1-(2,4,6-trichlorophenyl)acetone

Genetic toxicity: in vitro

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:

2013-10-15 to 2014-06-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD 473 (1997), EPA OPPTS 870.5375 (1998) and EC 440/2008 B. 10 (2008) without deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

1-(2,4,6-trichlorophenyl)acetone

EC Number:

810-702-6

Cas Number:

1228284-86-3

Molecular formula:

C9H7Cl3O

IUPAC Name:

1-(2,4,6-trichlorophenyl)acetone

Test material form:

other: not reported

Details on test material:

- Storage condition of test material: At room temperature, protected from light and humidity

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

- Blood samples were obtained from healthy donors not receiving medication. For this study, blood was collected from a female donor (37 years old) for Experiment I and from a male donor (20 years old) for Experiment II. Blood samples were drawn by venous puncture and collected in heparinized tubes by Dr. V. Theodor (64380 Rossdorf, Germany). The tubes were sent to Harlan CCR to initiate cell cultures within 24 h after blood collection. If necessary, the blood was stored before use at 4 °C.
- Type and identity of media:
Blood cultures were established by preparing a 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA (phytohemagglutinin) (3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
The following volumes were added to the flasks (per 10 mL):
7.60 mL culture medium
1.00 mL fetal bovine serum
0.10 mL antibiotic solution
0.05 mL phytohemagglutinin
0.05 mL heparin
0.10 mL HEPES
1.10 mL whole blood
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix: Phenobarbital/beta-naphthoflavone induced rat liver S9. Each batch of S9 is routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

The highest concentration used in the pre-test was chosen with regard to the current OECD guideline 473 for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced mitotic indices below 50 % of control, and/or the occurrence of precipitation. In the case of non-toxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulation in an appropriate solvent is possible.

With regard to the molecular weight of the test substance, 2375.0 µg/mL of the test item (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test substance concentrations between 15.4 and 2375.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, precipitation of the test substance was observed at the end of treatment at 144.7 µg/mL and above. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

Using reduced mitotic indices as an indicator for toxicity in Experiment I, no relevant cytotoxic effects were observed after 4 hours treatment in the absence and presence of S9 mix. Therefore, 2375.0 µg/mL was chosen as top treatment concentration for Experiment II.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (purity 99.9 %, supplier: Fisher Chemicals)
- Justification for choice of solvent/vehicle: On the day of the experiment (immediately before treatment), the test substance was dissolved in DMSO. The final concentration of DMSO in the culture medium was 1.0 % (v/v). The solvent was chosen due to its solubilisation properties and its relative non-toxicity to the cell cultures.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
On the day of the experiment (immediately before treatment), the test substance was dissolved in DMSO. The final concentration of DMSO in the culture medium was 1.0 % (v/v). The culture medium was replaced with serum-free medium containing the test substance. All cultures were incubated at 37 °C in a humidified atmosphere with 5.5 % CO2.

DURATION
- Preincubation period: about 48 h after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks (Nunc GmbH & Co. KG, 65203 Wiesbaden, Germany).
- Exposure duration:
-- Exposure time 4 hours: The culture medium was replaced with serum-free medium containing the test substance. For the treatment with metabolic activation 50 µL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 h the cells were spun down by gentle centrifugation for 5 minutes (approx. 900 x g). The supernatant with the dissolved test substance was discarded and the cells were re-suspended in "saline G". The washing procedure was repeated once as described. The "saline G" solution was composed as follows (per litre):
NaCl: 8000 mg
KCl: 400 mg
glucose x H2O: 1100 mg
Na2HPO4x 2H20: 192 mg
KH2PO4: 150 mg
pH was adjusted to 7.2
After washing the cells were re-suspended in complete culture medium and cultured until preparation.
-- Exposure time 22 hours (without S9 mix): The culture medium was replaced with complete medium (with 10 % FBS) containing the test substance without S9 mix. The culture medium at continuous treatment was not changed until preparation of the cells. Concurrent solvent and positive controls were performed.
- Fixation time (start of exposure up to fixation or harvest of cells): The cultures were treated with the metaphase-arresting substance Colcemid (final concentration: 0.2 µg/mL) approximately three hours before the requested harvest time. The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were resuspended in hypotonic solution (0.0375 M KCl). Then the cell suspension was allowed to stand at 37 °C for 20 minutes. After removal of the hypotonic solution by centrifugation (approx. 900 x g) the cells were fixed with a mixture of methanol and glacial acetic acid (3+1 parts, respectively). A small amount of cell suspension was then dropped onto clean, wet microscope slides and allowed to dry. The slides were stained with Giemsa, mounted after drying and covered with a cover slip. All slides were labelled with a computer-generated random code to prevent scorer bias.
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): Colcemid (final concentration: 0.2 µg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 well-spread metaphases per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment II without S9 mix, where only 50 metaphases were evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% cells in mitosis)

