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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames-Test: negative, according to OECD TG 471, no GLP, Salmonella typhimurium strains: TA 98, TA 100, TA 1535 and TA 1537, with and without metabolic activation, 1991, K2


 


HPRT: Read-across, negative, according to OECD TG 476, GLP compliant, Chinese hamster ovary (CHO), with and without metabolic activation, 2020, K1


Chromosomal Damage: Read-across, negative, according to OECD TG 473, GLP compliant, Chinese hamster lung fibroblasts (V79), with and without metabolic activation, 2006, K1

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Non-GLP and no E.coli strain tested, otherweise fully adequate for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Analytical purity: 100%
- Storage condition of test material: room temperature
- Lot/batch No.: 90-025
Target gene:
his+
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix
Test concentrations with justification for top dose:
0, 20, 100, 500, 2500 and 5000 µg/plate (standard plate test and preincubation test)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9 mix; all strains
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; TA 1535 and TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitri-o-phenylenediamine
Remarks:
without S9 mix; TA 98
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; TA 1537
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: 20 minutes at 37°C
- Exposure duration: ca. 48 hours at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
reduced his background growth
Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test 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 and TA 100
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:
Toxicity
No bacteriotoxic effect (reduced his background growth) was observed.

Solubility
Incomplete solubility of test substance in DMSO was observed from about 100 µg/plate onward.

Standard Plate Test:

TA 1535 TA 100 TA 1537 TA 98
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
0 18 16 95 108 9 19 24 39
20 16 19 99 118 10 15 24 35
100 20 21 111 112 10 12 24 41
500 23 19 80 82 10 13 17 37
2500 20 20 88 85 8 10 13 39
5000 25 19 77 85 7 11 22 38
2-AA 281 1250 283 1474
MNNG 2423 1508
AAC 488
NPD 1328

Preincubation Test:

TA 1535 TA 100 TA 1537 TA 98
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
0 14 14 124 123 11 12 28 42
20 16 18 111 86 11 12 25 31
100 19 18 118 94 11 14 19 29
500 19 16 85 79 10 14 17 30
2500 14 20 83 76 9 12 16 28
5000 14 14 75 81 7 10 14 31
2-AA 192 1161 123 794
MNNG 959 995
AAC 493
NPD 855

Controls:

2-AA: 2 aminoanthracene (10 µg/plate)

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

AAC: aminoacridine (100 µg/plate)

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

Conclusions:
Under the conditions chosen, the test substance was not mutagenic in the reverse bacteria mutation assay.
Executive summary:

The test article was tested in the Ames reverse mutation assay (according to OECD guideline 471, no GLP) using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 at 20 to 5000 µg/plate in the standard plate test and in the preincubation test (vehicle: DMSO) with and without metabolic activation. An increase in the number of his+ revertants was not observed in the standard plate test or in the preincubation test either without S-9mix or after the addition of S-9 mix. No bacteriotoxic effect (reduced his background growth) was observed. Incomplete solubility of test substance in DMSO was observed from about 100 µg/plate onward. Under the conditions tested, the test substance does not induce point mutations in bacteria.

Endpoint:
in vitro cytogenicity / chromosome aberration 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 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:
not applicable
Positive controls validity:
valid
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Oct 2005 - 13 Apr 2006
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:
(1997)
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Physical state: Solid, black powder
- Analytical purity: > 99%
- Storage condition of test material: Room temperature
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM medium with glutamine supplemented with − 10% (v/v) fetal calf serum (FCS),
− 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL)
− 1% (v/v) amphotericin B (250 μg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for plating efficiency (= colony forming ability) incl. vital staining: yes
Metabolic activation:
with and without
Metabolic activation system:
The S-9 fraction was prepared according to AMES et al. (Mut. Res. 31, 347-364, 1975) from the livers of at least 5 male Sprague-Dawley rats that had received a single ip injection of 500 mg Aroclor 1254/ kg bw 5 days earlier.
Test concentrations with justification for top dose:
first experiment:
4-hour exposure, 18-hour sampling time, without S-9 mix: 0; 1.56; 3.13; 6.25; 12.5; 25.0; 50.0; 75.0; 100.0 μg/mL
4-hour exposure, 18-hour sampling time, with S-9 mix: 0; 1.56; 3.13; 6.25; 12.5; 25.0; 50.0; 75.0; 100.0 μg/mL

Dose selection was based on the solubility of the test substance, i.e. doses > 12.5 μg/mL both with and without S-9 mix led to strong precipitation which interferes with evaluation of metaphases.

