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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is non mutagenic in bacteria and in mammalian cells and non clastogenic (OECD 471, 473 and 476)

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HGPRT)

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.

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.

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

liver S9 from rats induced with phenobarbital i.p. and β-naphthoflavone orally

Test concentrations with justification for top dose:

1.17 μg/mL - 600.00 μg/mL (depending on incubation conditions, see table for details)

Vehicle / solvent:

Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 - 30 hours
- Exposure duration: 4h or 18h
- Expression time (cells in growth medium): 10, 14 or 24 hours

- Fixation time (start of exposure up to fixation or harvest of cells): 28h


SPINDLE INHIBITOR (cytogenetic assays): colcemide
STAIN (for cytogenetic assays): 7.5% (v/v) Giemsa/Titrisol solution pH 7.2

NUMBER OF REPLICATIONS:2

NUMBER OF CELLS EVALUATED: 100 metaphases per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

The V79 in vitro cytogenetic assay is considered valid if the following criteria are met:
• The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable metaphases.
• The numbers of cells with structural/numerical aberrations in the negative control has to be within the range of the historical negative control data.
• The positive control substances both with and without S9 mix have to induce a distinct increase of structural chromosome aberrations.

The test substance is considered as “positive” if the following criteria are met:
• A statistically significant, dose-related and reproducible increase in the number of cells
with structural chromosome aberrations (excl. gaps).
• The number of aberrant cells (excl. gaps) exceeds both the concurrent negative/vehicle
control value and the historical negative control data range.
A test substance generally is considered as “negative” if the following criteria are met:
• The number of cells with structural aberrations (excl. gaps) in the dose groups is not
statistically significant increased above the concurrent negative/vehicle control value and
is within the historical negative control data range.

Statistics:

The statistical evaluation of the data was carried out using the MUCHAN program system (BASF SE). The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni- Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test are statistically significant compared with the respective vehicle control, labels (* p ≤ 0.05, ** p ≤ 0.01) are printed in the tables.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not valid

Positive controls validity:

