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EC number: 269-507-4 | CAS number: 68259-05-2
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Genetic toxicity: in vitro
Administrative data
- 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
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 021
- Report date:
- 2021
Materials and methods
Test guideline
- 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
Test material
- Reference substance name:
- Bis(2-chloroethyl) 3,3'-[(2,5-dimethyl-p-phenylene)bis[iminocarbonyl(2-hydroxy-1,3-naphthylene)azo]]di-p-toluate
- EC Number:
- 269-507-4
- EC Name:
- Bis(2-chloroethyl) 3,3'-[(2,5-dimethyl-p-phenylene)bis[iminocarbonyl(2-hydroxy-1,3-naphthylene)azo]]di-p-toluate
- Cas Number:
- 68259-05-2
- Molecular formula:
- C50 H42 Cl2 N6 O8
- IUPAC Name:
- 2-chloroethyl 2-[(2Z)-2-[3-[[4-[[(4E)-4-[[5-(2-chloroethoxycarbonyl)-2-methylphenyl]hydrazinylidene]-3-oxonaphthalene-2-carbonyl]amino]-2,5-dimethylphenyl]carbamoyl]-2-oxonaphthalen-1-ylidene]hydrazinyl]-3-methylbenzoate
- Test material form:
- solid: particulate/powder
- Details on test material:
- Name: Cromophtal Red K 3830
Batch Nr. 05970HM9
appearance: solid, red
storage at room temperature
water: 0.13 wt%
Constituent 1
Method
- Target gene:
- his, trp
Species / strainopen allclose all
- 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.
Controls
- 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.
Results and discussion
Test resultsopen allclose all
- 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
Any other information on results incl. tables
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 |
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. |
Applicant's summary and conclusion
- Conclusions:
- The test substance was not mutagenic in the Ames test (including Prival modification).
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