2,9-bis(2-phenylethyl)anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames-Test: negative, according to OECD TG 471 and 472, GLP compliant, Salmonella typhimurium strains: TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli strain WP2, with and without metabolic activation, 1991, K1

HPRT: Read-across, negative, according to OECD TG 476, GLP compliant, Chinese hamster lung fibroblasts (V79), with and without metabolic activation, 2012, K1

Chromosome aberration test: negative, according to OECD TG 473, GLP compliant, Chinese hamster ovary cells (CHO), with and without metabolic activation, 1989, K1

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

see attached justification

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 May 2020 - 13 Jul 2020

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 using the Hprt and xprt genes)

Version / remarks:

29 Jul 2016

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

Aug 1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Batch identification: 100071P040
CAS No.: 4948-15-6
Content: Sum.: 99.7 g/100 g
Date of production: 21 Dec 2010
Date of expiry: 30 Mar 2023
Molecular weight: 598.66 g/mol
Physical state, appearance: Solid, orange-red
Mass-specific surface area (BET): 97.1 m²/g
Storage conditions: Room temperature
Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance preparations.
Storage stability: The stability of the test substance under storage conditions was guaranteed until 30 Mar 2023 as indicated by the sponsor, and the sponsor holds this responsibility.

Target gene:

hprt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHO (Chinese hamster ovary)
- Suitability of cells: not specified

For cell lines:
- Absence of Mycoplasma contamination: each batch checked
- Number of passages if applicable: at least 2 passages before experiment; a further passage to prepare test cultures
- Methods for maintenance in cell culture: Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity up to approximate confluence and subcultured twice weekly (routine passage in 75 cm² plastic flasks).
- Cell cycle length, doubling time or proliferation index : high proliferation rate (doubling time of about 12 - 16 hours)
- Modal number of chromosomes: karyotype with a modal number of 20 chromosomes
- Periodically checked for karyotype stability: not specified
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- All media were supplemented with penicillin/streptomycin (100 IU / 100 μg/mL), amphotericine B (2.50 μg/mL)
- Culture medium/Treatment medium(without S9 mix): Ham's F12 medium with stable glutamine, hypoxanthine, 10% (v/v) fetal calf serum (FCS)
- Treatment medium (with S9 mix): Ham's F12 medium with stable glutamine, hypoxanthine
- Pretreatment medium ("HAT" medium): Ham's F12 medium with hypoxanthine (136 µg/mL), aminopterin (1.8 µg/mL), thymidine (38.8 µg/mL), 10% (v/v) FCS
- Selection medium ("TG" medium): Ham's F12 medium with stable glutamine, 6-thioguanine (10 μg/mL), 10% (v/v) FCS

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9
- method of preparation of S9 mix
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

The S9 fraction was prepared according to Ames et al. (1975):
- At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days.
- During this time, the animals were housed in polycarbonate cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 45 - 65%. The day/night rhythm was 12 hours: light from 6 am – 6 pm and darkness from 6 pm – 6 am. Standardized pelleted feed and drinking water from bottles were available ad libitum.
- 24 hours after the last administration, the rats were sacrificed and the livers were prepared using sterile solvents and glassware at a temperature of +4°C.
- The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.
- The S9 mix was prepared freshly prior to each experiment (Ames, 1975). For this purpose, a sufficient amount of S9 fraction was thawed at room temperature; 1 part S9 fraction was mixed with 9 parts S9 supplement (cofactors) in the pre-experiment and main experiments. This preparation, the S9 mix (10% S9 fraction), was kept on ice until used. The concentrations of the cofactors in the S9 mix were: 8 mM MgCl2, 33 mM KCL, 5 mM glucose-6-phosphate, 4 mM NADP, 15 mM phosphate buffer (pH 7.4).
- The phosphate buffer (DeMarini, 1989) is prepared by mixing a Na2HPO4 solution with a NaH2PO4 solution in a ratio of about 4:1.

Ames, B.N. et al. (1975): Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mut. Res. 31, 347-364.
DeMarini, D.M. et al. (1989): Cytotoxicity and effect on mutagenicity of buffers in a microsuspension assay. Ter. Carc. Mut. 9, 287-295.

Test concentrations with justification for top dose:

Based on the data and the observations from the pre-test and taking into account the current
guidelines, the following doses were selected in this study:
Dose selection (with and without S9 mix), 4 h exposure: 0.05, 0.08, 0.15, 0.26, 0.48, 0.86, 1.54, 2.78, 5.00 µg/mL

The highest tested concentration (5.00 μg/mL) was based on test substance precipitation in culture medium.