valid

					Genotoxicity				Cytotoxicity
Experiment	Exposure duration	Test group	S9-mix	Precipitation	incl. gaps#	excl. gaps#	with exchanges	Polyploid cells [%]	Cell number [%]	Mitotic index [%]
1	4/18 hrs	Vehicle control1	-	n.d.	7.0	4.5	0.5	1.5	100.0	100.0
		1.17 µg/mL	-	-	n.d.	n.d.	n.d.	n.d.	118.9	n.d.
		2.34 µg/mL	-	-	3.0	2.0	1.0	0.0	123.8	73.5
		4.69 µg/mL	-	-	2.5	1.0	0.0	0.0	118.1	90.2
		9.38 µg/mL	-	-	2.5	2.0	0.0	0.0	111.8	92.4
		75.00 µg/mL	-	+	9.5	6.5	3.0	0.0	74.8	22.7
		150.00 µg/mL	-	+	n.s.	n.s.	n.s.	n.s.	53.2	n.s.
		300.00 µg/mL	-	+	n.s.	n.s.	n.s.	n.s.	52.5	n.s.
		600.00 µg/mL	-	+	n.s.	n.s.	n.s.	n.s.	41.2	n.s.
		Positive control2x	-	n.d.	21.0s	19.0s	12.0s	0.0	n.t.	106.1
3	4/18 hrs	Vehicle control1	-	n.d.	3.5	1.0	0.5	0.0	100.0	100.0
		6.25 µg/mL	-	-	n.d.	n.d.	n.d.	n.d.	85.9	n.d.
		12.50 µg/mL	-	-	6.0	3.5	0.5	0.0	91.1	90.7
		25.00 µg/mL	-	+	7.0	2.0	1.0	0.0	101.2	92.5
		50.00 µg/mL	-	+	5.5	4.5	1.5	0.0	102.5	55.9
		100.00 µg/mL	-	+	n.s.	n.s.	n.s.	n.s.	76.0	n.s.
		Positive control2x	-	n.d.	26.0s	23.0s	16.0s	0.0	n.t.	74.0
2	18/18 hrs	Vehicle control1	-	n.d.	4.0	1.0	0.0	0.0	100.0	100.0
		1.17 µg/mL	-	-	n.d.	n.d.	n.d.	n.d.	126.0	n.d.
		2.34 µg/mL	-	-	6.0	3.0	1.5	0.0	100.3	82.5
		4.69 µg/mL	-	-	6.5	4.0	1.5	0.0	108.4	92.4
		9.38 µg/mL	-	-	3.0	1.5	1.5	0.0	135.6	72.5
		18.75 µg/mL	-	+	n.s.	n.s.	n.s.	n.s.	109.3	n.s.
		37.50 µg/mL	-	+	n.s.	n.s.	n.s.	n.s.	65.9	n.s.
		75.00 µg/mL	-	+	n.s.	n.s.	n.s.	n.s.	64.7	n.s.
		Positive control2x	-	n.d.	24.0s	24.0s	18.0s	0.0	n.t.	64.5
2	18/28 hrs	Vehicle control1	-	n.d.	3.5	2.5	1.0	0.0	100.0	100.0
		1.17 µg/mL	-	-	n.d.	n.d.	n.d.	n.d.	89.1	n.d.
		2.34 µg/mL	-	-	4.0	3.5	0.0	0.0	124.9	124.1
		4.69 µg/mL	-	-	3.0	2.0	1.0	0.0	127.7	126.9
		9.38 µg/mL	-	-	5.5	2.0	0.0	0.5	83.8	99.5
		18.75 µg/mL	-	+	n.s.	n.s.	n.s.	n.s.	108.4	n.s.
		37.50 µg/mL	-	+	n.s.	n.s.	n.s.	n.s.	115.9	n.s.
		75.00 µg/mL	-	+	n.s.	n.s.	n.s.	n.s.	77.9	n.s.
		Positive control2x	-	n.d.	27.0s	27.0s	25.0s	0.0	n.t.	85.4
1	4/18 hrs	Vehicle control1	+	n.d.	7.5	3.5	1.0	0.0	100.0	100.0
		1.17 µg/mL	+	-	n.d.	n.d.	n.d.	n.d.	108.5	n.d.
		2.34 µg/mL	+	-	7.0	4.5	2.0	1.5	102.1	107.6
		4.69 µg/mL	+	-	5.5	3.0	1.0	0.0	103.1	91.8
		9.38 µg/mL	+	-	5.0	4.0	2.0	0.0	92.3	95.1
		37.50 µg/mL	+	+	6.5	4.0	2.5	1.0	103.5	71.7
		75.00 µg/mL	+	+	n.s.	n.s.	n.s.	n.s.	87.4	n.s.
		150.00 µg/mL	+	+	n.s.	n.s.	n.s.	n.s.	48.9	n.s.
		300.00 µg/mL	+	+	n.s.	n.s.	n.s.	n.s.	66.0	n.s.
		Positive control2x	+	n.d.	23.0s	23.0s	17.0s	0.0	n.t.	76.6
3	4/18 hrs	Vehicle control1	+	n.d.	6.0	3.0	0.5	0.0	100.0	100.0
		3.13 µg/mL	+	-	n.d.	n.d.	n.d.	n.d.	120.6	n.d.
		6.25 µg/mL	+	-	8.0	3.0	1.0	0.0	135.4	84.8
		12.50 µg/mL	+	+	7.5	5.0	2.0	1.5	110.5	80.6
		25.00 µg/mL	+	+	4.0	3.0	2.0	0.5	123.6	87.6
		50.00 µg/mL	+	+	n.d.	n.d.	n.d.	n.d.	123.4	n.d.
		Positive control2x	+	n.d.	32.0s	30.0s	21.0s	0.0	n.t.	68.2
2	4/28 hrs	Vehicle control1	+	n.d.	3.5	2.0	0.5	0.0	100.0	100.0
		1.17 µg/mL	+	-	n.d.	n.d.	n.d.	n.d.	94.3	n.d.
		2.34 µg/mL	+	-	5.0	2.0	1.5	0.0	115.5	74.2
		4.69 µg/mL	+	+	2.5	1.5	0.0	0.0	87.3	89.8
		9.38 µg/mL	+	+	8.0	3.0	0.5	0.0	110.0	112.9
		18.75 µg/mL	+	+	7.0	2.0	0.5	0.0	104.0	80.7
		37.50 µg/mL	+	+	n.s.	n.s.	n.s.	n.s.	85.8	n.s.
		75.00 µg/mL	+	+	n.s.	n.s.	n.s.	n.s.	40.8	n.s.
		Positive control2x	+	n.d.	29.0s	29.0s	23.0s	0.0	n.t.	79.0

