Calcium 3-hydroxy-4-[(1-sulphonato-2-naphthyl)azo]-2-naphthoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxic properties of PR 63:1 have been investigated in three in vitro tests: An AMES test performed according to OECD guideline 471, a chromosome aberration test was performed according to OECD guideline 473 and an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells, performed according to OECD test guideline 490. All tests showed a negative results, both in absence and presence of etabolic activation. This means there are no indications that this analogue has genotoxic properties.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

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:

his, trp

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:

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

Test concentrations with justification for top dose:

33 μg - 5400 μg/plate (SPT)
33 μg - 5400 μg/plate (Prival)

Vehicle / solvent:

Due to the insolubility of the test substance in water, DMSO was used as vehicle, which has been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with rat liver S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

other: 2-aminoanthracene (2-AA)

Remarks:

with hamster liver S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 4-nitro-o-phenylenediamine, N-methyl-N'-nitro-N-nitrosoguanidine, N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Details on test system and experimental conditions:

DETAILS ON TEST SYSTEM
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 37°C for 48 - 72 hours
NUMBER OF REPLICATIONS: in triplicate
NUMBER OF CELLS EVALUATED: all revertants / colonies counted
DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
• decrease in the number of revertants
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
• reduction in the titer
is recorded for all test groups both with and without S9 mix in all experiments

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA
1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

Key result

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A biologically relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the prival preincubation test either without S9 mix or after the addition of a metabolizing system.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation of the test substance was found from 100 μg/plate onward with and without S9 mix.
- Toxicity: No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test up to the highest required concentration. In the prival preincubation assay bacteriotoxicity (slight decrease in the number of his+ or trp+ revertants) was occasionally observed depending on the strain and test conditions from about 2700 μg/plate onward.

RANGE-FINDING/SCREENING STUDIES: In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation. In this study, due to the purity of the test substance 5.4 mg/plate was used as top dose in all experiments.

Remarks on result:

other: all strains/cell types tested

Conclusions:

In a reverse mutation assay with S. typhimurium (TA 1535, TA 100, TA 1537, TA 98) and E.coli ( WP2 uvrA), performed according to OECD guideline 471 and GLP principles, the test item did not have genotoxic activity without and with metabolic activation.

Executive summary:

The test item was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. S. typhimurium ( TA 1535, TA 100, TA 1537, TA 98) and E.coli ( WP2 uvrA), in a reverse mutation assay (Ames standard plate test and Prival preincubation test) at concentrations of 33 μg - 5800 μg/plate (SPT) and 100 μg - 5400 μg/plate (Prival) with and without metabolic activation. Precipitation of the test substance was found depending on the test conditions from about 100 μg/plate onward. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2700 μg/plate onward. A biologically relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the prival preincubation test either without S9 mix or after the addition of a metabolizing system.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 May 2017 - 19 September 2017

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)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd. 03 November 2015

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: healthy adult, non-smoking volunteers
- Cell cycle length, doubling time or proliferation index: AGT = 13.1 hours (dose range finding/first cytogenic assay; 29 years old donor), 13.2 hours (second cytogenic assay; 22 years old donor), 13.2 hours (additional second cytogenic assay; 25 years)
- Sex, age and number of blood donors if applicable: 18-35 years old
- Whether whole blood or separated lymphocytes were used: whole blood
- Methods for maintenance in cell culture: per culture 0.1 mL phytohaemagglutinin was added.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) and 30 U/ml heparin.
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 prepared from male rats treated with phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Test concentrations with justification for top dose:

The highest tested concentration was determined by the solubility of the test item in the culture medium.
Dose range finding test (without S9): 9.8, 19.5, 39, 78, 156 and 313 μg/mL
First cytogenic assay (with and without S9): 19.5, 39 and 78 μg/mL
Second cytogenic assay (without S9, 24 h exposure time): 39, 78 and 156 μg/mL
Second cytogenic assay (without S9, 48 h exposure time): 19.5, 39 and 78 μg/mL
Additional second cytogenic assay (without S9, 48 h exposure time): 20, 40, 50, 60, 70 and 80 μg/mL

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: the test item was dissolved or suspended in dimethyl sulfoxide of spectroscopic quality. The choice for the vehicle was according to OECD 473.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

