Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity: Only bacteria-specific effects were noted in the bacteria reverse mutation assay, whereas the prediction for gene mutation in the OECD QSAR Toolbox and the mutagenicity study in mammalian cells with a structural analogue was negative.

Clastogenicity: The chromosome aberration test in V79 cells was negative with metabolic activation and showed a positive response at a concentration which showed distinct cytotoxicity and visible precipitation. However, the prediction by the OECD QSAR Toolbox and the chromosome aberration test in V79 cells with a structural analogue was negative was clearly negative.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read across is based on the same physico-chemical properties, a close structural similarity and the same mechanism of action during use processes.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
- Source: Disperse Blue 291 Br
- Target: Disperse Red 184

3. ANALOGUE APPROACH JUSTIFICATION
see attachment in endpoint summary

4. DATA MATRIX
see attachment in endpoint summary
Reason / purpose:
read-across source
Principles of method if other than guideline:
Plate incorporation test under oxidative conditions according to Maron and Ames (1983), with modifications, using sterile deionized water as the negative control.
All the experiments were performed in duplicate. The dye was tested under partially anaerobic (reducing) conditions, and the results were compared to the data obtained using the normal oxidative protocol. The assay under reductive conditions was performed according to Prival and Mitchell (1982).
The mutation spectra of CI Disperse Blue 291 were determined using the Salmonella TA7000 series described by Gee et al. (1994) using the microsuspension modification (Kado et al., 1983) under oxidative conditions. Overnight cultures of each strain (around 109 cells/ml) were concentrated 10 fold by centrifugation at 10,000g at 4 °C for 10 min and resuspended in 0.015 M sodium phosphate buffer. 50 µI of cell suspension, 50 µI of 0.015 M sodium phosphate buffer or S9 mix, and 7.5 µI of the test sample were added to a tube and incubated at 37 °C for 90 min without shaking. After incubation, 2 ml of molten agar was added, the mixture was poured onto a minimal agar plate, and the plates were incubated at 37 °C for 72 h. Colonies were counted using an automatic colony counter. When the number of colonies was less than ten, the counts were also confirmed by hand, using a light box with magnification.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
other: TA98 NR
Additional strain / cell type characteristics:
nitroreductase deficient
Species / strain / cell type:
other: TA98 DNP6
Additional strain / cell type characteristics:
acetyltransferase deficient
Species / strain / cell type:
other: YG1021
Additional strain / cell type characteristics:
other: nitroreductase overproducing enzyme activity
Species / strain / cell type:
other: YG1024
Additional strain / cell type characteristics:
acetyltransferase proficient
Species / strain / cell type:
other: YG1041
Additional strain / cell type characteristics:
other: acetyltransferase and nitroreductase overproducing enzyme activity
Species / strain / cell type:
other: TA7001, TA7002, TA7003, TA7004, TA7005, TA7006
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
S. typhimurium TA 98
Metabolic activation:
with and without
Metabolic activation system:
Sprague Dawley rat liver S9 (MolTox, Boone, NC) induced with an Arochlor 1254 mix
Species / strain:
other: TA1537, TA100, TA98, TA1538
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA98NR
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA98NR
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: YG1021, YG1024, YG1041
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA98DNP6
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA98DNP6
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
slight increase
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA7001, TA7002, TA7004, TA7005
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
TA7004, TA7005 at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA7003, TA7006
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
TA7006 at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA7000 series
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
under reductive cleavage conditions, the color of the tested dye did not change when using the modification of Prival and Mitchell (1982), and the mutagenicity remained the same as that seen with normal oxidative metabolism. This suggests that the azobond was not cleaved in the testing conditions.
Conclusions:
The study shows that the mitagenic effect of the test substance is due to bacterial nitroreductase and O-acetyltransferase activity
Executive summary:

Disperse Blue 291 was tested for mutagenic activity in the Salmonella assay with strains with different levels of nitroreductase andO-acetyltransferase (i.e., TA98DNP6, YG1024, and YG1041) as well as standard strains TA 1535, TA1537, TA1538, TA98 and TA100 and strains which provide more information on the base-pair substitution (TA 7001 to 7006). Disperse Blue 291 showed direct-acting mutagenic activity with all strains ofSalmonella typhimuriumtested, except forTA 1535.According to the classification of Claxton et al. (1991), the potency of this product can be considered moderate (10-100 revertants/µg). In the absence of S9, the nitroreduction is strongly related to the mutagenic activity, because the mutagenicity of Disperse Blue 291 was very low when tested with the strains lacking nitroreductase activity (TA98NR) and was increased with the nitroreductase overproducing strains, (YG1021 and YG1041). The same mutagenic pattern was observed for the acetyltransferase deficient and overproducing strains (TA98DNP6, YG1024, and YG1041) revealing also the importance of the acetyltransferase enzyme in the activation of Disperse Blue 291. Because of the remarkable increase in the response with the nitroreductase andO-acetyltransferase overproducing strain (YG1041), it is likely that the product of the nitroreductase is a substrate for theO-acetyltransferase.