P Precipitation occured at the end of exposure period

* Relative values compared with the respective vehicle control

# Inclusive cells carrying exchanges

n.d. Not determined

n.s. Not scorable due to poor metaphase quality / strong cytotoxicity / strong test substance precipitation

n.t. Not tested

S Aberration frequency statistically significant higher than corresponding control values

1 Acetone 1% (v/v)

2 CPP 0.5 μg/mL

x Evaluation of a sample of 100 metaphase only due to strong clastogenicity

Conclusions:

CAS 15680-42-9 is not clastogenic in-vitro (OECD 473, GLP).

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium: His (-/-)
Escherchia coli: Tryp (-/-)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from rats treated with Phenobarbitone/b-naphthoflavone

Test concentrations with justification for top dose:

Standard plate test: 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate (with and without metabolic activation)
Preincubation test: 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, THF was used as vehicle, which
had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available

Untreated negative controls:

yes

Remarks:

(sterillity control)

Negative solvent / vehicle controls:

yes

Remarks:

THF

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

4-nitroquinoline-N-oxide

9-aminoacridine

congo red

other: N-methyl-N'-nitro-N-nitrosoguanidine, 4-Nitro-o-phenylenediamine (NOPD)

Details on test system and experimental conditions:

METHOD OF APPLICATION: standard plate test (plate incorporation method) and preincubation

DURATION
- Incubation period: about 48-72 h at 37 °C in darkness
- Preincubation period: ca. 20 min

NUMBER OF REPLICATIONS:
- 3 test plates per dose or per control (2 independent experiments)


DETERMINATION OF CYTOTOXICITY
- decrease in the number of revertants
- clearing or diminution of the background lawn
- reduction in the titer
(for all test groups with and without S9-mix)

Titer:
The titer is generally determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

Evaluation criteria:

Acceptance criteria
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the
historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in
the number of revertant colonies within the range of the historical positive control data or
above.
• Fresh bacterial culture containing approximately 10E9 cells per mL were used. For approval
the titer of viable bacteria was ≥ 10E8 colonies per mL.

Assessment criteria
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about
doubling of the spontaneous mutation rate in at least one tester strain either without
S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control
range under all experimental conditions in at least two experiments carried out independently of each other.

Statistics:

not performed

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

above 2.5 mg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

A weak bacteriotoxic effect (decrease in the number of his+ or trp+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions from about 2 500 μg/plate onward.
The inconclusive values concerning the bacteriotoxicity observed in the 1st Standard plate test with tester strains TA 1535 and TA 1537 were not verified in a repeat experiment and have to be regarded as not relevant.
In the prival preincubation assay bacteriotoxicity (reduced his- background; decrease in the number of his+ revertants) was occasionally observed depending on the strain and test conditions at 5 000 μg/plate.

Test substance precipitation was found from about 33 μg/plate onward with and without S9 mix.

