[1-[[(2-hydroxyphenyl)imino]methyl]-2-naphtholato(2-)-N,O,O']copper

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:

2014-2015

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[1-[[(2-hydroxyphenyl)imino]methyl]-2-naphtholato(2-)-N,O,O']copper

EC Number:

239-763-1

EC Name:

[1-[[(2-hydroxyphenyl)imino]methyl]-2-naphtholato(2-)-N,O,O']copper

Cas Number:

15680-42-9

Molecular formula:

C17H11CuNO2

IUPAC Name:

[1-[[(2-hydroxyphenyl)imino]methyl]-2-naphtholato(2-)-N,O,O']copper

Test material form:

solid: nanoform

Details on test material:

- Substance type: pigment
- Physical state: Solid / greenish yellow
- Name of test material (as cited in study report): Irgazin Yellow L 0800
- Lot/batch No.: 0005629903
- Analytical purity: 99.0 %
- Purity test date: 2013
- Expiration date of the lot/batch: unlimited stability under storage conditions
- Stability under test conditions: stable
- Storage condition of test material: room temperature
- Date of manufacture: 01 Dec 2011

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HGPRT)

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Culture medium: Ham's F12 medium containing stable glutamine and hypoxanthinesupplemented with 10% (v/v) fetal calf serum (FCS).
- Treatment medium (without S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) fetal calf serum.
- Treatment medium (with S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine.
- Pretreatment medium ("HAT" medium): Ham's F12 medium supplemented with: hypoxanthine (13.6 x 10-3 mg/mL), aminopterin (0.18 x 10-3 mg/mL), thymidine (3.88 x 10-3 mg/mL), 10% (v/v) fetal calf serum (FCS).
- Selection medium ("TG" medium): Hypoxanthine-free Ham's F12 medium supplemented with: 6-thioguanine (10 μg/mL), 1% (v/v) stable glutamine (200 mM), 10% (v/v) fetal calf serum (FCS).
- All media were supplemented with: 1% (v/v) penicillin/streptomycin and 1% (v/v) amphotericine B
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes; During the week prior to treatment, any spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium.

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

1st Experiment
without S9 mix (4-hour exposure period)
0; (6.3); 12.5; 25.0; 50.0; 100.0; 200.0; 400.0; (800.0) μg/mL
with S9 mix (4-hour exposure period)
0; (3.1; 6.3); 12.5; 25.0; 50.0; 100.0; (200.0; 400.0) μg/mL

2nd Experiment
without S9 mix (4-hour exposure period)
0; 4.7; 9.4; 18.8; 37.5; 150.0; 300.0; (600.0) μg/mL
with S9 mix (4-hour exposure period)
0; 4.7; 9.4; 18.8; 37.5; 75.0; 150.0; (300.0) μg/mL

Concentrations in parentheses could not be analyzed because the substance was too cytotoxic.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, acetone was selected as the vehicle which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical data are available.

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:

300 μg/mL EMS (with S9 mix), 1.25 μg/mL DMBA (without S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without S9)
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): 6-7 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:

Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range of 0.00 – 16.43 mutants per 10E6 clonable cells
• The positive controls both with and without S9 mix have to induce distinctly increased mutant frequencies (historical positive control data).
• At least 4 dose levels should be tested ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

Assessment 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.
Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10E6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
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 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 p-value (probability value) is below 0.10 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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Table 1:  Summary of results - experimental parts with and without S9 mix
Exp.	period [h]	Test groups [µg/mL]	S9 mix	Prec.*	MFcorr.[per 106cells]	CE1 [%]	CE2 [%]
1	4	1% Acetone	-	n.d.	0.81	100.0	100.0
		6.3	-	-	n.c.1	103.3	n.c.1
		12.5	-	-	1.89	94.9	102.7
		25.0	-	+	1.69	103.5	99.7
		50.0	-	+	1.38	89.8	101.1
		100.0	-	+	0.81	84.2	101.3
		200.0	-	+	3.87	53.3	95.3
		400.0	-	+	1.87	11.2	104.8
		800.0	-	+	n.c.2	0.0	n.c.2
		EMS 400 μg/mL	-	n.d.	81.63	113.4	99.2
2	4	1% Acetone	-	n.d.	7.55	100.0	100.0
		4.7	-	-	7.08	99.9	91.0
		9.4	-	-	0.43	101.3	76.5
		18.8	-	+	1.63	82.3	79.9
		37.5	-	+	0.40	87.0	83.0
		150.0	-	+	1.09	81.6	90.2
		300.0	-	+	3.41	49.9	89.9
		600.0	-	+	n.c.2	1.8	n.c.2
		EMS 400 μg/mL	-	n.d.	134.24	78.6	75.0
1	4	1% Acetone	+	n.d.	6.05	100.0	100.0
		3.1	+	-	n.c.1	97.0	n.c.1
		6.3	+	-	n.c.1	103.4	n.c.1
		12.5	+	-	9.47	99.2	94.7
		25.0	+	+	2.65	104.0	101.4
		50.0	+	+	2.79	104.2	109.3
		100.0	+	+	3.04	93.0	112.7
		200.0	+	+	n.c.2	8.2	n.c.2
		400.0	+	+	n.c.2	0.0	n.c.2
		DMBA 1.25 μg/mL	+	n.d.	125.44	91.9	92.5
2	4	1% Acetone	+	n.d.	3.73	100.0	100.0
		4.7	+	-	2.29	104.0	87.8
		9.4	+	-	1.74	105.6	97.4
		18.8	+	+	0.40	123.4	90.1
		37.5	+	+	0.73	120.4	91.0
		75.0	+	+	2.31	118.0	102.7
		150.0	+	+	6.08	76.9	99.4
		300.0	+	+	n.c.2	3.8	n.c.2
		DMBA 1.25 μg/mL	+	n.d.	336.32	84.9	67.2

* 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.d. Not determined

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

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 article 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 following OECD guideline 476 and under GLP conditions. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). The substance showed cytotoxicity at high doses and was not mutagenic.