second experiment:
18-hour exposure, 18-hour sampling time, without S-9 mix: 0; 0.78; 1.56; 3.13; 6.25; 12.5; 25.0 μg/mL
18-hour exposure, 28-hour sampling time, without S-9 mix: 0; 3.13; 6.25; 12.5 μg/mL
4-hour exposure, 28-hour sampling time, with S-9 mix: 0; 1.56; 3.13; 6.25; 12.5; 25.0 μg/mL

Again doses ≥ 12.5 μg/mL led to strong test substance precipitation which interferes with the evaluation of metaphases.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was considered the most suitable. Therefore, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the V79 in vitro cytogenetic test and for which historical control data are available.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S-9: 500 µg/mL ethyl methanesulfonate (EMS); With S-9: 500 µg/mL cyclophosphamide (CPP)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium in Quadriperm dishes

DURATION
- Preincubation period: 24 - 30 hrs
- Exposure duration: 4 or 18 hrs

- Expression time (cells in growth medium): 14 hrs or 24 hrs; after continuous treatment, i.e. 18 hours without S-9 mix, cells were treated in culture medium supplemented with 10% FCS and in the case of a sampling time of 28 hrs incubated again for another 10 hours.

- Fixation time (start of exposure up to fixation or harvest of cells): 2 - 3 hours prior to harvesting the cells, 0.2 μg Colcemid/mL culture medium was added to each chamber in order to arrest mitosis in the metaphase. For hypotonic treatment, 5 mL of a 0.4% KCl solution which was at 37°C was added for about 20 min. Subsequently, 5 mL of fixative (methanol : glacial acetic acid/3 : 1) which was added at 4°C and kept for at least 15 min and then replaced. After about another 10 min, the fixative was replaced again and kept for at least 5 min at room temperature for complete fixation.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): solution of Giemsa and Titrisol (15 mL Giemsa, 185 mL Titrisol pH 7.2) for 10 minutes

NUMBER OF REPLICATIONS: 2 chambers of Quadriperm dishes were used per test culture.

NUMBER OF CELLS EVALUATED: 200 metaphases evaluated

CELL MORPHOLOGY
About 3 hours and 16 - 18 hours after test substance treatment, cultures of all test groups were checked for cell morphology, which is an indication of attachment of the cells to the slides.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
A mitotic index based on 1 000 cells/culture was determined for all evaluated test groups in both experiments.
For the determination of cytotoxicity, additional cell cultures (using 25 cm2 plastic flasks) were treated in the same way as in the main experiment. Growth inhibition was estimated by counting the number of cells in the dose groups in comparison with the concurrent vehicle control at the end of the culture period using a counter.

CELLCYCLE
The cell cycle of the untreated V79 cells lasted for about 13 - 14 hours under the selected culture conditions. Thus, the selected 1st sampling time of 18 hours was within the 1 - 1.5 x the normal cell cycle time, as recommended by the OECD TG 473. The later sampling time of 28 hours was chosen to cover a possible cell cycle delay.
Evaluation criteria:
If there is a clear increase in chromosomally damaged cells, the number of metaphases to be analyzed is reduced from the planned 200 mitoses/test group.
For details concerning evaluation criteria, please refer to "Any other information on materials and methods incl. tables".

Assessment criteria
The test chemical was assessed as “positive” in this assay if the following criteria were met:
• A dose-related and reproducible significant increase in the number of cells with structural / numerical chromosomal aberrations.
• The number of aberrant cells exceeded both the concurrent negative control range and the highest value of the negative historical control range.

A test substance generally was considered as “negative” if the following criteria were met:
• The number of cells with structural / numerical aberrations in the dose groups was not significantly above the concurrent negative control and was within the historical control data.
Statistics:
The proportion of metaphases with aberrations was calculated for each group.
A comparison of each dose group with the vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test were significant, labels (* p < 0.05, ** p < 0.01) are printed in the tables.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At concentrations of 25 µg/mL and above, evaluation was not possible due to strong test substance precipitation which interferes with metaphase evaluation.

Mitotic index:
According to the results of the determination of the mitotic index, no suppression of the mitotic activity was observed under any of the experimental conditions.

Cell counts:
According to the results of the cell count, no growth inhibition was observed under any of the experimental conditions.

Cell morphology:
Cell attachment was not influenced at any dose evaluated for structural chromosomal aberrations.

CHROMOSOME ANALYSIS - 1ST EXPERIMENT

 

- Clastogenic mode of action: After a treatment time of 4 hours no increase in the number of chromosomally damaged cells was observed either without S-9 mix or after the addition of a metabolizing system.