Without metabolic activation, standard plate test

Strain	Test group	Dose	Mean	Standard deviation		Factor	Individual revertant colony counts
TA 1535	THF	-	10.3	0.6		-	10, 11, 10
	Test item	33	9.7	4.7		0.9	6 P, 15 P, 8 P
		100	9.3	0.6		0.9	9 P, 10 P, 9 P
		333	6.3	2.1		0.6	8 P, 7 P, 4 P
		1000	6.7	3.2		0.6	8 P, 3 P, 9 P
		2500	5.0	1.7		0.5	4 P, 7 P, 4 P
		5000	4.0	0.0		0.4	4 P, 4 P, 4 P
	MNNG	5.0	6482.3	144.9		627.3	6420, 6648, 6379
TA 100	THF	-	51.7	4.0		-	48, 51, 56
	Test item	33	45.3	2.3		0.9	48 P, 44 P, 44 P
		100	67.0	12.5		1.3	71 P, 77 P, 53 P
		333	63.0	1.0		1.2	62 P, 63 P, 64 P
		1000	62.3	16.5		1.2	79 P, 46 P, 62 P
		2500	48.7	5.5		0.9	49 P, 54 P, 43 P
		5000	44.7	3.1		0.9	42 P, 44 P, 48 P
	MNNG	5.0	5819.7	182.0		112.6	5731, 6029, 5699
TA 1537	THF	-	8.3	1.5		-	8, 10, 7
	Test item	33	6.7	3.1		0.8	10 P, 6 P, 4 P
		100	4.0	1.7		0.5	6 P, 3 P, 3 P
		333	6.7	1.2		0.8	6 P, 8 P, 6 P
		1000	5.0	2.6		0.6	3 P, 8 P, 4 P
		2500	4.0	2.0		0.5	6 P, 2 P, 4 P
		5000	5.7	0.6		0.7	6 P, 6 P, 5 P
	AAC	100	1983.7	286.5		238.0	2157, 1653, 2141
TA 98	THF	-	23.3	2.9		-	20, 25, 25
	Test item	33	24.3	4.9		1.0	22 P, 30 P, 21 P
		100	23.3	5.5		1.0	17 P, 27 P, 26 P
		333	21.3	6.5		0.9	21 P, 15 P, 28 P
		1000	17.7	5.7		0.8	13 P, 16 P, 24 P
		2500	15.0	1.0		0.6	15 P, 16 P, 14 P
		5000	7.3	2.5		0.3	5 P, 7 P, 10 P
	NOPD	10	349.0	22.6		15.0	325, 352, 370

E. coli	THF	-	72.0	10.8	-	69, 84, 63
	Test item	33	67.7	7.6	0.9	59 P, 71 P, 73 P
		100	68.7	17.2	1.0	88 P, 63 P, 55 P
		333	62.7	3.1	0.9	60 P, 66 P, 62 P
		1000	69.0	3.5	1.0	65 P, 71 P, 71 P
		2500	66.7	4.7	0.9	63 P, 72 P, 65 P
		5000	42.0	13.1	0.6	51 P, 48 P, 27 P
	4-NQO	862.7	5	6.0	12.0	869, 862, 857

