Dichlone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Sep 2014 - 08 Apr 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dichlone

EC Number:

204-210-5

EC Name:

Dichlone

Cas Number:

117-80-6

Molecular formula:

C10H4Cl2O2

IUPAC Name:

dichlone

Details on test material:

- Name of test material (as cited in study report): Dichlone
- Physical state: Solid/ yellow
- Storage condition of test material: Room temperature

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F12 medium containing stable glutamine and hypoxanthine (Biochrom; Cat. No. FG 0815) supplemented with 10% (v/v) fetal calf serum (FCS).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

1st Experiment (discontinued due to technical reasons)
without S9 mix (4-hour exposure period): (0); (0.16); (0.31); (0.63); (1.25); (2.50); (5.00); (10.00); (20.00) μg/mL
with S9 mix (4-hour exposure period): (0); (0.16); (0.31); (0.63); (1.25); (2.50); (5.00); (10.00); (20.00) μg/mL
2nd Experiment
without S9 mix (4-hour exposure period): 0; 0.16; 0.31; 0.63; 1.25; (2.50); (5.00); (10.00); (20.00) μg/mL
with S9 mix (4-hour exposure period): 0; (0.16); (0.31); 0.63; 1.25; 2.50; 5.00; (10.00); (20.00) μg/mL
3rd Experiment
without S9 mix (4-hour exposure period): 0; (0.06); (0.13); 0.25; 0.50; 1.00; 2.00; (4.00); (8.00) μg/mL
with S9 mix (4-hour exposure period): 0; (0.13); (0.25); (0.50); 1.00; 2.00; 4.00; 8.00; (16.00) μg/mL
numbers in parantheses were not evaluated.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water and other commonly used solvents (acetone, ethanol, tetrahydrofurane), dimethyl sulfoxide (DMSO) was the most suitable one.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

with S9: 1.25 µg/ml DMBA; without S9: 400 µg/ml EMS

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 5-7 days
- Selection time (if incubation with a selection agent): 7-9 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 6-thioguanine (10 μg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS:
Duplicate cultures were used for all experimental groups.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.

The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective vehicle control groups. A trend is judged as statistically significant whenever the one-sided p-value (probability value) is below 0.05 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not influenced by test substance treatment
- Effects of osmolality: not influenced by test substance treatment
- Precipitation: no precipitation in culture medium was observed up to the highest applied test substance concentration.


RANGE-FINDING/SCREENING STUDIES:
In the pretest for toxicity based on the purity and the molecular weight of the test substance 2300 μg/mL (approx. 10 mM) test substance was used as top concentration both with and without S9 mix at 4-hour exposure time. After 4 hours treatment in the absence and presence of S9 mix cytotoxicity was observed as indicated by a reduced relative cloning efficiency of about or below 20% in all applied concentrations (9 µg/ml was the lowest dose level).

CELL MORPHOLOGY
In the absence and presence of S9 mix, after 4 hours treatment in nearly all experiments the morphology and attachment of the cells was adversely influenced (grade > 2) in at least the highest applied concentration. In detail, in the absence of S9 mix after 4 hours treatment the cell morphology and attachment of the cells was adversely influenced from 10 μg/mL onward in the 1st Experiment, from 5 μg/mL onward in the 2nd Experiment and at 8 μg/mL in the 3rd Experiment. Besides, in the 2nd and 3rd Experiment in the presence of S9 mix after 4 hours treatment the cell morphology and attachment of the cells was adversely influenced at 20 μg/mL and at 16 μg/mL, resepctively.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequencies of the vehicle control groups were within the historical negative control data range including all vehicles used in our laboratory and, thus, fulfilled the acceptance criteria of this study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective negative control values were observed in all evaluated experiments in the absence and presence of S9 mix, at least at the highest applied concentrations. Without S9 mix, there was a decrease in the number of colonies from 2.50 μg/mL onward after an exposure period of 4 hours in the 1st and 2nd Experiment (CE1 relative: 8.1% or 0.0%, respectively). Cytotoxic effects were observed in the 3rd Experiment from 4.00 μg/mL (CE1 relative: 0.0%) onward. The cell densities were distinctly reduced in the expression period. In contrast, with S9 mix, there was a decrease in the number of colonies from 10.00 μg/mL onward in the 2nd Experiment and at 16.00 μg/mL in the 3rd Experiment (CE1 relative: 0.0%, each). The cell densities were distinctly reduced in the expression period. Due to microbial contamination the cultures seeded for determination of survival (CE1) were not scorable in the 1st Experiment with S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of results – experimental parts without metabolic activation

