2,20-dichloro-13,31-diethyl-4,22-dioxa-13,18,31,36-tetraazanonacyclo[19.15.0.03,19.05,17.06,14.07,12.023,25.024,32.025,30]hexatriaconta-1(36),2,5,7,9,11,14,16,18,20,23,25,27,29,32,34-hexadecaene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Pigment Violet 23 did not induce gene mutations by frameshifts or base-pair substitutions in the genome of the strains used when tested in a bacterial reverse mutation assay (Ames test, test strains: Salmonella typhimurium TA 98, TA 100, TA 1535, TA1537; E. coli WP2 uvrA) with and without metabolic activation (rat liver S9 and hamster liver S9) at up to 5000 µg/plate (RCC, 2005). Since the test item did not induce gene mutations Pigment Violet 23 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Pigment Violet 23 was not genotoxic in the mammalian cell gene (HPRT) mutation test in V79 Chinese Hamster cells when tested with and without rat liver S9 metabolic activation at concentrations up to 600 µg/ml (RCC, 2008).

Pigment Violet 23 was not genotoxic in the mammalian cell chromosome aberration test in V79 Chinese Hamster cells when tested with and without rat liver S9 metabolic activation at concentrations up to 600 mg/ml (RCC, 2008).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed in compliance with GLP and OECD Test guideline 471

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his- for S. typhimurium strains
trp- for E. coli WP2 uvr A

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (phenobarbital/ß-naphthoflavone-induced rat liver S9)

Test concentrations with justification for top dose:

Experiment I (plate incorporation): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate both with and without metabolic activation
Experiment II (pre-incubation): 33, 100, 333, 1000, 2500 and 5000 µg/plate both with and without metabolic activation

Vehicle / solvent:

DMSO, purity >99% (Merck, Darmstadt, Germany). The solvent was chosen because of its solubility properties and its relative non-toxicity to bacteria

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below for additional information

Details on test system and experimental conditions:

METHOD OF APPLICATION:
plate incorporation (experiment I); preincubation (experiment II). Since experiment I gave a negative result, experiment II was performed as a
preincubation assay.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: existence of evaluable plates (> 0 colonies) at five concentrations or more

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Not mandatory according to OECD guideline 471

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

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:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Pre-experiment was reported as experiment I because the criterion (evaluable plates (>0 colonies) at five concentrations or more in all strains are used) was met.

COMPARISON WITH HISTORICAL CONTROL DATA:
yes, see below

ADDITIONAL INFORMATION ON CYTOTOXICITY:
see below

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in both experiments.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix) with the exception of strain TA 98 without metabolic activation in experiment II. This strain showed a minor increase in revertant colony numbers at all concentrations of the test item. The absolute numbers of colonies reached and exceeded the threshold of two times the number of the corresponding solvent control at 33 and 100 μg/plate. To verify the results of this experiment an independent repeat experiment was performed under identical conditions with strain TA 98 in the absence of metabolic activation. No increase in the number of revertant colonies occurred in the repeat experiment and the effect observed in the second experiment was judged as biologically irrelevant. The repeat experiment is reported as experiment II A (see below).

There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In experiment I, the data in the negative and solvent control of strain WP2 uvrA were slightly above our historical control range in the presence and absence of metabolic activation. The number of colonies did not quite reach the lower limit of our historical control range in the solvent control of strain TA 98 without S9 mix in experiment II. Since these deviations are rather small, these effects are considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In strains TA 1535 and WP2 uvrA of the first experiment with metabolic activation the historical range of positive controls was just not reached (200 versus 221 colonies). This minor effect was judged to represent fluctuations. The threshold of two times or three times the corresponding solvent control was exceeded (factor of 5.3 and 2.5), so the test was considered valid.

Exp. II: preincubation method without S9 mix

Concentrations given in µg/plate

Strain -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA98 -- 2.1 -- 2.0 -- 1.9 -- 1.7 -- 1.9 -- 1.2

Exp. IIa: preincubation method without S9 mix (repeat assay)

Concentrations given in µg/plate

Strain -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA98 -- 1.0 -- 0.6 -- 0.6 -- 0.7 -- 1.0 -- 0.6

Conclusions:

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

The test item (Pigment Violet 23) did not induce gene mutations by frameshifts or base-pair substitutions in the genome of the strains used when tested in a bacterial reverse mutation assay (Ames test, test strains: Salmonella typhimurium TA 98, TA 100, TA 1535, TA1537; E. coli WP2 uvrA) with and without metabolic activation (rat liver S9 and hamster liver S9) at up to 5000 µg/plate. Since the test item did not induce gene mutations Pigment Violet 23 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of Pigment Violet 23 to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Hostaperm-Violett RL spez at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline-conform study under GLP without deviations.