P = Precipitation

With metabolic activation, Standard plate test

Strain		Test group	Dose          Mean           Standard (µg/plate)  revertants   deviation				Factor		Individual revertant colony counts
TA 1535		THF	-	12.7	4.5		-		17, 8, 13
		Test item	33	10.7	1.5		0.8		11 P, 12 P, 9 P
			100	7.3	1.2		0.6		8 P, 8 P, 6 P
			333	7.0	1.0		0.6		7 P, 6 P, 8 P
			1000	8.7	4.6		0.7		6 P, 6 P, 14 P
			2500	10.0	3.6		0.8		13 P, 11 P, 6 P
			5000	9.7	2.3		0.8		11 P, 11 P, 7 P
		2-AA	2.5	291.0	10.6		23.0		287, 283, 303
TA 100		THF	-	60.0	2.6		-		59, 63, 58
		Test item	33	58.0	4.0		1.0		58 P, 62 P, 54 P
			100	60.7	10.2		1.0		65 P, 49 P, 68 P
			333	67.0	4.4		1.1		62 P, 70 P, 69 P
			1000	67.7	12.5		1.1		62 P, 82 P, 59 P
			2500	62.3	3.1		1.0		65 P, 59 P, 63 P
			5000	63.7	5.5		1.1		58 P, 69 P, 64 P
		2-AA	2.5	1532.0	147.2		25.5		1667, 1375, 1554
TA 1537		THF	-	14.0	4.0		-		18, 10, 14
		Test item	33	7.7	5.0		0.5		13 P, 3 P, 7 P
			100	7.7	1.5		0.5		6 P, 9 P, 8 P
			333	7.3	2.9		0.5		9 P, 9 P, 4 P
			1000	5.0	1.7		0.4		4 P, 4 P, 7 P
			2500	5.0	2.0		0.4		3 P, 7 P, 5 P
			5000	3.7	3.1		0.3		7 P, 3 P, 1 P
		2-AA	2.5	137.3	18.5		9.8		119, 137, 156
TA 98		THF	-	33.7	3.8		-		38, 31, 32
		Test item	33	26.0	2.6		0.8		29 P, 25 P, 24 P
			100	29.3	3.5		0.9		29 P, 33 P, 26 P
			333	25.0	2.6		0.7		23 P, 24 P, 28 P
			1000	18.0	3.6		0.5		15 P, 22 P, 17 P
			2500	23.3	5.1		0.7		29 P, 19 P, 22 P
			5000	12.0	3.5		0.4		10 P, 16 P, 10 P
		2-AA	2.5	1295.7	155.3		38.5		1190, 1223, 1474
E. coli	THF		-	92.7	13.5	-		106, 79, 93
	Test item		33	76.0	12.0	0.8		76 P, 64 P, 88 P
			100	84.3	7.4	0.9		87 P, 90 P, 76 P
			333	87.3	4.9	0.9		84 P, 85 P, 93 P
			1000	70.7	5.9	0.8		64 P, 73 P, 75 P
			2500	66.0	5.3	0.7		62 P, 64 P, 72 P
			5000	31.3	5.5	0.3		35 P, 25 P, 34 P
	2-AA		60	201.0	16.4	2.2		215, 183, 205

Without metabolic activation, Standard plate teset, 2nd experiment

Strain	Test group	Dose (ug/plate)	Mean	SD	Factor	Individual revertant colony counts
TA 1535	THF	-	9.0	2.6	-	7, 12, 8
	Test item	33	10.7	5.1	1.2	12 P, 15 P, 5 P
		100	6.7	3.8	0.7	4 P, 5 P, 11 P
		333	10.0	7.2	1.1	18 P, 4 P, 8 P
		1000	6.7	3.1	0.7	10 P, 4 P, 6 P
		2500	10.7	1.5	1.2	9 P, 11 P, 12 P
		5000	6.7	0.6	0.7	7 P, 6 P, 7 P
	MNNG	5.0	5778.3	214.3	642.0	5986, 5791, 5558
TA 1537	THF	-	7.7	3.5	-	8, 11, 4
	Test item	33	6.3	2.1	0.8	7 P, 4 P, 8 P
		100	7.0	2.0	0.9	9 P, 7 P, 5 P
		333	9.0	2.6	1.2	10 P, 11 P, 6 P
		1000	7.0	1.0	0.9	7 P, 6 P, 8 P
		2500	7.3	1.2	1.0	6 P, 8 P, 8 P
		5000	4.0	2.0	0.5	4 P, 2 P, 6 P
	AAC	100	1201.7	202.7	156.7	1132, 1430, 1043