In the presence of S9, the mutagenicity was slightly increased with TA98NR, TA98, YG1021, TA98DNP6, and YG1024 suggesting that, P450 enzymes also have a role in the activation of these compounds, besides the bacterial enzymes. This could be explained by the activation of other radicals of the molecule by the S9 enzymes, for example the —OCH3or the —N(CH2CH3)2of the CI Disperse Blue 291.

 

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
other: ETAD TOXICOLOGICAL METHOD NO. 005
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
4, 20, 100, 500, 2500 μg/plate
Vehicle / solvent:
For the purpose of this study the test material was dissolved in Dimethyl sulphoxide.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
other: M-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG); 4 Nitro-O-phenyldiamine (4NOP); 2-aminoanthracene (2AA)
Rationale for test conditions:
In accordance with the test procedures.
Evaluation criteria:
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than two fold at all dose levels employed, the intervals of which should be between 3 and 5 fold and extend to the limits imposed by toxicity or solubility.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The test material was found to be a mutagen at the dose levels employed in this study.
Executive summary:

This study was conducted according to ETAD Test Method 005 and was designed to assess the mutagenic potential of the test material using a bacterial/microsome test system . The study was based on the in vitro technique described by Ames and his co-workers and Garner et al in which mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium to various concentrations of the test compound.

The strains used in this assay were all mutants derived from Salmonella typhimurium and were those recommended for general screening:

TA 1535, TA 100, TA 1537, TA 98

 

In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test compound to the tester organisms. No cytotoxicity was observed at the tested concentrations of 4 to 2500 µg/plate.

Concentrations of 4, 20, 100, 500, and 2500 µg/plate were hence used for the main assay.

The test substance was negative in TA 1535 without metabolic activation and weakly positive in TA 100 and TA 1537 without metabolic activation. A stronger positive reaction was observed in TA 98 without metabolic activation and in all strains with metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
Data predicted by OECD Toolbox v4.0. OECD Toolbox uses a valid estimation method; the substance was found to fall in the applicability domain of this method and results are considered relevant for risk assessment
Justification for type of information:
1. SOFTWARE
QSAR Toolbox

2. MODEL (incl. version number)
QSAR Toolbox 4.0
Database version: 4.0//3.4

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CAS#: 24610-00-2

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: chromosome aberration
see attachment

5. APPLICABILITY DOMAIN
see attachment

6. ADEQUACY OF THE RESULT
the substance lies well within the aplicability domain
Qualifier:
according to
Guideline:
other: REACH guidance on QSARs: Chapter R.6. QSARs and grouping of chemicals
GLP compliance:
no
Type of assay:
other: QSAR prediction
Key result
Species / strain:
not specified
Metabolic activation:
with and without
Genotoxicity:
negative
Additional information on results:
Prediction summary
Predicted endpoint: chromosome aberration; No effect specified; No species specified; No duration specified; No guideline specified
Predicted value: Negative
Unit/scale: Chromosome aberration I (Oasis)
Data gap filling method: Read-across analysis

Calculation approach (OECD principle 2 - Unambiguous algorithm): takes the highest mode value from the nearest 5 neighbours
Active descriptor: log Kow (calculated)
Data usage: All values
Uncertainty of the prediction (OECD principle 4 - Uncertainty of the prediction):
The prediction is based on 5 values, 4 of them (80%) equal to predicted value
Prediction confidence is measured by the p-value: 0.375

Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
Based on the QSAR prediction results, the test substance has no clastogenic properties
Executive summary:

Prediction summary

Predicted endpoint: chromosome aberration; No effect specified; No species specified; No duration specified; No guideline specified

Predicted value: Negative

Unit/scale: Chromosome aberration I (Oasis)

Data gap filling method: Read-across analysis

Calculation approach (OECD principle 2 - Unambiguous algorithm): takes the highest mode value from the nearest 5 neighbours