The following experiments were performed:
- Dose-range finding study (24 and 48 hours, reported as part of the first experiment): with and without 1.8% (v/v) S9.
- Experiment 1 (3 hours): with and without 1.8% (v/v) S9
- Experiment 2 (24 and 48 hours): without S9 (as clear negative results were obtained in the presence of metabolic activation, the repetition of the experiment was not considered necessary)
- Experiment 2A (48 hours): without S9

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 +/- 2 hours culture period
- Exposure duration: 3h, 24 h and 48 h
- Fixation time: 3 h and 24 h exposure period: 24 h fixation time; 48 h exposure period: 48 h fixation time.

SPINDLE INHIBITOR: colchicine (0.5 μg/mL medium)

STAIN: 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. Slides were marked and allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper.

NUMBER OF CELLS EVALUATED: 1000 cells

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 150

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, onesided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

ACCEPTABILITY CRITERIA
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analysed by the Fisher’s exact test (one-sided, p < 0.05).

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

Key result

Species / strain:

lymphocytes: peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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: no
- Effects of osmolality: no
- Evaporation from medium: no
- Precipitation: at and above 78 μg/mL

RANGE-FINDING/SCREENING STUDIES: No statistically significant increase in the number of cells with chromosome aberrations and no increase in the number of polyploid cells and cells with endoreduplicated chromosomes was observed.

Since the quality control of one of the duplicate cultures of the highest concentration tested (48-hour exposure) in the second assay was scored above the 95% control limits of the historical data range, the 48-hour exposure experiment was repeated. The concentration spacing was adjusted in the repeat assay to be able to investigate a dose response of the test item.

NUMBER OF METAPHASES OBSERVED
- Dose range finding test (without S9) 24 h exposure: 46-121% of control; 48 h exposure: 49-109% of control
- First cytogenetic assay (without S9): 3 h exposure: 85-97% of control
- First cytogenetic assay (with S9): 3 h exposure: 93-100% of control
- Second cytogenetic assay (without S9): 24 h exposure: 69-88% of control; 48 h exposure: 82-122% of control
- Second additional cytogenetic assay (without S9): 48 hours exposure: 83-96% of control

HISTORICAL CONTROL DATA (see Table 1 and Table 2)
- The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database.
- The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No biologically relevant effects of the test item on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Table 1 Mitotic Index of Human Lymphocyte Cultures Treated with C.I. Pigment Red 63:1 in the Dose-Range Finding Test

24 h exposure time, 24 h fixation time
		Number of metaphases
C.I. Pigment Red 63:1 concentration (µg/ml)		Absolute	No. of cells scored	% of control
Control^a)		39	1021	100
9.8		37	1023	95
19.5		32	1018	82
39		47	1000	121
78		28	1001	72
156^b)		26	1011	67
313^b)		18	1013	46
48 h exposure time, 48 h fixation time
		Number of metaphases
C.I. Pigment Red 63:1 concentration (µg/ml)		Absolute	No. of cells scored	% of control
Control^a)		45	1009	100
9.8		49	1001	109
19.5		45	1013	100
39		46	1017	102
78	^b)	42	1002	93
156	^b)	35	1006	78
313	^b)	22	1013	49

^a)   Dimethyl sulfoxide; ^b)  C.I. Pigment Red 63:1 precipitated in the culture medium.

Table 2 Mitotic Index of Human Lymphocyte Cultures Treated with C.I. Pigment Red 63:1 in the First Cytogenetic Assay

3 h exposure time, 24 h fixation time
				Number of metaphases
C.I. Pigment Red 63:1 concentration (µg/ml)		Absolute			Number of cells scored			% of control
Control^b)		75	-	68	1008	-	1006	100
19.5		77	-	61	1011	-	1002	97
39		38	-	63	1011	-	1008	71
78	^c)	61	-	60	1008	-	1013	85
MMC-C; 0.5 µg/ml		32	-	34	1007	-	1011	46
MMC-C; 0.75 µg/ml		21	-	26	1010	-	1006	33
3 h exposure time, 24 h fixation time
C.I. Pigment Red 63:1 concentration (µg/ml)		Absolute			Number of cells scored			% of control
Control^b)		62	-	67	1013	-	1018	100
19.5		61	-	68	1005	-	1010	100
39		53	-	64	1015	-	1010	91
78^c)		60	-	60	1012	-	1012	93
CP; 10 µg/ml		40	-	41	1011	-	1011	63

a) Duplicate cultures;b) Dimethyl sulfoxide;c) C.I. Pigment Red 63:1 precipitated in the culture medium