With metabolic activation, standard plate experiment, 2nd repeat

Strain	Test group	Dose (ug/plate)	Mean	SD	Factor	Individual revertant colony counts
TA 1535	THF	-	9.3	3.2	-	13, 8, 7
	Test item	33	9.3	2.9	1.0	11 P, 6 P, 11 P
		100	9.3	4.2	1.0	8 P, 14 P, 6 P
		333	12.0	2.6	1.3	15 P, 10 P, 11 P
		1000	10.7	1.2	1.1	12 P, 10 P, 10 P
		2500	11.7	2.5	1.3	9 P, 14 P, 12 P
		5000	8.0	3.0	0.9	5 P, 8 P, 11 P
	2-AA	2.5	258.7	30.3	27.7	272, 280, 224
TA 1537	THF	-	9.7	2.3	-	7, 11, 11
	Test item	33	11.7	2.3	1.2	13 P, 13 P, 9 P
		100	10.0	4.6	1.0	6 P, 15 P, 9 P
		333	8.0	5.2	0.8	5 P, 5 P, 14 P
		1000	7.7	1.5	0.8	8 P, 9 P, 6 P
		2500	7.3	0.6	0.8	8 P, 7 P, 7 P
		5000	5.0	1.7	0.5	6 P, 3 P, 6 P
	2-AA	2.5	169.0	29.5	17.5	199, 168, 140

Without metabolic activation, Prival incubation assay

Strain		Test group	Dose (ug/plate)		Mean	SD	Factor	Individual revertant colony counts
TA 1535		THF	-		11.3	3.5	-	8, 15, 11
		Test item	33		8.3	2.5	0.7	8, 11, 6
			100		11.0	3.0	1.0	8 P, 11 P, 14 P
			333		10.7	0.6	0.9	11 P, 10 P, 11 P
			1000		11.3	3.5	1.0	8 P, 11 P, 15 P
			2500		10.0	2.0	0.9	8 P, 12 P, 10 P
			5000		7.0	2.0	0.6	7 P, 5 P, 9 P
		MNNG	5.0		523.7	36.7	46.2	558, 528, 485
TA 100		THF	-		91.0	6.6	-	84, 92, 97
		Test item	33		77.3	5.0	0.8	72, 82, 78
			100		84.0	14.7	0.9	97 P, 87 P, 68 P
			333		94.0	6.2	1.0	92 P, 101 P, 89 P
			1000		71.3	6.4	0.8	74 P, 64 P, 76 P
			2500		70.0	5.0	0.8	70 P, 75 P, 65 P
			5000		21.3	2.5	0.2	21 P B, 24 P B, 19 P B
		MNNG	5.0		1304.7	368.8	14.3	893, 1605, 1416
TA 1537		THF	-		13.7	0.6	-	14, 13, 14
		Test item	33		15.0	1.7	1.1	14, 14, 17
			100		11.0	5.6	0.8	6 P, 10 P, 17 P
			333		12.0	1.0	0.9	13 P, 11 P, 12 P
			1000		12.0	2.0	0.9	14 P, 10 P, 12 P
			2500		12.7	1.2	0.9	12 P, 12 P, 14 P
			5000		6.3	0.6	0.5	6 P, 6 P, 7 P
		AAC	100		1047.7	451.4	76.7	527, 1329, 1287
TA 98		THF	-		22.3	1.5	-	22, 24, 21
		Test item	33		23.0	3.6	1.0	27, 20, 22
			100		26.7	4.0	1.2	31 P, 23 P, 26 P
			333		23.7	1.5	1.1	24 P, 25 P, 22 P
			1000		18.3	3.5	0.8	18 P, 15 P, 22 P
			2500		16.3	3.2	0.7	20 P, 15 P, 14 P
			5000		4.0	2.6	0.2	2 P B, 7 P B, 3 P B
		NOPD	10		505.0	33.0	22.6	472, 538, 505
E. coli		THF	-		78.0	10.1	-	76, 69, 89
		Test item	33		81.0	4.4	1.0	83, 76, 84
			100		73.3	16.5	0.9	57 P, 73 P, 90 P
			333		73.7	11.2	0.9	86 P, 71 P, 64 P
			1000		57.0	3.6	0.7	61 P, 54 P, 56 P
			2500		57.0	4.6	0.7	61 P, 58 P, 52 P
			5000		51.3	5.0	0.7	46 P, 56 P, 52 P
		4-NQO	5		1014.7	50.1	13.0	1066, 1012, 966