Active descriptor: log Kow (calculated): 5.94

Data usage: All values

Uncertainty of the prediction (OECD principle 4 - Uncertainty of the prediction):

The prediction is based on 5 values, 4 of them (80%) equal to predicted value

Prediction confidence is measured by the p-value: 0.375

Analogue(s) selection

(OECD principle 3 - Applicability domain)

Database(s) used:

- Carcinogenic Potency Database (CPDB)

- Micronucleus OASIS

- Toxicity Japan MHLW

- Genotoxicity OASIS

- Transgenic Rodent Database

- ECVAM Genotoxicity & Carcinogenicity

- ECHA CHEM

- Bacterial mutagenicity ISSSTY

- Carcinogenicity&mutagenicity ISSCAN

- Micronucleus ISSMIC

Category boundaries (applicability domain):

- Active descriptor(s) range:

- log Kow: from -2.86 to 6.78 target chemical is in domain

- Response range:

- Chromosome aberration: from Negative to Positive

Profilers:

- DNA alerts for CA and MNT by OASIS v.1.1 (primary grouping) target chemical is in domain

Additional data pruning:

none

Manually eliminated data points:

none

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
23 November 1998 to 15 January 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read across is based on the same physico-chemical properties, a close structural similarity and the same mechanism of action during use processes.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
- Source: see attachment in endpoint summary
- Target: Disperse Red 184

3. ANALOGUE APPROACH JUSTIFICATION
see attachment in endpoint summary

4. DATA MATRIX
see attachment in endpoint summary
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Source of cells: cell bank of "Genetic Toxicology", HMR Germany
Test organism: cell line V79 of Chinese hamster lung fibroblasts
Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

MAIN EXPERIMENT TOXICITY TABLES

 

Table 2a (3 hours treatment time without S9-mix)

 

Dose

μg/ml

S9-mix

Extinction in microwell plates mean less blank values

Standard deviation

Relative survival*

Solvent control

0

-

0.485

0.03

100.0

+

0.402

0.03

100.0

Test item

25

-

0.427

0.01

88.1

+

0.376

0.03

93.9

50

-

0.402

0.03

82.9 #

+

0.338

0.02

84.0 #

75

-

0.378

0.03

78.0 #

+

0.340

0.02

84.6 #

125

-

0.343

0.08

70.6 #

+

0.308

0.02

78.8 #

250

-

0.291

0.04

60.0 #

+

0.277

0.02

69.0 #

500

-

0.236

0.02

48.6 #§

+

0.250

0.02

62.2 #§

750

-

0.204

0.03

42.0 #§

+

0.220

0.02

54.7 #§

1285

-

0.318

0.02

65.6 #§

+

0.268

0.02

66.7 #§

 

Table 2b (20 hours treatment time without S9-mix)

 

Doseμg/ml

S9-mix

Extinction in microwell plates mean less blank values

Standard deviation

Relative survival*

Solvent control

0

-

0.489

0.04

100.0

Test item

5

-

0.480

0.06

98.1

 

10

-

0.461

0.02

94.3

 

25

-

0.421

0.02

86.0

 

50

-

0.378

0.02

77.4 #

 

75

-

0.328

0.02

67.1 #

 

125

-

0.248

0.03

50.7 #

 

250

-

0.195

0.03

39.8 #

 

* = relative survival (mean value/mean value corresponding control x 100)

Solvent control = DMSO

# = microscopical visible precipitation of the test compounds

§= macroscopical visible precipitation of the test compounds

 

TABLE OF THE MITOTIC INDEX

First experiment

Doseμg/ml

S9-Mix

Treatment time (h)

Mitotic index

Mean

Relative mitotic index percent*

Test group

1

2

Solvent control DMSO

0.0

-

3

5.1

5.7

5.9

100.0

Test item

125.0

-

3

4.5

4.3

4.4

74.8

500.0

-

3

2.8

4.3

3.6

61.0

1285.0

-

3

4.6

3.2

3.9

66.1

Positive control EMS

1500.0

-

3

5.7

3.3

4.5

76.3

 

Solvent control DMSO

0.0

+

3

7.0

8.2

7.6

100.0

Test item

125.0

+

3

7.3

7.2

7.3

96.1

500.0

+

3

5.4

6.5

6.0

79.0

1

1285.0

+

3

4.3

5.0

4.7

61.8

Positive control CPA

3.0

+

3

4.1

3.8

4.0

52.6

 

