2,2'-[6-(4-methoxyphenyl)-1,3,5-triazine-2,4-diyl]bis{5-[(2-ethylhexyl)oxy]phenol}

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:

October 08, 1997 through January 06, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

CCR Cytotest Cell Research GmbH & Co KG, 64380 Roßdorf, Germany

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): CGF-C-1607
- Physical state: solid yellow
- Analytical purity: >98%
- Lot/batch No.: 6
- Expiration date of the lot/batch: July 30, 1999
- Storage condition of test material: room temperature

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimal Essential Medium supplemented with fetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Cytokinesis block (if used):

Colcemid (0.2 μg/ml culture medium)

Metabolic activation:

with and without

Metabolic activation system:

liver S9-mix from phenobarbital/B-naphthoflavone treated rats

Test concentrations with justification for top dose:

Concentrations analysed for CA
Experiment I without S9: 26.3, 52.5, 105.0, 210.0 µg/ml
Experiment II without S9: 26.3, 52.5, 105.0, 210.0 µg/ml
Experiment I with S9: 13.1, 26.3, 52.5, 210.0 µg/ml
Experiment II with S9: 13.1, 26.3, 52.5, 210.0 µg/ml
1.6 and 210 µg/ml (with and without S9 mix) for the assessment of the cytotoxic potential
Test concentrations were chosen based on pretest data.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: its solubility properties and its non-toxicity to the cells

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethanesulphonate (EMS); cyclophosphamide (CPA)

Remarks:

EMS = without S9; CPA = with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h in exp. I and 18 h and 28 h in exp. II
15.5 and 25.5 h after the start of the treatment colcemid was added to the cultures. 2.5 h later, the cells were fixed.


SPINDLE INHIBITOR (cytogenetic assays): colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: 100 per culture; 2 cultures per treatment group

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

The chromosome aberration assay performed in our laboratory is considered acceptable if it meets the following criteria:
a) The number of structural aberrations found in the negative and/or solvent controls falls within the range of our historical laboratory control data: 0.00 % - 4.00 %.
b) The positive control substances should produce significant increases of the number of cells with structural chromosome aberrations.

A test article is classified as mutagenic if it induces either a significant and concentration related increase of the number of structural chromosome aberrations or a significant and reproducible positive response for at least one of the test points. A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosome aberrations nor a significant and reproducibly positive response at anyone of the test points is considered non-mutagenic in this system.

Statistics:

Statistical significance was confirmed by means of the Fischer's exact test

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effect
- Effects of osmolality: no effect
- Precipitation: Precipitation of the test article in culture medium was observed after treatment with 105 µg/ml and above in the absence of S9 mix and 52.5 ug/ml and above in the presence of S9 mix.

Any other information on results incl. tables

Table 3. Summary of Results

Exp.	Exposure Period	S9 Mix	Concentration of CGF-C-1607 in ug/ml	Polyploid cells in %	Mitotic index in % of control	Incl. gaps	Aberrant cells in % excl. gaps*	exchanges
I	18 h	-	Solvent control	2.5	100.0	2.0	1.5	0.5
		-	26.3	2.0	84.9	0.0	0.0	0.0
		-	52.5	2.5	87.2	1.0	0.5	0.5
		-	105.0^P	2.0	100.3	1.0	1.0	0.5
		-	210.0^P	2.0	100.3	0.5	0.5	0.0
II	18 h	-	Solvent control	2.0	100.0	5.0	3.0	0.5
		-	26.3	2.5	78.4	1.0	0.5	0.0
		-	52.5	0.5	83.3	1.5	1.5	0.0
		-	105.0^P	4.0	70.8	1.0	0.5	0.0
		-	210.0^P	2.5	62.8	2.0	2.0	0.5
II	28 h	-	Solvent control	3.5	100.0	2.0	0.5	0.5
		-	52.5	3.5	100.7	2.5	2.5	0.0
		-	210.0^P	4.5	113.6	4.0	3.5^S	1.0
I	18 h	+	Solvent control	4.0	100.0	1.0	1.0	0.0
		+	13.1	3.5	90.9	2.0	1.5	0.0
		+	26.3	2.0	102.2	2.0	1.0	0.0
		+	52.5^P	2.5	111.6	3.5	3.0	0.5
		+	210.0^P	3.0	94.0	1.5	1.5	0.0
II	18 h	+	Solvent control	1.5	100.0	2.5	2.5	0.5
		+	13.1	3.0	115.6	3.0	2.5	0.5
		+	26.3	2.0	110.1	4.0	2.5	1.0
		+	52.5^P	3.5	98.6	1.0	1.0	0.0
		+	210.0^P	2.0	77.4	3.0	2.0	0.0
II	28 h	+	Solvent control	2.5	100.0	3.5	2.5	0.5
		+	26.3	2.0	121.8	2.0	2.0	0.0
		+	52.5^P	4.0	85.8	1.5	1.0	0.0
		+	210.0^P	2.0	93.1	0.0	0.0	0.0

* inclusive cells carrying exchanges

^SAberration frequency statistically significant higher than corresponding solvent control values

^PPrecipitation occurred

Table 4. Statistical significance in experiment I was evaluated by means of the Fisher’s exact test. Evaluation was performed only for cells carrying aberrations exclusive of gaps.