Vehicle / solvent:

Due to the insolubility of the test substance in culture medium, 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. The final concentration of the vehicle acetone in culture medium was 1% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Time schedule:
- Day 1: Seeding of the cells pretreated with "HAT" medium: in 300 cm² flasks (20x10^6 cells in 40 mL)
- Day 2: Test substance incubation (20 – 24 hours after seeding); exposure period (4 hours); removal of test substance by intense washing; 1st passage of the treated cells in 175 cm² flasks (2x10^6 cells in 20 mL medium) and seeding of the cloning efficiency 1 (survival) in 60 mm petri dishes (200 cells in 5 mL medium).
- Day 5: 2nd passage of the treated cells (seeding of 2x10^6 cells in 20mL medium)
- Day 7 - 9: Drying, fixation, staining and counting of the cloning efficiency 1; 3rd passage of the treated cells; addition of selection medium ("TG" medium); and seeding of the cloning efficiency 2 (viability)
- From day 16: Drying, fixation, staining and counting of the selected colonies and cloning efficiency 2

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 2
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 20x10^6 cells in 40 mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Attachment period of the cells: 20 - 24 hours
- Exposure duration/duration of treatment: 4 hours

FOR GENE MUTATION:
- Expression time: 7-9 days (from day 2)
- Selection time: 6-7 days (from day 7 to 9)
- Selective agent: 6-thioguanine (10 μg/mL)
- Fixation time: from day 7-9 and 16
- Each test group were fixed with methanol, stained with Giemsa and counted.

METHODS FOR MEASUREMENT OF CYTOTOXICITY

- Cloning efficiency 1: For the determination of the influence of the test substance after the exposure period, 200 cells per concentration were reserved from the treated cells and were seeded in petri dishes (60 mm diameter) and coated with 5 mL Ham's F12 medium incl. 10% (v/v) FCS in parallel to the 1st passage directly after test substance incubation.
- Cloning efficiency 2: For the determination of the mutation rate after the expression period, two aliquots of 200 cells each were reserved from the transfer into selection medium (after 7 – 9 days) and seeded in two petri dishes (60 mm diameter) containing 5 mL Ham's F12 medium incl. 10% (v/v) FCS.
- In all cases, after seeding the flasks or petri dishes were incubated for 5 - 8 days to form colonies. These colonies were fixed, stained and counted.

The cloning efficiency (CE, %) was calculated for each test group as follows:

total number of colonies in the test group
CEabsolute = ————————————————————— x 100
total number of seeded cells in the test group

CEabsolute of the test group
CErelative = —————————————— x 100
CEabsolute of the vehicle control


The number of colonies in every petri dish was counted and recorded. Using the formula above the values of absolute cloning efficiencies (CEabsolute, CE1 absolute and/or CE2 absolute) were calculated. Based on these values the relative cloning efficiencies (CErelative, CE1 relative and/or CE2 relative) of the test groups were calculated and reported as a percentage of the respective CEabsolute value of the corresponding vehicle control (vehicle control = 100%).
In addition, with regard to cell loss while exposure period, relative survival (RS) is calculated based on CE of cells plated immediatedly after treatment adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in vehicle controls.

number of cells at the end of treatment
Adjusted CE = —————————————————— x CE1
number of seeded cells

adjusted CE of the test group
RS = ———————————————————— x 100
adjusted CE of the vehicle control


METHODS FOR MEASUREMENTS OF GENOTOXICIY

The number of colonies in each flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized per every 10^6 cells seeded. The uncorrected mutant frequency (MFuncorr.) per 10^6 cells was calculated for each test group as follows:

total number of mutant colonies
MFuncorr. = —————————————–— x 10^6
number of seeded cells

The uncorrected mutant frequency was corrected with the absolute cloning efficiency 2 for each test group to get the corrected mutant frequency (MFcorr.):

MFuncorr.
MFcorr. = —–——–— x 100
CE2 absolute

OTHER:
- Check or determination of further parameters: pH, osmolality, solubility, cell morpology

Evaluation criteria:

Acceptance criteria:

- The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the vehicle controls should be within our historical negative control data range (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• Concurrent positive controls both with and without S9 mix should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase in mutant frequencies compared with the concurrent vehicle control.