Second experiment

Doseμg/ml

S9-mix

Treatment time (h)

Mitotic index

Mean

Relative mitotic index percent*

Test group

1

2

Solvent control DMSO

0.0

-

20

5.0

5.4

5.2

100.0

Test item

25.0

-

20

5.6

4.2

4.9

94.2

75.0

-

20

4.9

4.7

4.8

92.3

250.0

-

20

2.3

2.9

2.6

50.0

Positive control EMS

400.0

-

20

3.1

2.9

3.0

57.7

 

Third experiment

Doseμg/ml

S9-mix

Treatment time (h)

Mitotic index

Mean

Relative mitotic index percent*

Test group

1

2

Solvent control DMSO

0.0

+

3

8.4

9.1

8.8

100.0

Test item

1285.0

+

3

4.0

6.6

 

 

 

 

 

4.8

5.2

5.2

59.1

Positive control CPA

3.0

+

3

5.4

3.2

4.3

48.9

* The mitotic index was determined in 1000 cells

 

TABLE OF THE NUMBER OF POLYPLOID CELLS

First experiment

Doseμg//ml

S9-mix

Treatment time (h)

Polyploid cells*

Mean

Test group

1

2

Solvent control DMSO

0.0

-

3

2

1

1.5

Test item

125.0

-

3

0

3

1.5

500.0

-

3

0

2

1.0

1285.0

-

3

0

0

0.0

Positive control EMS

1500.0

-

3

1

1

1.0

 

 

 

 

 

 

 

Solvent control DMSO

0.0

+

3

4

1

2.5

Test item

125.0

+

3

0

1

0.5

500.0

+

3

2

0

1.0

1285.0

+

3

0

4

2.0

Positive control CPA

3.0

+

3

0

1

0.5

 

Second experiment

Doseμg/ml

S9-mix

Treatment time (h)

Polyploid cells*

Mean

Test group

1

2

Solvent control DMSO

0.0

-

20

0

4

2.0

Test item

25.0

-

20

6

6

6.0

75.0

-

20

66

2

4.0

250.0

-

20

0

1

0.5

Positive control EMS

400.0

-

20

0

1

0.5

 

Third experiment

Doseμg/ml

S9-mix

Treatment time (h)

Polyploid cells*

Mean

Test group

1

2

Solvent control DMSO

0.0

+

3

3

2

2.5

Test item

1285.0

+

3

1

7

 

 

 

 

5

6

4.8

Positive control CPA

3.0

+

3

2

0

1.0

* The number of polyploid cells was determined in 1000 cells

Conclusions:
The test substance was not mutagenic in this chromosome aberration test system in vitro with cells of the V79 Chinese hamster cell line under the conditions described in this report.
Executive summary:

The present study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 473 "Genetic Toxicology:In Vitro Mammalian Chromosome Aberration Test". Adopted: July 21st, 1997 and U.S. EPA: OPPTS 870.5375 Health Effects Test Guidelines In Vitro Mammalian Chromosome Aberration Test, August 1998 and EEC Directive 92/69, L 383 A, Annex B. 10, p. 148 -150. This study was conducted in compliance with the Principles of Good Laboratory Practice (GLP).

 

In this study the potential of the test substance to induce chromosome aberrations was investigated in V 79 cells of the Chinese hamster lung in vitro. For each experiment duplicate cultures were used for each concentration.

 

The test compound was suspended in DMSO and tested at the following concentrations:

- First experiment with 3 h treatment time:

without S9-mix: 25, 50, 75,125*, 250, 500*, 750 and 1285*μg/ml

with S9-mix:   25, 50, 75, 125*, 250, 500*, 750 and 1285*μg/ml

- Second experiment with 20 h treatment time:

without S9-mix: 5, 10, 25*, 50, 75*. 125 and 250*μg/ml

- Third experiment with 3 h treatment time:

with S9-mix: 1285*μg/ml

*= evaluated slides

 

The concentration ranges were based on the results of preliminary testing for solubility and toxicity. The highest concentration produced a distinct lowering of the mitotic index.

 

At concentrations of 500 μg/ml and above macroscopic visible precipitation of the test substance onto the slides was observed. Microscopically precipitation of the test compound was observed at 50 μg/ml and above.

 

There was an enhancement of the aberration rates at the 3 h treatment time with 1285 μg/ml with S9-mix, but only in one culture. These data were found significantly enhanced in the Fisher's exact-test.

