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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Pigment Red 220 was not mutagenic in the Ames test (OECD 471, GLP, both in the standard and the Prival setting).

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 26th 2021 - 15th Oct 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
26 Jun 2020 / Prival modification
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Rheinland-Pfalz, Deutschland
Type of assay:
bacterial reverse mutation assay
Target gene:
his, trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:

- source of S9 :
a) liver of 5 adult male Wistar rats induced by a combination of phenobarbital and ß-naphthoflavone
- method of preparation of S9 mix: 24 hours after the last administration, the rats were sacrificed, and the livers were prepared. The livers were weighed, washed and homogenized in KCl solution. After centrifugation of the homogenate, portions of the supernatant (S9 fraction) were stored at -70°C to -80°C. The S9 mix was prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction was thawed at room temperature and 1 part of S9 fraction is mixed with 9 parts of S9 supplement (cofactors). This mixture of both components (S9 mix) was kept on ice until used.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with benzo(a)pyrene.

Cofactors of the rat liver S9 mix
The concentrations of the cofactors in the rat liver S9 mix are:
MgCl2 8 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 15 mM

b) Uninduced hamster liver S9 fraction
- male Syrian golden hamsters (7 - 8 weeks old, liver microsomal fraction )
After centrifugation of the hamster liver 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

Cofactors of the hamster liver S9 mix (reductive S9 mix)
The concentrations of the cofactors in the hamster liver S9 mix are (9):
MgCl2 8.0 mM
KCl 33.0 mM
glucose-6-phosphate 20.0 mM
glucose-6-phosphate dehydrogenase 2.8 units/mL
NADP 4.0 mM
NADH 2.0 mM
FMN 2.0 mM
phosphate buffer (pH 7.4) 15.0 mM

The efficacy of the hamster liver S9 mix is demonstrated by testing the positive control Congo red.
Test concentrations with justification for top dose:
0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
congo red
other: 2-aminoanthracene (2.5 μg/plate or 60 μg/plate; dissolved in DMSO; with S9-mix) and (10 μg/plate, dissolved in DMSO; with hamster S9-mix for all strains). N-methyl-N'-nitro-N-nitrosoguanidine and 9-aminoacridine without S9
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration : triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Fresh cultures of bacteria were grown up to late exponential or early stationary phase of growth (approximately 10^9 cells per mL). These cultures grown overnight were kept in iced water from the beginning of the experiment until the end in order to prevent further growth.
- Test substance added: Standard plate test, Preincubation Test

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: about 20 minutes
- Exposure duration/duration of treatment: 48 – 72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Rationale for test conditions:
The substance contains an azo bond which triggers the Prival modifaction.
Evaluation criteria:
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies compatible with the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used.

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: limit concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: limit concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: limit concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: limit concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: limit concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
other: limit concentration
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no data
- Data on osmolality: no data
- Water solubility: insoluble
- Precipitation and time of the determination: Test substance precipitation was observed at and above 8.33 μg/plate with and without S9 mix.

RANGE-FINDING/SCREENING STUDIES (if applicable):
1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control

STUDY RESULTS
- Concurrent vehicle negative and positive control data : see Tab. 1, 2 and 3

Ames test:
- Signs of toxicity :
A bacteriotoxic effect (decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions at and above 278 μg/plate. The overview is shown in table 6.

- Individual plate counts : see Tab. 1, 2 and 3
- Mean number of revertant colonies per plate and standard deviation : see Tab. 1, 2 and 3

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
see Tab. 4 and 5

Tab. 1A Assay conditions: Standard plate test ( Experiment: 1st_SPT, without metabolic activation)