Assessment criteria:

- A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).

- Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

- A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation
frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent
vehicle control value and within the range of our laboratory’s historical negative control data
(95% control limit).

Statistics:

A linear dose-response was evaluated by testing for linear trend. The dependent variable was the corrected mutant frequency and the independent variable was the dose.
The calculation was performed using EXCEL function RGP.
The used model is one of the proposed models of the International Workshop on Genotoxicity
Test procedures Workgroup Report (Moore, 2003).
A pair-wise comparison of each test group with the control group was carried out using Fisher's exact test with Bonferroni-Holm correction (Holm, 1979 and Siegel, 1956). The calculation was performed using EXCEL function HYPGEOM.VERT.
If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables.
However, both, biological and statistical significance are considered together.


- Moore, M.M. et al. (2003) Mouse Lymphoma Thymidine Kinase Gene Mutation Assay: International Workshop on Genotoxicity Tests (IWGT) Workgroup Report – Plymouth, UK 2002. Mut. Res. 540, 127–140.
- Holm, S. (1979) A Simple Sequentially Rejective Multiple Test Procedure. Scand J Statist 6, 65-70.
- Siegel, S. (1956) Nonparametric statistics for the behavioral sciences. New York, NY, US: McGraw-Hill

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not affected
- Data on osmolality: not affected
- Possibility of evaporation from medium: no
- Water solubility: insoluble in water and cell culture medium
- Precipitation and time of the determination: test substance precipitation was observed at 1.25 μg/mL and above after 4 hours in the absence and presence of S9 mix
- Cell morphology and attachment of the cells: not adversely influenced (grade > 2) in any test group

RANGE-FINDING/SCREENING STUDIES:
An initial range-finding cytotoxicity test for the determination of the experimental doses was conducted.
The pre-test was performed following the method described for the main experiment. The relative survival (RS) was determined as a toxicity indicator for dose selection and various parameters were checked for all, or at least some, selected doses.
In the pre-test for toxicity based on the solubility properties of the test substance 10.00 μg/mL (approx. 0.02 mM) was used as top concentration both with and without S9 mix at 4 hour exposure time.

STUDY RESULTS
- The mutation frequencies of the vehicle control groups were within our historical negative control data range (95% control limit) and, thus, fulfilled the acceptance criteria of this study.
- The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method and/or of the metabolic activity of the S9 mix employed.

For all test methods and criteria for data analysis and interpretation:
- At least four concentrations were evaluated to describe a possible dose response relationship.
- Statistical analysis; statistical significance for p ≤ 0.05
- see table 1 in section "Any other information on results incl. tables"

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements and genotoxicity results: see table 2 in section "Any other information on results incl. tables"

HISTORICAL CONTROL DATA
please see table 3 and 4 in section "Any other information on results incl. tables"

Table 1: Linear trend-test

 

Linear trend-test	Slope*	One-sided p-value*
Corrected Mutation frequency without S9 mix	-0.71162	0.7360
Corrected Mutation frequency with S9 mix	-1.32571	0.8616

* The linear trend-test testing for an increased mutant frequency is significant (significance level of 5%), if the one-sided p-value is lower than 0.05 and the slope is greater than 0.

 

Table 2: Summary of results

 

Exp.	Exposure period	Test groups	S9 mix	Prec.*	Genotoxicity** MFcorr.	Cytotoxicity***
	[h]	[µg/mL]			[per 10^6 cells]	RS [%]	CE2 [%]
1	4	VC^1	-	n.d.	1.05	100.0	100.0
		0.05	-	-	n.c.	125.6	n.c.
		0.08	-	-	n.c.	134.3	n.c.
		0.15	-	-	n.c.	135.3	n.c.
		0.26	-	-	3.39	128.5	103.5
		0.48	-	-	0.00	124.5	135.8
		0.86	-	-	0.68	132.5	103.5
		1.54	-	+	0.61	128.7	114.7
		2.78	-	+	n.c.^1	n.c.^1	n.c.^1
		5.00	-	+	n.c.^1	n.c.^1	n.c.^1
		PC^2	-	n.d	42.12^s	113.2	102.5
1	4	VC^1	+	n.d.	1.42	100.0	100.0
		0.05	+	-	n.c.	91.9	n.c.
		0.08	+	-	n.c.	89.1	n.c.
		0.15	+	-	n.c.	103.8	n.c.
		0.26	+	-	3.13	107.9	102.5
		0.48	+	-	1.69	68.1	105.3
		0.86	+	-	1.53	96.1	92.9
		1.54	+	+	0.72	82.6	98.9
		2.78	+	+	n.c.^1	n.c.^1	n.c.^1
		5.00	+	+	n.c.^1	n.c.^1	n.c.^1
		PC^3	+	n.d.	48.52^s	72.6	84.3