Table 3 Mitotic Index of Human Lymphocyte Cultures Treated with C.I. Pigment Red 63:1 in the Second Cytogenetic Assay

24 h exposure time, 24 h fixation time		Number of metaphases^a^)
C.I. Pigment Red 63:1 concentration (µg/ml)		Absolute			No. of cells scored			% of control
Control^b)		37	-	46	1000	-	1000	100
39		39	-	34	1001	-	1000	88
78		28	-	36	1000	-	1000	77
156	^c)	25	-	32	1000	-	1002	69
MMC-C; 0.2 µg/ml		15	-	12	1000	-	1000	33
MMC-C; 0.3 µg/ml		6	-	12	1001	-	1001	22
48 h exposure time, 48 h fixation time
C.I. Pigment Red 63:1 concentration (µg/ml)
Control^b)		49	-	41	1001	-	1000	100
19.5		53	-	57	1000	-	1000	122
39		44	-	41	1000	-	1000	94
78	^c)	40	-	34	1000	-	1000	82
MMC-C; 0.1 µg/ml		42	-	29	1000	-	1000	79
MMC-C; 0.15 µg/ml		19	-	21	1000	-	1000	44

a) Duplicate cultures; b) Dimethyl sulfoxide; c) C.I. Pigment Red 63:1 precipitated in the culture medium

Table 4 Mitotic Index of Human Lymphocyte Cultures Treated with C.I. Pigment Red 63:1 in Cytogenetic Assay 2A

48 h exposure time, 48 h fixation time		Number of metaphases^a^)
C.I. Pigment Red 63:1 concentration (µg/ml)		Absolute			No. of cells scored			% of control
Control^b)		66	-	70	1002	-	1000	100
20		63	-	67	1003	-	1002	96
40		60	-	60	1000	-	1006	88
50		59	-	62	1004	-	1004	89
60		54	-	60	1000	-	1005	84
70		56	-	59	1004	-	1009	85
80	^c)	58	-	55	1000	-	1000	83
MMC-C; 0.1 µg/ml		66	-	69	1006	-	1006	99
MMC-C; 0.15 µg/ml		60	-	63	1000	-	1006	90

a) Duplicate cultures; b) Dimethyl sulfoxide; c) C.I. Pigment Red 63:1 precipitated in the culture medium

Table 5 Historical control data for in vitro chromosome aberration studies of the positive controls

	3 hours exposure time				24 hours exposure time		48 hours exposure time
	Gaps included		Gaps excluded		Gaps included	Gaps excluded	Gaps included	Gaps excluded
	+ S9-mix	- S9-mix	+ S9-mix	- S9-mix	- S9-mix	- S9-mix	- S9-mix	- S9-mix
Mean number of aberrant cells per 100 cells	32.63	29.33	31.98	28.86	30.90	30.00	35.95	34.96
SD	13.05	13.32	13.09	13.39	13.79	14.09	15.06	15.19
n	280	280	280	280	276	276	268	268
Upper control limit (95% control limits)	57.86	53.16	57.12	52.57	58.23	57.87	66.11	65.15
Lower control limit (95% control limits)	7.41	5.50	6.84	5.15	3.58	2.13	5.79	4.78

SD = Standard deviation; n = Number of observations

Distribution historical positive control data from experiments performed between January 2012 and November 2016. 