Exp.	Exposure period [h]	Test group [µg/ml]	S9 mix	Prec.*	Genotoxicity** Mfcorr [per 106 cells]	Cytotoxicity*** CE1 [%]	Cytotoxicity*** CE2 [%]
1	4	vehicle control^1	-	n.d.	n.c.(3)	100	n.c.(3)
		0.16	-	-	n.c.(3)	95.1	n.c.(3)
		0.31	-	-	n.c.(3)	102.7	n.c.(3)
		0.63	-	-	n.c.(3)	91.9	n.c.(3)
		1.25	-	-	n.c.(3)	55	n.c.(3)
		2.5	-	-	n.c.(3)	8.1	n.c.(3)
		5	-	-	n.c.(3)	0	n.c.(3)
		10	-	-	n.c.(3)	0	n.c.(3)
		20	-	-	n.c.(3)	0	n.c.(3)
		positive control^2	-	n.d.	n.c.(3)	100.1	n.c.(3)
2	4	vehicle control^1	-	n.d.	1.56	100	100
		0.16	-	-	0.62	107.6	105.3
		0.31	-	-	0.65	104.2	96.9
		0.63	-	-	0	104.2	105.3
		1.25	-	-	2.17	81.2	100.3
		2.5	-	-	n.c. (2)	0	n.c. (2)
		5	-	-	n.c. (2)	0	n.c. (2)
		10	-	-	n.c. (2)	0	n.c. (2)
		20	-	-	n.c. (2)	0	n.c. (2)
		positive control^2	-	n.d.	82.17	93.6	84.2
3	4	vehicle control^1	-	n.d.	1.78	100	100
		0.06	-	-	n.c. (1)	106.5	n.c. (1)
		0.13	-	-	n.c. (1)	99.2	n.c. (1)
		0.25	-	-	0	97.8	107.3
		0.5	-	-	4.14	104.2	115.8
		1	-	-	0.27	97.8	119.1
		2	-	-	1.63	53.2	116.3
		4	-	-	n.c. (2)	0	n.c. (2)
		8	-	-	n.c. (2)	0	n.c. (2)
		positive control^2	-	n.d.	114.05	94.5	103.7

Summary of results – experimental parts with metabolic activation

Exp.	Exposure period [h]	Test group [µg/ml]	S9 mix	Prec.*	Genotoxicity** Mfcorr [per 106 cells]	Cytotoxicity*** CE1 [%]	Cytotoxicity*** CE2 [%]
1	4	vehicle control^1	+	n.d.	n.c.(3)	n.c.(3)	n.c.(3)
		0.16	+	-	n.c.(3)	n.c.(3)	n.c.(3)
		0.31	+	-	n.c.(3)	n.c.(3)	n.c.(3)
		0.63	+	-	n.c.(3)	n.c.(3)	n.c.(3)
		1.25	+	-	n.c.(3)	n.c.(3)	n.c.(3)
		2.5	+	-	n.c.(3)	n.c.(3)	n.c.(3)
		5	+	-	n.c.(3)	n.c.(3)	n.c.(3)
		10	+	-	n.c.(3)	n.c.(3)	n.c.(3)
		20	+	-	n.c.(3)	n.c.(3)	n.c.(3)
		positive control^3	+	n.d.	n.c.(3)	n.c.(3)	n.c.(3)
2	4	vehicle control^1	+	n.d.	1.4	100	100
		0.16	+	-	n.c. (1)	95.2	n.c. (1)
		0.31	+	-	n.c. (1)	110.2	n.c. (1)
		0.63	+	-	3.52	110.4	99.1
		1.25	+	-	1.23	109.2	84.8
		2.5	+	-	1.78	108.4	92.1
		5	+	-	2.17	98.2	96.7
		10	+	-	n.c. (2)	0	n.c. (2)
		20	+	-	n.c. (2)	0	n.c. (2)
		positive control^3	+	n.d.	130.29	105	77.3
3	4	vehicle control^1	+	n.d.	4.84	100	100
		0.13	+	-	n.c. (1)	105.1	n.c. (1)
		0.25	+	-	n.c. (1)	100.5	n.c. (1)
		0.5	+	-	n.c. (1)	110.5	n.c. (1)
		1	+	-	0.37	105.4	95.2
		2	+	-	1.46	110.6	109.9
		4	+	-	0	105.2	113.5
		8	+	-	0	63.5	119.9
		16	+	-	n.c. (2)	0	n.c. (2)
		positive control^3	+	n.d.	140.91	106.2	114.9

* Precipitation in culture medium at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per 106 cells corrected with the CE2 value

*** Cloning efficiency related to the respective vehicle control

n.c. (1) Culture was not continued since a minimum of only four analysable concentrations are required

n.c. (2) Culture was not continued due to strong cytotoxicity

n.c. (3) Culture was not continued due to technical reasons (microbial contamination)

n.d. Not determined

¹ DMSO 1% (v/v)

² EMS 400 μg/mL

³ DMBA 1.25 μg/mL

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out, with and without the

addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test the concentrations ranged from 0.16 to 8.0 µg/ml. Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence of metabolic activation and for 4 hours in the presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguaninecontaining medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations. In this study, in the 2nd and 3rd Experiment, at least the highest concentrations tested for gene mutations were clearly cytotoxic in the absence and presence of metabolic activation. The 1st Experiment was discontinued due to technical reasons. Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.