 

* Microscopically visible 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

^s Mutant frequency statistically significantly higher than corresponding control values (p ≤ 0.05)

n.c. Culture was not continued since a minimum of only four analysable concentrations is required

n.c.¹ Culture was not continued since only one concentration beyond the solubility limit is required

n.d. Not determined

VC vehicle control

PC positive control

¹ Acetone 1% (v/v) ² EMS 400 μg/mL ³ DMBA 1.25 μg/mL

 

 

Table 3: HISTORICAL NEGATIVE CONTROL DATA

Summary (all vehicles)

Period: March 2016 - December 2019

	Without S9 mix all vehicles*	With S9 mix all vehicles*
Corrected Mutant Frequency**
Exposure period	4 hrs	4 hrs
Mean	2.76	2.93
Minimum	0.00	0.00
Maximum	8.00	9.93
Standard Deviation	1.73	2.09
95% Lower Control Limit	0.00	0.00
95% Upper Control Limit	6.21	7.08
No. of Experiments	130	132

* = culture medium, water 10% (v/v), DMSO 1% (v/v), acetone 1% (v/v)

** = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE2)

 

 

Table 4: HISTORICAL POSITIVE CONTROL DATA

Summary (all vehicles)

Period: March 2016 - December 2019

	Without S9 mix 400 µg/mL ethyl methanesulfonate (EMS)	With S9 mix 1.25 µg/mL 7,12-Dimethylbenz[a]anthracene (DMBA)
	Corrected Mutant Frequency*	Corrected Mutant Frequency*
Exposure period	4 hrs	4 hrs
Mean	160.94	126.62
Minimum	42.47	21.52
Maximum	438.29	270.48
Standard Deviation	73.52	54.78
No. of Experiments	124	129

* = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE2)

Conclusions:

Under the experimental conditions of this study, the test substance is not mutagenic in an in vitro mammalian cell gene mutation test (HPRT-locus) in absence and in the presence of metabolic activation.

Executive summary:

The test substance was evaluated for genotoxic potential in a HPRT locus assay using CHO cells according to OECD TG 476 (GLP compliant). In one experiment a dose range from 0.05 to 5 µg/ml was tested, both with and without the addition of liver S9 mix from phenobarbital and β-naphthoflavone induced rats. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without or after the addition of the metabolizing system (S9 mix). No cytotoxicity could be observed, but a precipitation of the test materials was seen after 4 h exposure at dose 1.54 and above. Overall, the test item was considered to be non-mutagenetic under the conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Physical state: black powder
- Analytical purity: 99 %
- Storage condition of test material: room temperature
- Lot/batch No.: 96-2363

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:

Aroclor 1254 induced rat liver S-9 mix

Test concentrations with justification for top dose:

20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix: TA100 and TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

without S9 mix: TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix: TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix: E. coli WP2 uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
Plate incorporation method:
- Exposure duration: ca. 48 hours at 37°C
Preincubation method:
- Preincubation period: 30 minutes at 30°C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS:
- 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
reduced his- or trp- background growth

Evaluation criteria:

In general, a substance to be characterized as positive in the bacterial tests has to fulfill the following requirements:
- doubling of the spontaneous mutation rate ( control)
- dose-response relationship
- reproducibility of the results

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: from about 2500 µg/plate onward

Standard Plate Test Result:

	TA 1535		TA 100		TA 1537		TA 98		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	10	12	88	102	10	10	27	35	36	47
20	10	13	92	115	11	12	22	35	26	39
100	9	13	74	98	9	10	31	33	29	38
500	15	8	67	94	9	10	29	40	33	37
2500	14	12	90	102	9	9	26	38	29	34
5000	12	16	77	92	9	12	26	41	36	34
2-AA		320		908		121		2350		173
MNNG	1336		1327
AAC					463
NPD							1817
ENNG									199

Preincubation Test Result:

	TA 1535		TA 100		TA 1537		TA 98		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	17	18	116	113	11	11	29	35	22	24
20	17	15	123	120	9	10	24	39	25	28
100	16	18	97	119	9	10	27	42	26	27
500	20	18	79	107	10	12	28	40	23	30
2500	14	17	85	89	10	10	21	40	25	30
5000	16	15	69	81	12	10	22	37	22	24
2-AA		151		661		109		1137		127
MNNG	879		897
AAC					230
NPD							1067
ENNG									399

Controls:

2-AA: 2-aminoanthracene (10 µg/plate for TA strains, 60 µg/plate for WP2uvrA)

MNNG: N-methyl-N'-nitro-N-nitrosoguanidine (5 µg/plate)

AAC: 9-aminoacridine (100 µg/plate)

NPD: 4-nitro-o-phenylendiamine (10 µg/plate)

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidin (10 µg/plate)

Conclusions:

According to the results of the present study, the test substance is not mutagenic in the Ames test and in the Escherichia coli reverse mutation assay under the experimental conditions chosen here.

Executive summary:

The test item was tested in a GLP compliant Ames reverse mutation assay (according to OECD guidelines 471 and 472) using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA at 20 to 5000 µg/plate with and without metabolic activation using DMSO as the vehicle. In the toxicity test, the test compound proved to be not toxic to the bacteria up to 5000 µg/plate. Incomplete solubility of the test substance in DMSO was observed from about 2500 µg/plate onward. In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of the metabolic activation system, treatment of the cells with test material did not resultant in relevant increases in the number of revertant colonies. Under the conditions tested, the test article is not mutagenic in bacteria.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

other: Toxicity Test Guideline, (Japan) 1984

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Physical state: black powder
- Analytical purity: 99 %
- Storage condition of test material: room temperature
- Lot/batch No.: 96-2363

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S-9 liver microsomal fraction

Test concentrations with justification for top dose:

without and with S9 mix:
7 h : 1.0 ; 5.0* ; 10.0* ; 20.0* μg/ml
24 h : 0.1 ; 0.5 ; 1.0 ; 5.0* ; 10.0* ; 20.0* μg/ml
30 h : 1.0 ; 5.0* ; 10.0* ; 20.0* μg/ml
(* precipitation of the test compound in the culture medium during incubation and not evaluated)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative non-toxicity for the cells. The final concentration of the solvent in the culture medium did not exceed 1 % v/v.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix Migrated to IUCLID6: final Concentration : 0.72 mg/ml = 5.76 mM

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix Migrated to IUCLID6: final Concentration: 4.20 μg/ml = 15.00 μM

Details on test system and experimental conditions:

PRE-EXPERIMENT FOR TOXICITY: The toxicity of the test compound was determined in a pre-experiment (0.1 - 50 µg/ml) in order to establish a concentration dependent plating efficiency relationship. The experimental conditions in this pre-experiment were the same as described below for the experiment.

DOSE SELECTION
According to the results from this pre-experiment at least six concentrations to be applied in the chromosomal aberration assay were chosen.
The highest dose level used was 10 mM unless limited by the solubility of the test article or that producing some indication of cytotoxicity (reduced plating efficiency and/or partial inhibition of mitosis).
In case of toxic effects the highest dose level should reduce if possible the plating efficiency if possible to approximately 20 - 50 %. In addition, this concentration should suppress if possible mitotic activity (% cells in mitosis) by approximately 50 %, but not so great a reduction that insufficient scorable mitotic cells can be found.
Treatment was performed with the following concentrations:
without and with S9 mix:
7 h : 1.0 ; 5.0* ; 10.0* ; 20.0* μg/ml
24 h : 0.1 ; 0.5 ; 1.0 ; 5.0* ; 10.0* ; 20.0* μg/ml
30 h : 1.0 ; 5.0* ; 10.0* ; 20.0* μg/ml
(* precipitation of the test compound in the culture medium during incubation).
According to the criteria mentioned above, one (7, 30 h) and three concentrations (24 h) were selected to evaluate metaphases for cytogenetic damage.
In the pre-experiment for toxicity the colony forming ability of the CHO cells was only slightly reduced after treatment with 1.0 μg/ml in the presence of S9 mix. However, higher concentrations precipitated in the culture medium during incubation.
In the main experiment, cells after treatment with 1.0 μg/mL as highest dose level could be evaluated for cytogenetic damage. With these concentrations applied in the absence and presence of S9 mix the mitotic index was suppressed.