Strain           Test group       Dose (µg/plate) Mean revertants Standard Deviation Factor  Individual revertant colony counts
TA 1535 DMSO - 16.3 4.0 - 21, 14, 14
Test item 33 17.3 0.6 1.1 17 P, 17 P, 18 P
100 14.0 2.6 0.9 17 P, 13 P, 12 P
333 13.7 3.5 0.8 17 P, 14 P, 10 P
1000 9.0 2.6 0.6 6 P, 10 P, 11 P
2500 7.7 2.5 0.5 8 P, 10 P, 5 P
5000 5.0 1.0 0.3 5 P, 6 P, 4 P
  MNNG 5.0 7963.0 342.5 487.5 7783, 7748, 8358
TA 100 DMSO - 136.3 21.7 - 156, 140, 113
Test item 33 149.7 11.6 1.1 144 P, 163 P, 142 P
100 158.0 11.4 1.2 166 P, 163 P, 145 P
333 158.3 13.0 1.2 145 P, 159 P, 171 P
1000 117.0 6.2 0.9 115 P, 112 P, 124 P
2500 105.3 20.5 0.8 128 P, 100 P, 88 P
5000 61.0 6.6 0.4 60 P, 68 P, 55 P
  MNNG 5.0 5718.0 108.6 41.9 5754, 5596, 5804
TA 1537 DMSO - 11.0 1.0 - 10, 11, 12
Test item 33 14.7 7.6 1.3 18 P, 6 P, 20 P
100 9.0 2.0 0.8 7 P, 11 P, 9 P
333 11.3 2.1 1.0 12 P, 9 P, 13 P
1000 6.0 1.0 0.5 6 P, 7 P, 5 P
2500 6.3 0.6 0.6 6 P, 6 P, 7 P
5000 3.7 2.1 0.3 6 P, 2 P, 3 P
  AAC 100 1653.3 177.8 150.3 1629, 1489, 1842
TA 98 DMSO - 27.7 3.8 - 26, 25, 32
Test item 33 20.7 5.7 0.7 19 P, 27 P, 16 P
100 18.0 3.6 0.7 19 P, 14 P, 21 P
333 21.0 6.0 0.8 15 P, 21 P, 27 P
1000 10.7 1.5 0.4 9 P, 11 P, 12 P
2500 12.3 4.2 0.4 11 P, 17 P, 9 P
5000 8.7 3.2 0.3 11 P, 5 P, 10 P
  NOPD 10 421.7 33.8 15.2 413, 459, 393
E. coli DMSO - 37.3 3.1 - 38, 34, 40
Test item 33 39.7 3.8 1.1 37 P, 38 P, 44 P
100 42.7 6.1 1.1 36 P, 44 P, 48 P
333 32.0 10.4 0.9 38 P, 20 P, 38 P
1000 29.7 18.6 0.8 21 P, 51 P, 17 P
2500 18.3 3.8 0.5 14 P, 21 P, 20 P
5000 12.7 3.2 0.3 14 P, 9 P, 15 P
  4-NQO 5 1235.7 62.9 33.1 1272, 1272, 1163

Key to plate postfix codes

P Precipitation

Tab. 1B Assay conditions: Standard plate test ( Experiment: 1st_SPT, with metabolic activation)