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

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: minimum essential medium containing 10% fetal calf serum
- 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:

S9-mix (rat liver protein with cofactors)

Test concentrations with justification for top dose:

0.3, 0.6, 1.2, 2.4, 4.7, 9.4, 150, 300, 600 μg/ml
(Exp. I up to 600, Exp. II only up to 9.4μg/ml)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrathydrofuran

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: w/o metabolic activation; cyclophosphamide with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: -
- Exposure duration: 4, 18 and 28 h
- Recovery time (cells in growth medium): 14/24 h, respectively (only the 4 h-exposure group, other groups no recovery after substance treatment)
- Fixation time (start of exposure up to fixation or harvest of cells): 18/28 h, respectively


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: 2/experiment; 2 experiments


NUMBER OF CELLS EVALUATED: 100/slide


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cell number in defined field


OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

A test item is classified as non-clastogenic if:
a) the number of induced structural chromosome aberrations in all scored dose groups is in
the range of the laboratory’s historical control data range (0.0 - 4.0 % aberrant cells,
excluding gaps).
and/or
b) no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if:
1) the number of induced structural chromosome aberrations is not in the range of the
laboratory’s historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps).
and
2) either a concentration-related or a significant increase of the number of structural
chromosome aberrations is observed.
Statistical significance was confirmed by means of the Fisher’s exact test (9) (p < 0.05).
However, both biological and statistical significance should be considered together. If the
criteria mentioned above for the test item 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.
Although the inclusion of the structural chromosome aberrations is the purpose of this study,
it is important to include the polyploids and endoreduplications. The following criterion is
valid:
A test item can be classified as aneugenic if:
the number of induced numerical aberrations is not in the range of the laboratory’s
historical control data range (0.0 – 5.2 % polyploid cells).

Statistics:

Fisher's Exact Test (only on cells with aberrations excluding gaps)

Key result

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Exp I: >0.6 μg/ml
Exp II: >2.4/>4.7 μg/ml



COMPARISON WITH HISTORICAL CONTROL DATA: identified significances are within historical control data

Table 1: Summary of Chromosome Aberrations in V79 cells

Test item concentration [μg/ml ]	Preperation interval [h]	Mitotic Index  [% control]	aberrant cells [% control]	Gaps [%]	Chromatid-type aberrations [%]	Chromosome-type aberrations[%]	Multiple aberrations  [%]	Chromosomal disintegration [%]	Polyploid Cells[%]
Exposure 4 hours without Metabolic Activation
Solvent control°	18	100.0	1.0	0.5	0.5	0.0	0.0	0.0	2.2
0.6	18	105.8	0.5	0.0	0.5	0.0	0.0	0.0	1.8
1.2^P	18	104.8	1.0	0.5	0.0	0.5	0.0	0.0	1.8
2.4^P	18	109.2	2.5	1.5	1.0	0.0	0.0	0.0	1.8
Positive control°°	18	100.5	13.0	1.5	11.5	1.5	0.5	0.0	1.8
Exposure 4 hours with Metabolic Activation
Solvent control°	18	100.0	1.0	0.0	1.0	0.0	0.0	0.0	1.4
0.6	18	100.9	5.0	1.5	2.5	1.5	0.0	0.0	1.9
1.2^P	18	109.5	1.5	0.0	1.0	0.5	0.0	0.0	2.1
2.4^P	18	90.0	2.5	0.0	2.5	0.0	0.0	0.0	2.2
Positive control**	18	62.3	8.5	0.5	6.0	1.5	0.0	0.0	1.7
Exposure 18 hours without Metabolic Activation
Solvent control°	18	100.0	0.5	0.5	0.0	0.0	0.0	0.0	2.9
0.6	18	78.0	4.0	1.0	3.5	1.0	0.0	0.0	2.8
1.2	18	85.2	3.0	0.0	3.0	0.0	0.0	0.0	2.3
2.4^P	18	97.7	1.5	0.0	1.0	1.0	0.0	0.0	2.6
Positive control °°	18	69.3	11.0	1.0	9.5	1.0	0.0	0.0	2.2
Exposure 28 hours without Metabolic Activation
Solvent control°	28	100.0	0.5	0.0	0.0	0.5	0.0	0.0	3.1
2.4	28	82.4	1.5	0.0	1.0	0.5	0.0	0.0	2.8
4.7^P	28	111.0	1.5	0.5	0.0	1.0	0.0	0.0	3.2
Positive control °°	28	69.3	36.0	0.5	24.5	2.0	0.0	0.0	3.2
Exposure 4 hours with Metabolic activation
Solvent control°	28	100.0	1.0	0.0	1.0	0.0	0.0	0.0	3.2
1.2	28	103.7	1.0	0.0	0.5	0.5	0.0	0.0	3.1
2.4	28	78.3	2.0	0.5	2.5	0.0	0.0	0.0	3.4
4.7^P	28	95.4	0.0	0.0	0.0	0.0	0.0	0.0	2.7
Positive control**	28	94.3	10.5	0.0	4.5	5.0	1.0	0.0	2.9

°   tetrahydrofuran                  °°EMS

* CPA                            ^P Precipitation

Conclusions:

Interpretation of results (migrated information):
negative

The test item (Pigment Violet 23) was not genotoxic in the mammalian cell chromosome aberration test in V79 Chinese Hamster cells when tested with and without rat liver S9 metabolic activation at concentrations up to 600 mg/ml.