EXPERIMENTAL PERFORMANCE:
METHOD OF APPLICATION: in medium;
Seeding of the Cultures: Two days old logarithmically growing stock cultures more than 50 % confluent were trypsinised and a single cell suspension
was prepared. The trypsin concentration was 0 .2 % in Ca-Mg-free salt solution.
The cells were seeded into Quadriperm dishes (Heraeus, Hanau, D.) which contained microscopic slides (2 chambers per dish and test group). The medium was DMEM/F12 (1 :1) + 10 % FCS.
Approximately 0.6 - 1.5 x 10 5 cells were seeded into each chamber with regard to fixation interval.
After 48 h the medium was replaced with serum-free medium containing the test compound, either without S9 mix or with 20 μl/ml S9 mix. After 4 h this medium was replaced with normal medium after rinsing twice with "saline G".
All incubations were done at 37° C in a humidified atmosphere with 11.0 % CO2.
5, 21, 27 h after start of the treatment spindle inhibitor is added to the cultures. 2.0 h (7 h interval) or 3.0 h later (24 h and 30 h interval) the cells on the slides in the chambers were treated with hypotonic solution (0.4 % KCl) at 37 °C for 20 min. After incubation in the hypotonic solution the cells were fixed with 3:1 absolute methanol:glacial acetic acid. All two slides per group were prepared. After fixation the cells were stained with giemsa.

NUMBER OF REPLICATIONS: 2

SPINDLE INHIBITOR (cytogenetic assays): colcimid (0.2 µg/ml culture medium).

ANALYSIS
- Analysis of Metaphase Cells: The slides were evaluated using NIKON microscopes with 100x oil immersion objectives. Gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations are recorded as structural chromosome aberrations.

NUMBER OF CELLS EVALUATED: At least 100 well spread metaphases per slide (200 per test group) were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome number of 20 ± 1 are included in the analysis.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% cells in mitosis)

Evaluation criteria:

A test substance is classified as mutagenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points.
A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant positive response at any one of the test points is considered non-mutagenic in this system.

Statistics:

A statistical evaluation of the results was not necessary to perform. The aberration rates of the test groups after treatment with the test article were in the range of the control values.

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:

valid

Positive controls validity:

valid

Additional information on results:

In the pre-experiment on toxicity (colony forming ability) in the presence of S9 mix after treatment with 1.0 μg/ml the colony forming ability was only slightly reduced. However, higher concentrations precipitated in the culture medium during incubation.
In the main experiment, the mitotic index was reduced after treatment with 1.0 μg/ml indicating that the substance had cytotoxic properties.

Summarv of results

Fixation Interval: 7 h				% aberrant cells
article	number of cells analyzed	concentration /ml	S9-Mix	incl. Gaps	excl. Gaps	exchanges
solvent control	200	0	-	0.5	0	0
test article	200	1	-	3.5	2.5	0.5
solvent control	100*	0	+	7	4	1
test article	100*	1	+	8	4	0
Fixation Interval: 24 h				% aberrant cells
article	number of cells analyzed	concentration /ml	S9-Mix	incl. Gaps	excl. Gaps	exchanges
negative control	200	0	-	2.5	2.5	1
solvent control	200	0	-	0.5	0	0
positive control EMS	200	0.72	-	26.5	23.5	12.5
test article	200	0.1	-	1.5	1	0.5
test article	200	0.5	-	4	2.5	0.5
test article	200	1	-	4	3	0
negative control	200	0	+	4.5	4	1.5
solvent control	100*	0	+	9	5	2
positive control CPA	200	4.2	+	15	10.5	8
test article	200	0.1	+	9.5	5	1.5
test article	200	0.5	+	5	2.5	0.5
test article	200	1	+	7	2	0
						1
Fixation Interval: 24 h				% aberrant cells
article	number of cells analyzed	concentration /ml	S9-Mix	incl. Gaps	excl. Gaps	exchanges
solvent control	200	0	-	4.5	1	0
test article	200	1	-	4.5	2.5	0.5
solvent control	200	0	+	4	3.5	1
test article	200	1	+	7.5	4	1.5

*one slide out of two was not scorable

Conclusions:

In the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosomal aberration test in the CHO Chinese Hamster cell line and is therefore considered to be non-mutagenic in this chromosomal aberration test.