Strain           Test group       Dose (µg/plate) Mean revertants Standard Deviation Factor  Individual revertant colony counts
TA 1535 DMSO - 14.3 2.1 - 15, 16, 12
Test item 33 14.3 2.5 1.0 12 P, 17 P, 14 P
100 12.3 2.3 0.9 11 P, 11 P, 15 P
333 10.0 1.7 0.7 9 P, 12 P, 9 P
1000 8.3 0.6 0.6 8 P, 9 P, 8 P
2500 8.0 2.0 0.6 10 P, 8 P, 6 P
5000 6.3 1.5 0.4 6 P, 8 P, 5 P
  2-AA 2.5 317.3 38.1 22.1 320, 354, 278
TA 100 DMSO - 130.7 23.9 - 120, 158, 114
Test item 33 152.0 8.9 1.2 142 P, 155 P, 159 P
100 163.0 13.7 1.2 148 P, 166 P, 175 P
333 154.7 7.1 1.2 156 P, 147 P, 161 P
1000 148.0 2.0 1.1 148 P, 146 P, 150 P
2500 107.3 13.6 0.8 112 P, 118 P, 92 P
5000 76.0 6.1 0.6 79 P, 80 P, 69 P
  2-AA 2.5 2920.0 166.2 22.3 2759, 3091, 2910
TA 1537 DMSO - 9.3 0.6 - 9, 10, 9
Test item 33 12.7 3.5 1.4 13 P, 9 P, 16 P
100 12.3 5.1 1.3 18 P, 11 P, 8 P
333 9.0 1.0 1.0 9 P, 10 P, 8 P
1000 5.3 0.6 0.6 5 P, 6 P, 5 P
2500 2.0 1.0 0.2 1 P, 3 P, 2 P
5000 2.0 0.0 0.2 2 P, 2 P, 2 P
  2-AA 2.5 159.3 11.2 17.1 151, 155, 172
TA 98 DMSO - 24.0 1.0 - 23, 24, 25
Test item 33 32.7 5.5 1.4 39 P, 30 P, 29 P
100 27.0 5.6 1.1 32 P, 21 P, 28 P
333 24.3 6.7 1.0 32 P, 21 P, 20 P
1000 12.7 4.0 0.5 17 P, 12 P, 9 P
2500 9.7 3.2 0.4 6 P, 11 P, 12 P
5000 9.0 4.6 0.4 8 P, 14 P, 5 P
  2-AA 2.5 2129.7 138.1 88.7 2137, 2264, 1988
E. coli DMSO - 34.3 4.0 - 32, 39, 32
Test item 33 38.7 4.6 1.1 36 P, 36 P, 44 P
100 34.0 0.0 1.0 34 P, 34 P, 34 P
333 30.7 10.0 0.9 27 P, 42 P, 23 P
1000 25.7 5.0 0.7 31 P, 21 P, 25 P
2500 17.3 4.0 0.5 18 P, 21 P, 13 P
5000 14.3 1.2 0.4 15 P, 13 P, 15 P
  2-AA 60 155.0 27.6 4.5 152, 129, 184

Key to plate postfix codes

P Precipitation

 

Tab. 2A: Assay conditions: Standard plate test ( Experiment: 2nd-SPT, without metabolic activation)

Strain           Test group       Dose (µg/plate) Mean revertants Standard Deviation Factor  Individual revertant colony counts
TA 1535 DMSO - 10.3 2.1 - 8, 11, 12
Test item 8.33 11.0 1.0 1.1 10 P, 12 P, 11 P
27.8 11.0 2.6 1.1 8 P, 12 P, 13 P
83.3 11.0 3.0 1.1 11 P, 14 P, 8 P
278 9.3 1.5 0.9 8 P, 9 P, 11 P
833 8.0 1.7 0.8 9 P, 9 P, 6 P
2500 5.0 2.0 0.5 3 P, 5 P, 7 P
  MNNG 5.0 2962.0 426.3 286.6 3379, 2527, 2980
TA 100 DMSO - 100.7 12.2 - 114, 98, 90
Test item 8.33 128.0 13.1 1.3 140 P, 114 P, 130 P
27.8 116.3 11.7 1.2 114 P, 106 P, 129 P
83.3 111.7 5.9 1.1 116 P, 114 P, 105 P
278 104.3 6.5 1.0 111 P, 104 P, 98 P
833 70.7 9.5 0.7 60 P, 78 P, 74 P
2500 78.0 17.8 0.8 64 P, 98 P, 72 P
  MNNG 5.0 1473.5 256.7 14.6 1655, 1292, - T
TA 1537 DMSO - 7.0 1.0 - 6, 7, 8
Test item 8.33 6.7 3.1 1.0 6 P, 4 P, 10 P
27.8 4.7 2.1 0.7 4 P, 3 P, 7 P
83.3 5.7 1.5 0.8 6 P, 7 P, 4 P
278 4.7 2.1 0.7 4 P, 3 P, 7 P
833 2.3 0.6 0.3 3 P, 2 P, 2 P
2500 1.7 1.2 0.2 1 P, 3 P, 1 P
  AAC 100 2064.0 663.8 294.9 2828, 1736, 1628
TA 98 DMSO - 10.7 2.1 - 10, 9, 13
Test item 8.33 15.0 2.0 1.4 17 P, 13 P, 15 P
27.8 14.7 2.5 1.4 17 P, 12 P, 15 P
83.3 10.7 4.0 1.0 7 P, 10 P, 15 P
278 13.7 2.3 1.3 15 P, 11 P, 15 P
833 11.0 2.6 1.0 13 P, 12 P, 8 P
2500 7.7 1.2 0.7 7 P, 9 P, 7 P
  NOPD 10 475.0 1.7 44.5 474, 474, 477
E. coli DMSO - 30.3 3.2 - 34, 28, 29
Test item 8.33 23.0 3.6 0.8 22 P, 27 P, 20 P
27.8 19.7 2.1 0.6 22 P, 19 P, 18 P
83.3 22.0 5.6 0.7 16 P, 27 P, 23 P
278 19.3 5.5 0.6 19 P, 25 P, 14 P
833 21.3 4.9 0.7 19 P, 27 P, 18 P
2500 19.7 4.5 0.6 24 P, 15 P, 20 P
  4-NQO 5 256.0 46.5 8.4 264, 206, 298