- Aneugenic mode of action: No increase in the number of cells with changes in the number of chromosomes was demonstrated either without S-9 mix or after the addition of a metabolizing system.

 

 

CHROMOSOME ANALYSIS - 2ND EXPERIMENT

 

- Clastogenic mode of action: Both with and without S-9 mix, no increase in the number of structurally damaged metaphases was observed either after a treatment time of 4 hours or after a continuous treatment of 18 hours at both sampling times, i.e. 18 hours and 28 hours.

- Aneugenic mode of action: No increase in the number of cells with changes in the number of chromosomes was demonstrated either without S-9 mix or after the addition of a metabolizing system.

 

SUMMARY TABLE

Metaphases with Aberrations
Incl Gaps Exc. Gaps
Exposure Time Sampling Time Dose (µg/ml) S9-Mix number of metaphases n % n %
4 h 18 h Vehicle - 200 11 5.5 4 2
4 h 18 h 3.13 - 200 10 5 5 2.5
4 h 18 h 6.25 - 200 7 3.5 2 1
4 h 18 h 12.5 - 200 5 2.5 4 2
4 h 18 h EMS 500 - 100 19 19 15 15
4 h 18 h Vehicle + 200 9 4.5 3 1.5
4 h 18 h 3.13 + 200 12 6 7 3.5
4 h 18 h 6.25 + 200 15 7.5 4 2
4 h 18 h 12.5 + 200 8 4 3 1.5
4 h 18 h CPP 0.5  + 100 31 31 26 26
18 h 18 h Vehicle - 200 6 3 2 1
18 h 18 h 3.13 - 200 13 6.5 7 3.5
18 h 18 h 6.25 - 200 6 3 2 1
18 h 18 h 12.5 - 200 4 2 0 0
18 h 18 h EMS 500 - 100 19 19 19 19
18 h 28 h Vehicle - 200 9 4.5 8 4
18 h 28 h 12.5 - 200 6 3 3 1.5
18 h 28 h EMS 500 - 100 22 22 19 19
4 h 28 h Vehicle + 200 7 3.5 3 1.5
4 h 28 h 3.13 + 200 8 4 4 2
4 h 28 h 6.25 + 200 11 5.5 9 4.5
4 h 28 h 12.5 + 200 7 3.5 4 2
4 h 28 h CPP 0.5  + 1000 17 17 15 15

EMS = ethyl methanesulfonate

CPP = cyclophosphamide

 

Discussion and conclusion:

 

According to the results of the present in vitro cytogenetic study, the test substance did not lead to an increase in the number of structural chromosomal aberrations incl. and excl. gaps either without S-9 mix or after the addition of a metabolizing system in two experiments performed independently of each other selecting different exposure times (4 hours treatment and continuous treatment) and sampling times (18 and 28 hours); the types and frequencies of aberrations were close to the range of that of the concurrent negative control values at both sampling times and in the range of the historical control data.

No increase in the number of cells containing numerical chromosomal aberrations was demonstrated either.

The increase in the frequencies of chromosomal aberrations induced by the positive control agents EMS and CPP clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S-9 mix employed.

 

Thus, under the experimental conditions chosen here, the conclusion is drawn that the test article is neither a clastogenic (chromosome-damaging) nor an aneugenic agent under in vitro conditions using V79 cells.

Conclusions:
Under the experimental conditions chosen here, the conclusion is drawn that the test article is neither a clastogenic (chromosome-damaging) nor an aneugenic agent under in vitro conditions using V79 cells.
Executive summary:

In an GLP and OECD guideline compliant study (No. 473) the test substance was assessed for its potential to induce structural chromosomal aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in V79 cells in vitro both in the presence and in the absence of a metabolizing system. According to an initial range-finding cytotoxicity test for the determination of the highest experimental doses, the test substance did not exhibit any pronounced toxicity up to the highest possible dose, i.e. 3 000 μg/mL, at which distinct test substance precipitation was observed. However, due to strong test substance precipitation, which interfers with metaphase evaluation, lower doses must be selected and the test groups in bold type were evaluated:

• 1st experiment

4-hour exposure, 18-hour sampling time, without S-9 mix

0; 1.56;3.13; 6.25; 12.5; 25.0; 50.0; 75.0; 100.0 μg/mL

4-hour exposure, 18-hour sampling time, with S-9 mix

0; 1.56;3.13; 6.25; 12.5; 25.0; 50.0; 75.0; 100.0 μg/mL

• 2nd experiment

18-hour exposure, 18-hour sampling time, without S-9 mix

0; 0.78; 1.56;3.13; 6.25; 12.5; 25.0 μg/mL

18-hour exposure, 28-hour sampling time, without S-9 mix

0; 3.13; 6.25;12.5μg/mL

4-hour exposure, 28-hour sampling time, with S-9 mix

0; 1.56;3.13; 6.25; 12.5; 25.0 μg/mL

Doses > 12.5 μg/mL both with and without S-9 mix led to strong precipitation which interferes with evaluation of metaphases. About 2 - 3 hours prior to harvesting the cells, Colcemid was added to arrest cells at a metaphase-like stage of mitosis (c-metaphases). After preparation of the chromosomes and staining with Giemsa, 100 metaphases for each culture in the case of the test substance and vehicle controls, or 50 cells for each culture in the case of the concurrent positive controls, were analyzed for chromosomal aberrations. The negative controls (vehicle controls) gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive control chemicals, i.e. EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. On the basis of the results of the present study, the test substance did not cause any increase in the number of structurally aberrant metaphases incl. and excl. gaps at both sampling times either without S-9 mix or after adding a metabolizing system in two experiments performed independently of each other. No increase in the frequency of cells containing numerical aberrations was demonstrated either. Thus, under the experimental conditions of this assay, the test material is considered not to be either a clastogenic or an aneugenic agent under in vitro conditions in V79 cells.

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
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 106 cells]



RS [%]



CE[%]



1



4


VC1

-



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



 



 


PC2

-



n.d



42.12s



113.2



102.5



1



4


VC1

+



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



 



 


PC3

+



n.d.



48.52s



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.

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 article was tested in the Ames reverse mutation assay (according to OECD guideline 471, no GLP) using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 at 20 to 5000 µg/plate in the standard plate test and in the preincubation test (vehicle: DMSO) with and without metabolic activation (BASF AG, 1991). An increase in the number of his+ revertants was not observed in the standard plate test or in the preincubation test either without S-9mix or after the addition of S-9 mix. No bacteriotoxic effect (reduced his background growth) was observed. Incomplete solubility of test substance in DMSO was observed from about 100 µg/plate onward. Under the conditions tested, the test substance does not induce point mutations in bacteria.


In a second, supporting Ames reverse mutation assay (study comparable to OECD guideline 471, no GLP) Pigment Red 179 was tested using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 and Escherichia coli WP2 uvr A with and without metabolic activation using the plate incorporation test (Hoechst AG, 1984). The test compound was tested at doses of 4 to 10000 µg/plate (vehicle: DMSO) and proved to be not toxic to the bacterial strains at doses of 5000 µg/plate. For mutagenicity testing 4 to 5000 µg/plate was chosen as concentration range. Visible precipitation of the test compound on the plates has been observed at 500 µg/plate. 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


As no data for the test substance is available regarding chromosomal damage a read-across to the category member EC 479-300-2 was performed.


An in vitro chromosome aberration assay was conducted in V79 cells according to OECD TG 473 and GLP regulations (BASF, 2006). The substance was assessed for its potential to induce structural chromosomal aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in V79 cells both in the presence and in the absence of a metabolizing system (Aroclor-induced rat liver S-9 mix). The study comprised two independent experiments. In the first experiment, an exposure time of 4 hours and a sampling time of 18 hours were employed. Doses were between 1.56 µg/mL and 100 µg/mL, with and without S-9 mix. However, due to strong test substance precipitation, which interfers with metaphase evaluation, lower doses were selected for evaluation (3.13; 6.25; 12.5 µg/mL).


The second experiment comprised three assays:



  1. 18-hour exposure, 18-hour sampling time, without S-9 mix; doses were 0.78 – 25 µg/mL.

  2. 18-hour exposure, 28-hour sampling time, without S-9 mix; doses were 3.13 – 12.5 µg/mL.

  3. 4-hour exposure, 28-hour sampling time, with S-9 mix; doses were 1.56 – 25 µg/mL.


Again doses ≥ 12.5 μg/mL led to strong test substance precipitation which could not be evaluated for chromosome aberrations. Both of the positive control chemicals, i.e. ethyl methanesulfonate and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. On the basis of the results of the present study, the test substance did not cause any increase in the number of structurally aberrant metaphases incl. and excl. gaps at both sampling times either without S-9 mix or after adding a metabolizing system in two experiments performed independently of each other. No increase in the frequency of cells containing numerical aberrations was demonstrated either. Thus, under the experimental conditions of this assay, the substance is considered not to be either a clastogenic or an aneugenic agent under in vitro conditions in V79 cells.


 


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.