Table 6 Historical control data for numerical aberrations for in vitro chromosome aberration studies of the solvent control

	3 hours exposure time				24 hours exposure time		48 hours exposure time
	Poly		Endo		Poly	Endo	Poly	Endo
	+ S9-mix	- S9-mix	+ S9-mix	- S9-mix	- S9-mix	- S9-mix	- S9-mix	- S9-mix
Mean number numerical aberrations per 100 cells	0.08	0.08	0.01	0.02	0.09	0.01	0.09	0.01
SD	0.31	0.28	0.09	0.14	0.35	0.11	0.32	0.09
n	284	282	284	282	278	278	270	270
Upper control limit (95% control limits)	0.47	0.51	0.06	0.14	0.49	0.08	0.53	0.06
Lower control limit (95% control limits)	-0.32	-0.34	-0.04	-0.10	-0.31	-0.06	-0.35	-0.04

SD = Standard deviation; n = Number of observations

Poly = polyploidy; Endo = endoreduplication

Distribution historical negative control data from experiments performed between January 2012 and November 2016. 

Conclusions:

The results of a chromosome aberration study, performed according to OECD guideline 473, showed that C.I. Pigment Red 63:1 is not clastogenic in human lymphocytes.

Executive summary:

A chromosome aberration study was performed, according to OECD guideline 473 and GLP principles, to assess the possible clastogenicity of C.I. Pigment Red 63:1. Cultured peripheral human lymphocytes were used as a test system. The test item was tested in the presence and absence of a metabolic activation system (S9 -mix) in two independent experiments, up to a dose level of 78 μg/mL (precipitation dose level) for a 3 -hour exposure time, up to 156 μg/mL for a 24 -hour exposure time and up to 78 and 80 μg/mL for a 48 -hour exposure time. Fixation times were 24 hours for the 3 -hour and 24 -hour exposure times and 48 hours for the 48 -hour exposure time. The test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No effects of C.I. Pigment Red 63:1 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. The acceptability criteria were met and therefore it was concluded that the test conditions were adequate and the metabolic activation system functioned properly. It is concluded that C.I. Pigment Red 63:1 is not clastogenic in human lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October 2017 - 05 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd. 03 November 2015

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells from American Type Culture Collection, (ATCC, Manassas, USA), (2001)
- Suitability of cells: recommended test system in international guidelines

MEDIA USED
- Type and identity of media
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin
Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium: 3 hr exposure: basic medium, supplemented with 5% (v/v) heat-inactivated horse serum (=R5 medium); 24 hr exposure: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/ml trifluorothymidine
Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium)
- Properly maintained: yes, stock cultures of the cells were stored in liquid nitrogen (-196°C).
- Periodically checked for Mycoplasma contamination: yes
- Cleansed against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose-range finding test (with and without S9-mix, 3 hour treatment; without S9-mix, 24 hour treatment): 9.8, 19.5, 39, 78, 156* μg/mL
Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 156* μg/mL exposure medium.

Experiment 1
3 hour treatment (without and with S9): 1.2, 2.4, 4.9, 9.8, 19.5, 39, 78, 156* μg/mL
Experiment 2
24 hour treatment (without S9): 1.2, 2.4, 4.9, 9.8, 19.5, 39, 78, 156* μg/mL

*precipitation observed.

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: as recommended in OECD test guideline 490

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9, in DMSO, 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9: 7.5 µg/mL in Hanks’ balanced salt solution without calcium and magnesium.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: below 1 x 10^6 cells/mL

TEST ITEM PREPARATION
The test item was dissolved in DMSO. Test item concentrations were used within 2 hours of preparation. The final concentration of the solvent in the exposure medium was 0.5% (v/v).

DURATION
- Cleansing period: Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.
- Exposure duration: 3 hours (experiment 1; with and without S9-mix); 24 hours (experiment 2; without S9-mix).
- Expression time: 2 days in which at least 4 x 10^6 cells were subcultured every day
- Selection time (if incubation with a selection agent): 11 or 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)
STAIN: 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

NUMBER OF REPLICATIONS:
- test concentrations: 1
- positive control: 1
- solvent control: 2

DETERMINATION OF THE MUTATION FREQUENCY
For determination of the cloning efficiency the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 105 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for cloning efficiency and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well. The plates for the cloning efficiency and MF were scored with the naked eye or with the microscope.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controlsis between 65 and 120%. An acceptable number of surviving cells (10^6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 10^6 survivors, and for CP not below 700 per 10^6 survivors.

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with thehistorical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Statistics:

The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Precipitation: The test item precipitated directly in the exposure medium at concentrations of 156 μg/mL and above. After 3 hours, the test item precipitated in the exposure medium at concentrations of 78 μg/mL and above. The test item was tested beyond the limit of the solubility to obtain adequate cytotoxicity data. The concentration used as the highest test item concentration for the dose-range finding test was 156 μg/mL.

