5 % aberrant cells, excluding gaps) at concentration 78.8 µg/mL slightly exceeded the laboratory’s historical control range (0.0 – 3.0 % aberrant cells, excluding gaps). Since the value was not statistically significant compared to the respective solvent control and no dose-related response was observed, the finding was regarded as biologically irrelevant.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

It is concluded that Citrathal is not clastogenic in human lymphocytes when tested up to cytotoxic concentrations with and without S9 -mix.