Executive summary:

The genotoxic potential of the test item was evaluated in a mammalian cell chromosome aberration assay according to OECD guideline 473. Chinese Hamster V79 cells were exposed to the test item over a period of either 4, 18 or 28 hours (for the 4 hours exposure, with and without metabolic activation by rat liver S9 mix, for 18 and 28 hours without metabolic activation). After short-term (4 h) exposure, cells were maintained for another 14/24 hours under substance-free conditions before harvest.2.5 hours before harvest, Colchemid was added to the cell culture medium to stop cell division in the stadium of metaphase. At harvest, cells were fixed by treatment with hypertonic solution and subsequent treatment with methanol/acetic acid. Cells were stained with Giemsa stain and analysed under the microscope at 100x magnification.

In Experiment I and II no cytotoxicity was observed up to the highest evaluated concentration where test item precipitation occured. In both experiments no clastogenicity was observed at the concentrations evaluated, either with or without metabolic activation. In Experiment II in the absence of S9 mix at preparation interval 18 hours two statistically significant increases in the number of aberrant cells (each 3 %) were observed after treatment with 0.6 and 1.2 Lg/mL. These values are clearly within the range of the laboratory’s historical control data range (0.0 – 4.0 % aberrant cells, excluding gaps) and therefore without biological relevance. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline-conform study under GLP without deviations.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: minimum essential medium containing 10% fetal calf serum
- 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:

S9-mix (rat liver protein with cofactors)

Test concentrations with justification for top dose:

1.2, 2.3, 4.7, 9.4, 18.8, 37.5 and 600 μg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrathydrofuran

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

THF only

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: w/o metabolic activation; 7,12-Dimethylbenz(a)anthracene with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (4h-incubation without serum, 24 h incubations with serum)


DURATION
- Preincubation period: none
- Exposure duration: 4 and 24 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution


NUMBER OF REPLICATIONS: 5/experiment, 2 experiments


NUMBER OF CELLS EVALUATED: colonies >50 cells


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

test item classified as positive for mutagenicity if induces either concentration-related increase of mutant frequency or reproducible and positive response at one of the test points (at least three times above the spontaneous mutation frequency)

Statistics:

linear regerssion on dose-dependeny of mutant frequencies using SYSTAT statistics software

Key result

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: negligible decrease (-0.02pH-units) at highest dose
- Effects of osmolality: negligible increase (+9 mOsm) at highest dose

Table 2: Results of V79/HPRT assay

Concentration [microgram/mL]	mutant colonies per 10^-6 cells**	mutant colonies per 10^6 cells**	Cytotoxicity (yes/no)	Remarks
	— MA	+ MA
0*	7.35	4.65	no
1.2	2.35	3.75	no
2.3	7.45	5.4	no
4.7	3.55	6.2	no
9.4	7.6	6.85	no	precipitation
600	3.5	6.8	no	precipitation
Positive control***	114.6	358	-MA: no; +MA: yes
0*	11.05	-	no
4.7	15.05	-	no
9.4	12.7	-	no
18.8	12.85	-	no
37.5	6.4	-	no	precipitation
600	9.7	-	no	precipitation
Positive control***	171.45	-	yes

*solvent control with Tetrahydrofuran

** mean of 10 plates (5/culture, 2 indep. cultures)

***without MA: EMS, with MA: DMBA

Conclusions:

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

The test item (Pigment Violet 23) was not genotoxic in the mammalian cell gene (HPRT) mutation test in V79 Chinese Hamster cells when tested with and without rat liver S9 metabolic activation at concentrations up to 600 µg/ml.

Executive summary:

The genotoxic potential of the test item was evaluated in a mammalian cell gene mutation assay according to OECD guideline 476. The target for gene mutation was the gene coding for hypoxanthine-guanine phosphoribosyl transferase (HPRT), an enzyme that can convert 6 -thioguanine (6 -TG) to its toxic ribophosphorylated derivative. Chinese Hamster V79 cells were exposed to the test item over a period of either 4 or 24 hours (for the 4 hours exposure, with and without metabolic activation by rat liver S9 mix). After exposure, cells were maintained for 7 days test substance-free under normal growth conditions to deplete HPRT in mutated cells. After this period, cells were maintained for 8 days in 6 -TG containing medium, that is toxic to non-mutated (HPRT-expressing) cells. After this selection period, the number of colonies remaining (each representing a mutated cell) were counted and their number compared to those achieved the solvent controls and positive control substances known to be mutagenic.

The test subsatnce was not cytotoxic up to precipitation. No substancial and reproducible dose-dependent increase in mutant frequency was observed, neither with nor without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Pigment Violet 23 does not have to be classified for mutagenicity according to the criteria laid down in the EU Dangerous Substances Directive (67/548/EEC) and in the EU Classification Labelling and Packaging Regulation (1272/2008/EC) because Pigment Violet 23 did not reveal any mutagenic effect in the bacterial reverse mutation assay in the presence or absence of metabolic activation at concentrations up to 5000 µg/plate, in an in vitro gene mutation study in mammalian cells (HPRT assay in V79 cells) at up to a precipitating concentration of 600 µg/ml and in a mammalian cell chromosome aberration test in V79 cells at up to a precipitating concentration of 600 µg/ml.