RANGE-FINDING/SCREENING STUDIES
- The test item precipitated in the culture medium at a dose level of 156 μg/mL culture medium in the absence and presence of S9-mix. No toxicity was observed.

EXPERIMENT 1 AND 2
No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls. Precipitation was observed in both experiments at 156 μg/mL. No toxicity was observed.

ACCEPTABILITY
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database (See table 1 and 2). It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
The suspension growth over the two-day expression period for cultures treated with DMSO was between 13 and 17 (3 hour treatment) and 100 and 105 (24 hour treatment).

Table 1 :Historical Control Data of the Spontaneous Mutation Frequencies of the Solvent Controls for the Mouse Lymphoma Assay

	Mutation frequency per 10^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	96	92	96
SD	29	30	29
n	268	248	285
Upper control limit (95% control limits)	160	152	160
Lower control limit (95% control limits)	32	31	32

SD = Standard deviation n = Number of observations

 Table 2: Historical Control Data of the Mutation Frequencies of the Positive Controls for the Mouse Lymphoma Assay

	Mutation frequency per 10^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	808	684	1669
SD	239	206	843
n	136	124	146
Upper control limit (95% control limits)	1465	1222	4196
Lower control limit (95% control limits)	152	146	-859

SD = Standard deviation n = Number of observations

Distribution historical positive control data from experiments performed between November 2014 and November 2017.  

Conclusions:

Based on the results of an in vitro mammalian cell gene mutation test, performed according to OECD guideline 490 and GLP principles, C.I. Pigment Red 63:1 is not mutagenic with and without metabolic activation in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

An in vitro mammalian cell gene mutation test was performed according to OECD guideline 490 and GLP principles. The dose levels that were tested in the dose finding study and experiments were up to and including precipitating concentrations. Precipitation occurred at 156 μg/mL. No toxicity was observed during the experiments. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In the absence and presence of S9-mix, C.I. Pigment Red 63:1 did not induce a significant increase in the mutation frequency. In conclusion C.I. Pigment Red 63:1 is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The test item was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. S. typhimurium ( TA 1535, TA 100, TA 1537, TA 98) and E.coli ( WP2 uvrA), in a reverse mutation assay (Ames standard plate test and Prival preincubation test) at concentrations of 33 μg - 5800 μg/plate (SPT) and 100 μg - 5400 μg/plate (Prival) with and without metabolic activation. Precipitation of the test substance was found depending on the test conditions from about 100 μg/plate onward. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2700 μg/plate onward. A biologically relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the prival preincubation test either without S9 mix or after the addition of a metabolizing system.

A chromosome aberration study was performed, according to OECD guideline 473 and GLP principles, to assess the possible clastogenicity of C.I. Pigment Red 63:1. Cultured peripheral human lymphocytes were used as a test system. The test item was tested in the presence and absence of a metabolic activation system (S9 -mix) in two independent experiments, up to a dose level of 78 μg/mL (precipitation dose level) for a 3 -hour exposure time, up to 156 μg/mL for a 24 -hour exposure time and up to 78 and 80 μg/mL for a 48 -hour exposure time. Fixation times were 24 hours for the 3 -hour and 24 -hour exposure times and 48 hours for the 48 -hour exposure time. The test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No effects of C.I. Pigment Red 63:1 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. The acceptability criteria were met and therefore it was concluded that the test conditions were adequate and the metabolic activation system functioned properly. It is concluded that C.I. Pigment Red 63:1 is not clastogenic in human lymphocytes.

An in vitro mammalian cell gene mutation test was performed according to OECD guideline 490 and GLP principles. The dose levels that were tested in the dose finding study and experiments were up to and including precipitating concentrations. Precipitation occurred at 156 μg/mL. No toxicity was observed during the experiments. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In the absence and presence of S9-mix, C.I. Pigment Red 63:1 did not induce a significant increase in the mutation frequency. In conclusion C.I. Pigment Red 63:1 is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Justification for classification or non-classification

Based on the current data-set, the substance is not classified for genotoxic properties according to Regulation (EC) No. 1272/2008.