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates.

Evaluation criteria:

A test substance is classified as non-clastogenic if:
- the number of induced structural chromosomal aberrations in all evaluated concentration groups is in the range of the laboratory historical control data.
- no statistically significant increase of the rate of structural chromosomal aberrations is observed in comparison to the respective solvent control.
A test substance is classified as clastogenic if:
- the number of induced structural chromosomal aberrations is not in the range of the historical laboratory control data
and
- either a concentration-related or a statistically significant increase in the number of cells carrying structural chromosomal aberrations is observed.
A test substance not meeting the criteria for classification as non-mutagenic or mutagenic may be considered equivocal in this assay and may be subject to further investigation.

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. If the above mentioned criteria for the test substance are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
An increase in the number of polyploid cells or endoreduplicated cells may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical chromosomal aberrations or to inhibit cell cycle progression.

Results and discussion

Test results

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

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
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: DMSO was used as solvent.
- Precipitation: yes
In Experiment I and II n the absence amd presence of S9 mix precipitation of the test substance in the culture medium was observed at 144.7 µg/mL and above at the end of treatment.

RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described below for the mutagenicity assay. The pre-test phase was performed with 10 concentrations of the test substance and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 h (with and without S9 mix). The preparation interval was 22 h after start of the exposure. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA:
Either with or without metabolic activation, no clastogenicity was observed at the concentrations evaluated. the aberration rate of the cells after treatment with the test substance (0.5-3.3 % aberrant cells, exlcuding gaps) were close to the solvent control data (0.0 – 3.0 % aberrant cells, exlcuding gaps) and within the range of the laboratory historical solvent control data. However, in Experiment II in the presence of S9 mix, statistically significant increases were observed due to the low response of the solvent control (0.0 % aberrant cells, excluding gaps) after treatment with 82.7, 144.7, 253.2 and 443.1 µg/mL (3.3, 1.8, 2.0 and 2.5 % aberrant cells, excluding gaps). These values were in the range of the laboratory historical solvent control data (0.0-3.5 % aberrant cells, excluding gaps) and therefore considered as being biologically irrelevant.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment I in the absence and presence of S9 mix and in Experiment IIA in the presence of S9 mix, no clear cytotoxicity was observed up to the highest applied concentration. However, the mitotic indices were markedly reduced to about or below 60 % of control. In Experiment II in the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration (51.3 % of control). In Experiment II in the presence of S9 mix concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Tabe 1 Summary of results of the chromosomal aberration study with the test item without S9 mix

Exp.	Preparation interval	Test substance concentration	Mitotic indices	Aberrant cells
		(µg/mL)	(% of control)	(% incl. gaps*)	(% excl. gaps*)	(% carrying exchanges)
Exposure period 4 hrs without S9 mix
I	22 hrs	Solvent control^1	100	0.5	0.5	0
		Positive control^2	91.4	9	8.5^S	1.5
		82.7	86.8	1	1	0
		144.7^P	103.4	0.5	0.5	0
		443.1^P	74.4	1	1	0
		775.5^P	61.3	1	1	0
Exposure period 22 hrs without S9 mix
II	22 hrs	Solvent control^1	100	1	1	0
		Positive control^3#	32.9	45	45.0^S	8
		47.2	105.8	2	1.5	0.5
		82.7	79.2	2.5	1.5	0
		144.7^P	51.3	3	2.5	0