Key to plate postfix codes

P Precipitation

T Technical fault

 

 

Tab. 2B: Assay conditions: Standard plate test ( Experiment: 2nd-SPT, with metabolic activation)

Strain           Test group       Dose (µg/plate) Mean revertants Standard Deviation Factor  Individual revertant colony counts
TA 1535 DMSO - 11.0 2.6 - 10, 14, 9
Test item 8.33 9.7 0.6 0.9 10 P, 10 P, 9 P
27.8 13.3 0.6 1.2 13 P, 14 P, 13 P
83.3 10.0 2.6 0.9 9 P, 8 P, 13 P
278 9.7 3.5 0.9 6 P, 13 P, 10 P
833 6.7 2.9 0.6 5 P, 10 P, 5 P
2500 6.3 3.1 0.6 3 P, 9 P, 7 P
  2-AA 10 360.7 127.3 32.8 214, 442, 426
TA 100 DMSO - 100.7 20.5 - 77, 112, 113
Test item 8.33 119.3 8.6 1.2 121 P, 110 P, 127 P
27.8 105.0 16.0 1.0 89 P, 121 P, 105 P
83.3 106.7 19.1 1.1 114 P, 121 P, 85 P
278 102.3 10.8 1.0 110 P, 90 P, 107 P
833 99.3 3.1 1.0 100 P, 96 P, 102 P
2500 83.3 2.3 0.8 86 P, 82 P, 82 P
  2-AA 10 954.7 174.0 9.5 1098, 761, 1005
TA 1537 DMSO - 7.3 2.5 - 7, 10, 5
Test item 8.33 9.3 1.5 1.3 9 P, 8 P, 11 P
27.8 9.3 2.5 1.3 7 P, 9 P, 12 P
83.3 10.0 3.6 1.4 6 P, 13 P, 11 P
278 7.0 1.7 1.0 9 P, 6 P, 6 P
833 11.0 1.7 1.5 9 P, 12 P, 12 P
2500 4.0 1.7 0.5 3 P, 3 P, 6 P
  2-AA 10 89.7 80.8 12.2 182, 32, 55
TA 98 DMSO - 23.3 4.5 - 23, 28, 19
Test item 8.33 19.7 3.1 0.8 23 P, 17 P, 19 P
27.8 27.3 1.2 1.2 26 P, 28 P, 28 P
83.3 20.0 3.5 0.9 24 P, 18 P, 18 P
278 19.3 5.5 0.8 14 P, 25 P, 19 P
833 18.0 3.0 0.8 15 P, 21 P, 18 P
2500 14.7 2.1 0.6 13 P, 17 P, 14 P
2-AA 10 573.7 77.2 24.6 500, 567, 654
  CoR 210 735.0 124.6 31.5 740 P, 857 P, 608 P
E. coli DMSO - 27.3 3.1 - 24, 28, 30
Test item 8.33 22.3 4.0 0.8 20 P, 27 P, 20 P
27.8 18.0 1.0 0.7 19 P, 17 P, 18 P
83.3 18.3 3.1 0.7 21 P, 15 P, 19 P
278 22.7 4.0 0.8 27 P, 19 P, 22 P
833 17.3 8.3 0.6 20 P, 8 P, 24 P
2500 14.7 3.2 0.5 11 P, 17 P, 16 P
  2-AA 10 317.0 146.2 11.6 255, 212, 484