With metabolic activation, Prival assay

Strain		Test group	Dose (ug/plate)		Mean	SD		Factor	Individual revertant colony counts
TA 1535		THF	-		14.0	0.0		-	14, 14, 14
		Test item	33		13.0	1.7		0.9	12, 15, 12
			100		14.7	3.2		1.0	17 P, 11 P, 16 P
			333		12.3	5.5		0.9	15 P, 6 P, 16 P
			1000		12.0	2.0		0.9	14 P, 10 P, 12 P
			2500		11.0	1.0		0.8	11 P, 10 P, 12 P
			5000		7.0	1.0		0.5	8 P, 7 P, 6 P
		2-AA	10		222.0	11.4		15.9	235, 217, 214
TA 100		THF	-		83.3	7.0		-	84, 76, 90
		Test item	33		85.7	10.2		1.0	74, 90, 93
			100		100.7	11.9		1.2	114 P, 97 P, 91 P
			333		95.3	12.9		1.1	106 P, 81 P, 99 P
			1000		95.7	9.0		1.1	87 P, 105 P, 95 P
			2500		89.3	2.1		1.1	90 P, 87 P, 91 P
			5000		74.0	3.0		0.9	77 P, 71 P, 74 P
		2-AA	10		893.7	283.4		10.7	1114, 993, 574
TA 1537		THF	-		11.3	4.0		-	15, 12, 7
		Test item	33		9.0	5.3		0.8	5, 7, 15
			100		12.3	2.3		1.1	15 P, 11 P, 11 P
			333		10.0	2.0		0.9	8 P, 10 P, 12 P
			1000		10.3	3.1		0.9	7 P, 13 P, 11 P
			2500		9.3	1.5		0.8	8 P, 9 P, 11 P
			5000		6.3	0.6		0.6	6 P, 7 P, 6 P
		2-AA	10		116.0	6.1		10.2	123, 112, 113
TA 98		THF	-		33.0	7.8		-	37, 24, 38
		Test item	33		30.0	4.6		0.9	29, 26, 35
			100		34.0	3.6		1.0	31 P, 33 P, 38 P
			333		34.7	11.6		1.1	24 P, 33 P, 47 P
			1000		32.0	8.5		1.0	41 P, 24 P, 31 P
			2500		28.0	7.2		0.8	26 P, 22 P, 36 P
			5000		32.0	7.2		1.0	24 P, 34 P, 38 P
		2-AA	10		990.0	7.0		30.0	985, 998, 987
		CoR	210		530.3	50.9		16.1	548, 570, 473
E. coli		THF	-		84.3	15.9		-	92, 66, 95
		Test item	33		82.0	3.5		1.0	84, 84, 78
			100		90.7	9.0		1.1	90 P, 100 P, 82 P
			333		84.7	9.3		1.0	77 P, 95 P, 82 P
			1000		87.0	11.3		1.0	94 P, 74 P, 93 P
			2500		83.0	4.6		1.0	88 P, 79 P, 82 P
			5000		69.7	9.1		0.8	78 P, 71 P, 60 P
		2-AA	10		240.3	66.6		2.8	251, 301, 169

Conclusions:

Pigment Yellow 129 is not mutagenic in bacteria (OECD 471 with Prival modification for azo compunds).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No mutagenicity was observed in a Prival Ames test (OECD 471, GLP) (BASF 2014) and in the mammalian gene mutation assay (OECD 476, GLP) (BASF 2015). No clastogenicity was observed in-vitro (OECD 473, GLP) (BASF 2015). All studies fulfilled the validity critiera.

Therefore, the substance is considered to be non genotoxic. The test material was characterized for its particles in the nano-size range.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008.

No mutagenicity was observed in bacteria and in mammalian cells in vitro. No clastogenicity was observed in mammalian cells in vitro.

As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.