Executive summary:

The test article was assessed for its potential to induce structural chromosome aberrations in CHO cells in vitro. Preparation of chromosomes was done 7 h (high dose), 24 h (low, medium and high dose) and 30 h (high dose) after start of treatment with the test article. The treatment interval was 4 h. In each experimental group two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations. The following dose levels were evaluated with and without S9 mix: 7 h: 1.0 μg/ml; 24 h: 0.1; 0.5; 1.0 μg/ml; 30 h: 1.0 μg/ml. The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response. Treatment of the cells with 1.0 µg/ml reduced only slightly the plating efficiency. Higher concentrations than 1.0 µg/ml precipitated in the culture medium during incubation. However, the mitotic index was reduced with the highest concentration in the absence and presence of S9 mix. There was no relevant increase in cells with structural aberrations after treatment with the test article at any fixation interval either without or with metabolic activation by S9 mix. Appropriate reference mutagens were used as positive controls and showed distinct increases of cells with structural chromosome aberrations.

Therefore, the test article is considered to be non-mutagenic in this chromosomal aberration test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial Mutagenicity

The test item was tested in a GLP compliant Ames reverse mutation assay (according to OECD guidelines 471 and 472) using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 at 20 to 5000 µg/plate with and without metabolic activation using DMSO as the vehicle (BASF AG, 1991). In the toxicity test, the test compound proved to be not toxic to the bacteria up to 5000 µg/plate. Incomplete solubility of the test substance in DMSO was observed from about 2500 µg/plate onward. In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of the metabolic activation system, treatment of the cells with test material did not resultant in relevant increases in the number of revertant colonies. Under the conditions tested, the test article is not mutagenic in bacteria.

In a second, supporting Ames reverse mutation assay (study comparable to OECD guideline 471) the test article was tested using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 at 1 to 5000 µg/plate (vehicle: DMSO) with and without metabolic activation (BASF AG, 1985). The test material was not mutagenic in any of the S. typhimurium strains. Under the conditions tested, the test article is not mutagenic in the Ames test.

 

Mammalian Mutagenicity

As no data for the test substance is available regarding mutagenicity in mammalian cells a read-across to the category member CAS 4948-15-6 was performed.

The test substance was evaluated for genotoxic potential in a HPRT locus assay using CHO cells according to OECD TG 476 with GLP compliance (BASF, 2020). In one experiment a dose range from 0.05 to 5 µg/ml was tested, both with and without the addition of liver S9 mix from phenobarbital and β-naphthoflavone induced rats. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without or after the addition of the metabolizing system (S9 mix). No cytotoxicity could be observed, but a precipitation of the test materials was seen after 4 h exposure at dose 1.54 and above. Overall, the test item was considered to be non-mutagenetic under the conditions of the test.

 

Chromosomal Damage

The test article was studied in a GLP-compliant in vitro mammalian chromosome aberration test with Chinese hamster ovary cells (CHO) following OECD guideline 473 (CCR, 1989). The treatment interval was 4 hours. Preparation of chromosomes was done 7 hours (1.0 µg/L), 24 hours (0.1, 0.5 and 1.0 µg/L) and 30 hours (1.0 µg/L) after start of treatment with the test article in absence and presence of S9-mix. Treatment of the cells with 1.0 μg/ml reduced only slightly the plating efficiency. Higher concentrations than 1.0 μg/ml precipitated in the culture medium during incubation. The mitotic index was reduced with the highest concentration in the absence and presence of S9-mix. There was no relevant increase in cells with structural aberrations after treatment with the test article at any fixation interval either without or with metabolic activation by S9-mix. Therefore, the substance is considered to be non-mutagenic in this chromosomal aberration test.

 

Further toxicological data of category members:

The test article belongs to the "perylene based organic pigments" category (see attached document for details on category members and for read across justification). Regarding the genetic toxicity, additional reliable data are available for other category members. All of the studies are taken into account for the evaluation and assessment of the toxicity of the test article.

At least one Ames tests per substance is available for all other category members. None of these tests gave any rise to concern for genotoxicity. Consequently, all substances of this category have been regarded as not genotoxic in the bacterial reverse mutation test.

The additional HPRT and CA assays, performed for other category members, each with and without metabolic activation, were also negative and lead no evidence for a mutagenic potential of the test substances.

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 indication of genotoxicity was observed in the Ames test (OECD 471, GLP), the HPRT Test (OECD 476, GLP) and the in vitro chromosome aberration assay (OECD 473, GLP). As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.