*: Including cells carrying exchanges

P: Precipitation occurred at the end of treatment

S: Aberration frequency statistically significant higher than corresponding control values

#: 50 metaphases per culture were evaluated

1: DMSO 1.0 % (v/v)

2: EMS 660.0 µg/mL

3 EMS 770.0 µg/mL

Table 2 Summary of results of the chromosomal aberration study with the test item with S9 mix

Exp.	Preparation interval	Test substance concentration	Mitotic indices	Aberrant cells
		(µg/mL)	(% of control)	(% incl. gaps*)	(% excl. gaps*)	(% carrying exchanges)
Exposure period 4 hrs with S9 mix
I	22 hrs	Solvent control^1##	100	3	3	0.3
		Positive control^2	62.1	10	9.5^S	2
		82.7	88.7	2.5	2.5	0
		144.7^P	98.4	0.5	0.5	0
		443.1^P	80.1	1	1	0
		775.5^P	59	3	2.5	0
		1357.1^P	55.9	2	1.5	0
II	22 hrs	Solvent control^1	100	0	0	0
		Positive control^3#	56.5	39	39.0^S	8
		82.7^##	91.1	3.3	3.3^S	0
		144.7^P##	107.9	2	1.8^S	0.3
		253.2^P##	93	2.3	2.0^S	0.3
		443.1^P##	56.1	2.8	2.5^S	0.3

*: Including cells carrying exchanges

P: Precipitation occurred at the end of treatment

S: Aberration frequency statistically significant higher than corresponding control values

#: 50 metaphases per culture were evaluated

##: 200 metaphases per culture were evaluated

1: DMSO 1.0 % (v/v)

2: CPA 2.5 µg/mL

3: CPA 15.0 µg/mL

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

In a valid, conclusive and reliable study according to OECD 473 (1997), EPA OPPTS 870.5375 (1998) and EC 440/2008 B. 10 (2008), the test substance did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, the substance is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating or the highest evaluable concentrations.

Executive summary:

This in vitro assay was performed to assess the potential of the test item to induce structural chromosomal aberrations in cultured human lymphocytes in the absence and presence of an exogenous metabolic activation system (liver S9 mix from phenobarbital/beta-naphthoflavone treated male rats). In each experimental group two parallel cultures were analysed. Per culture at least 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, where only 50 metaphases were evaluated. The highest applied concentration in this study (2375.0 µg/mL of the test substance, approx. 10 mM) was chosen with regard to the molecular weight of the test substance and with respect to the current OECD Guideline 473. Concentration selection for the cytogenetic experiments was performed considering the toxicity data and test substance precipitation and in accordance with OECD Guideline 473.

In Experiment I in the absence and presence of S9 mix, no clear cytotoxicity was observed up to the highest applied concentration. However, the mitotic indices were markedly reduced to about or below 60 % of control. In Experiment II in the absence of S9 mix clear cytotoxic effects were observed at the highest evaluated concentration. In Experiment II in the presence of S9 mix concentrations showing clear cytotoxicity were not evaluable for cytotoxic damage. Either with or without metabolic actication, no clastogenicity was observed at the concentrations evaluated. However, in Experiment II with the presence of S9 mix, statistically significant increases were observed after treatment with 82.7, 144.7, 253.2 and 443.1 µg/mL (3.3, 1.8, 2.0 and 2.5 % aberrant cells, excluding gaps) and lacked a concentration -response. Furthermore, when assessing statistical significance of the test substance treatment, the aberration frequencies were compared to a very low concurrent solvent control value (0.0 %). Therefore, the statistically significant values were considered as being biologically irrelevant.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test substance as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, the substance is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic and/or precipitating concentrations.