Solvent Control versus		Exposure period	S9 mix	p-value
Test group	26.3 ug/ml	18 h	-	n.c.
Test group	52.5 ug/ml	18 h	-	n.c.
Test group	105.0 ug/ml	18 h	-	n.c.
Test group	210.0 ug/ml	18 h	-	n.c.
Test group	13.1 ug/ml	18 h	+	0.34
Test group	26.3 ug/ml	18 h	+	n.c.
Test group	52.5 ug/ml	18 h	+	0.09
Test group	210.0 ug/ml	18 h	+	0.34
Negative Control versus Positive Control
EMS	600 ug/ml	18 h	-	< 0.001^S
CPA	0.71 ug/ml	18 h	+	< 0.001^S

n.c. = not calculated as the aberration rate is equal or lower than the control rate

^Saberration rate is statistically significantly higher than the control rate

Table 5. Statistical significance in experiment II was evaluated by means of the Fisher’s exact test. Evaluation was performed only for cells carrying aberrations exclusive of gaps.

Solvent Control versus		Exposure period	S9 mix	p-value
Test group	26.3 ug/ml	18 h	-	n.c.
Test group	52.5 ug/ml	18 h	-	n.c.
Test group	105.0 ug/ml	18 h	-	n.c.
Test group	210.0 ug/ml	18 h	-	n.c.
Test group	13.1 ug/ml	18 h	+	n.c.
Test group	26.3 ug/ml	18 h	+	n.c.
Test group	52.5 ug/ml	18 h	+	n.c.
Test group	210.0 ug/ml	18 h	+	n.c.
Test group	52.5 ug/ml	28 h	-	0.06
Test group	210.0 ug/ml	28 h	-	0.02^S
Test group	26.3 ug/ml	28 h	+	n.c.
Test group	52.5 ug/ml	28 h	+	n.c.
Test group	210.0 ug/ml	28 h	+	n.c.
Negative Control versus Positive Control
EMS	600 ug/ml	18 h	-	< 0.001^S
CPA	0.71 ug/ml	18 h	+	< 0.001^S

n.c. = not calculated as the aberration rate is equal or lower than the control rate

^Saberration rate is statistically significantly higher than the control rate

Applicant's summary and conclusion

Conclusions:

Interpretation of results:negative with and without metabolic activation
In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, CGF-C-1607 is considered to be non-mutagenic in this chromosome aberration test.

Executive summary:

The test article CGF -C-1607, dissolved in acetone, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The chromosomes were prepared 18 h (exp. I and II) and 28 h (exp. II) after start of treatment with the test article. In experiment I, the exposure period was 4 h with and without metabolic activation. In experiment II the cultures were exposed for 4 h with S9 mix and 18 h and 28 h without S9 mix. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test on toxicity was chosen with regard to the solubility properties of the test article. Test article concentrations between 1.6 and 210 ug/ml (with and without S9 mix) were applied for the assessment of the cytotoxic potential using cell growth inhibition as indicator for toxicity. Neither in the absence nor in the presence of S9 mix clearly dose related toxic effects were observed. Precipitation of the test article in culture medium was observed after treatment with 105 ug/ml and above in the absence of S9 mix and 52.5 ug/ml and above in the presence of S9 mix. No influence of the test article on the pH value or osmolarity was observed. In the cytogenetic experiments, test article concentrations within a range of 6.5 - 210 ug/ml (without S9 mix) and 3.3 - 210 ug/ml (with S9 mix) were applied for the investigation of the potential to induce cytogenetic damage. In the absence and the presence of S9 mix, in neither experiment, reduced mitotic indices or cell numbers were observed, except in experiment II at interval 18 h in the absence of S9 mix after treatment with 210 ug/ml. In this study, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test article. A single significant increase (3.5 % aberrant cells exclusive gaps) in the absence of S9 mix in experiment II at exposure period 28 h after treatment with 210 ug/ml has to be regarded as being biologically irrelevant. In addition, no increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls. 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 in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

Therefore, CGF-C-1607 is considered to be non-mutagenic in this chromosome aberration test.