Key to plate postfix codes

P Precipitation

Tab. 3A: Assay conditions: Preincubation test ( Experiment: 3rd-PIT, without metabolic activation)

Strain           Test group       Dose (µg/plate) Mean revertants Standard Deviation Factor  Individual revertant colony counts
TA 100 DMSO - 116.3 15.9 - 126, 125, 98
Test item 33 114.3 15.4 1.0 132 P, 104 P, 107 P
100 106.3 15.8 0.9 89 P, 120 P, 110 P
333 119.7 16.1 1.0 138 P, 113 P, 108 P
1000 112.7 5.7 1.0 111 P, 108 P, 119 P
2500 108.7 11.0 0.9 96 P, 116 P, 114 P
5000 99.3 9.0 0.9 90 P, 108 P, 100 P
  MNNG 5.0 2082.0 123.0 17.9 2223, 2026, 1997
TA 98 DMSO - 20.3 2.9 - 17, 22, 22
Test item 33 21.3 5.0 1.0 16 P, 22 P, 26 P
100 15.7 4.0 0.8 15 P, 12 P, 20 P
333 12.7 3.2 0.6 9 P, 14 P, 15 P
1000 18.7 2.5 0.9 21 P, 19 P, 16 P
2500 12.7 3.2 0.6 14 P, 9 P, 15 P
5000 5.7 3.8 0.3 4 P, 3 P, 10 P
  NOPD 10 498.0 9.8 24.5 490, 509, 495

Key to plate postfix codes

P Precipitation

Tab. 3B: Assay conditions: Preincubation test ( Experiment: 3rd-PIT, with metabolic activation)

 

Strain           Test group       Dose (µg/plate) Mean revertants Standard Deviation Factor  Individual revertant colony counts
TA 100 DMSO - 113.3 12.1 - 112, 126, 102
Test item 33 111.3 10.3 1.0 114 P, 100 P, 120 P
100 122.0 7.2 1.1 124 P, 114 P, 128 P
333 125.3 9.2 1.1 120 P, 136 P, 120 P
1000 117.3 14.2 1.0 120 P, 102 P, 130 P
2500 105.3 7.0 0.9 106 P, 112 P, 98 P
5000 99.3 10.1 0.9 98 P, 110 P, 90 P
  2-AA 10 2886.7 622.2 25.5 2369, 3577, 2714

Key to plate postfix codes

P Precipitation

Tab. 4: Historical Negative Controls

Strain                        S9 Mix

 Vehicle

   No. of
    
Plates

   No. of

   Values

  Min

    Max

   Mean

     SD

 

TA 1535                    Without

(All)

222

84

8

19

13

2.3

With

(All)

222

84

7

19

12

2.3

 

 

 

 

 

 

 

 

TA 100                       Without

(All)

231

84

18

133

110

14.3

With

(All)

231

86

82

145

110

12.0

 

 

 

 

 

 

 

 

TA 1537                     Without

(All)

228

84

5

15

10

2.1

With

(All)

225

84

6

16

10

1.9

 

 

 

 

 

 

 

 

TA 98                         Without

(All)

228

85

14

30

20

3.4

With

(All)

234

85

16

37

26

4.4

 

 

 

 

 

 

 

 

E. coli                         Without

(All)

216

83

13

40

27

4.7

With

(All)

216

83

17

39

27

3.7

Historical negative control
(Prival modification, 2004 - 2020)
Strain S9 Mix
(3:7)
Vehicle No. of
Plates
No. of
Values
Min Max Mean SD
                 
TA 1535 with (all) 78 26 8 18 14 3.5
                 
TA 100 with (all) 84 28 38 138 105 21.6
                 
TA 1537 with (all) 84 28 6 18 10 3.1
                 
TA 98 with (all) 84 28 23 49 33 6.4
                 
E.coli with (all) 75 25 14 75 37 16.2

Tab. 5: Historical Positive Control

Strain

S9 Mix

Positive

No. of

No. of

Min

Max

Mean

SD

 

 

control

Plates

Values

 

 

 

 

TA 1535                     Without

MNNG

180

66

1517

6912

4122

1569.6

With

2-AA

180

66

97

421

207

76.5

 

 

 

 

 

 

 

 

TA 100                       Without

MNNG

183

66

573

6005

3058

1305.3

With

2-AA

186

68

816

3693

1826

687.5

 

 

 

 

 

 

 

 

TA 1537                    Without

AAC

183

66

187

1400

829

198.7

With

2-AA

180

66

66

270

151

50.7

 

 

 

 

 

 

 

 

TA 98                        Without

NOPD

186

67

531

1148

857

109.2

With

2-AA

192

67

392

2791

1457

641.4

 

 

 

 

 

 

 

 

E. coli                        Without

4-NQO

174

66

348

1638

1090

408.3

With

2-AA

171

66

81

278

215

39.2

 

Historical positive control
(Prival modification, 2004 - 2020)           
Strain S9 Mix
(3:7)
Positive
control
No. of
Plates
No. of
Values
Min Max Mean SD
                 
TA 1535 with 2-AA 72 24 100 918 298 234.4
                 
TA 100 with 2-AA 78 26 361 2835 1132 590.1
                 
TA 1537 with 2-AA 78 26 70 453 154 77.9
                 
TA 98 with 2-AA 78 26 355 3545 979 656.3
    CoR 78 26 251 852 524 153.0
                 
E.coli with 2-AA 69 23 165 1879 645 524.2

Table 6: Indicators of toxicity: decreased revertant numbers were observed at following concentrations (μg/plate):

Experiment S9 TA 1535 TA 100 TA 1537 TA 98 E.coli
1st-SPT Without 1000 – 5000 5000 1000 – 5000 1000 – 5000 2500 – 5000
With 1000 – 5000 5000 1000 – 5000 1000 – 5000 2500 – 5000
2nd-Prival Without 2500 - 833 – 2500 - 278; 2500
With 833 – 2500 - 2500 2500 833 – 2500
3rd-Prival Without n.t. - n.t. 2500 – 5000 n.t.
With n.t. - n.t. n.t. n.t.
Conclusions:
The test substance was not mutagenic in the Ames test (including Prival modification).

Additional information

Ames tests

The GLP compliant study was performed using the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 following OECD testing guideline 471 (Ciba-Geigy 1980).

The test was performed with and without liver microsomal activation. The test material was tested at the following concentrations: 1.58, 5, 15.8, 50, 150,500,1580 and 5000 micrograms per plate. No toxic effect of the test material was observed.

Up to the highest investigated dose, no relevant increase of the revertant colony numbers was obtained in any Salmonella typhimurium strain used when compared with the corresponding controls. The presence of microsomal activation did not influence these findings. Positive control incubations confirmed the validity of the study. Pigment Brown was found to be non mutagenic.

In 2021, Pigment Red 220 was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (Ames standard plate test and Prival preincubation test) (BASF 2021, GLP, OECD 471). The modified Bacterial Reverse Mutation Test according to Prival facilitates azo reduction and is therefore the most appropriate method for the investigation of azo-dyes and diazo compounds. The strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA were used. The dose ranges were 33 μg - 5000 μg/plate (SPT) and 8.33 μg - 5000 μg/plate (Prival), respectively. Precipitation of the test substance was observed at and above 8.33 μg/plate with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions at and above 278 μg/plate. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the prival preincubation test without S9 mix or after the addition of a metabolizing system.

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. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008.