Because of the different results between the duplicate cultures a third independent experiment with two slides and duplicate cultures was performed. In this experiment the enhancement of the aberration rate was not reproduced.

Without S9-mix up to the highest investigated dose the test compound induced no significant increase in the number of chromosome aberrations.

 

Appropriate reference mutagens used as positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay, and the efficacy of the S9-mix.

 

In conclusion, the test substance does not induce chromosome mutations (=aberrations) in V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described. It is therefore considered to be non-mutagenic in this chromosome aberration assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
chromosomal aberrations in cultured mammalian cells
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Source of cells: cell bank of "Genetic Toxicology", HMR Germany
Test organism: cell line V79 of Chinese hamster lung fibroblasts
Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
The test compound was suspended in DMSO and tested at the following concentrations:

without S9-mix:
20 h: 3.16#, 10.0#, 31.6, 100.0, 316.0 and1000.0* µg/mL
28 h: 31.6#, 100.0#, 316.0 and 1000.0* µg/mL

with S9-mix:
20 h: 31.6#, 100.0, 316.0 and 1000.0 µg/mL
28 h: 316.0# and 1000.0 µg/mL

* not evaluated because of high toxicity
# not used because higher concentrations were evaluated
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Evaluation criteria:
Analysis of metaphases
The slides were coded and 25 - 100 metaphases per experimental group and cell culture were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed. Only metaphases with 22 ±2 chromosomes are included in the analysis. The metaphases were examined for the following aberrations: chromatid gap, chromosome gap, chromatid break, chromosome break, minute, double minute, chromatid deletion, chromosome deletion, chromatid exchanges including intrachanges, chromosome exchanges including intrachanges, dicentrics, pulverization and ring formation. Furthermore the incidence of polyploid metaphases was determined in 1000 cells of each cell culture.
Additionally a mitotic index was determined by counting the number of cells undergoing mitosis in a total of 1000 cells. The mitotic index is given in per cent.
After the metaphases had been evaluated, the code was broken. The values for the control group were compared with the results from the dose groups and the positive control at each sampling time.

Criteria for a valid assay
The assay was considered valid if the following criteria are met:
the solvent control data were within the laboratory's normal control range for the spontaneous mutant frequency
the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range

Criteria for a positive response
The evaluation of the results was performed as follows:
The test compound is classified as mutagenic if it induces a statistically significant increase in the aberration rate (without gaps) with one or more of the concentrations tested as compared with the solvent controls.
The test compound is classified as mutagenic if there is a concentration-related increase in the aberration rate (without gaps).
The test compound is classified as non-mutagenic if the tests are negative both with and without metabolic activation.
Statistics:
Statistics
The Biometry of the results was performed with a one-sided Fisher - Exact test.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
There was an enhancement of the aberration rates inclusive and exclusive gaps 20 h and 28 h after the start of the treatment with the concentration of 316 µg/mL (relative mitotic index 19.4% and 44%, respectively) without S9-mix. In addition, an increased number of cells with break events was found in these groups. In the presence of S9-mix no relevant reproducible enhancement of metaphases over the range of the solvent control was found with any of the concentrations used.

As the increase in aberration rates were only observed at 316 µg/mL without metabolic activation and in the presence of cytotoxicity and visible test substance precipitation, the relevance and reliability of this result is questionable.
Executive summary:

In this study the potential of Disperse Red 184 to induce chromosome aberrations was investigated in V 79 cells of the Chinese hamster lung in vitro. For each experiment two cell cultures were used.

Disperse Red 184 was suspended in DMSO. Evaluation of the solubility of that suspension in cell culture medium showed that 1000 µg/mL was the highest practicable concentration and produced precipitate. Accordingly, a preliminary toxicity study was carried out using a maximum concentration of 1000 µg/mL and a range of lower dose levels down to 10 µg/mL.

Following treatment in the absence of S9 metabolic activation, severe toxicity was observed at 500 µg/mL and above. Survival declined in a dose-related manner reaching 29.2 % of the solvent control value at the highest dose level, 1000 µg/mL.

In the presence of metabolic activation (S9-mix) there was only a slight indication of toxicity up to the limit of solubility.

Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test compound solutions did not have any effect on these parameters.

Hence, the test compound was suspended in DMSO at the following concentrations:

without S9-mix:

20 h: 3.16#, 10.0#, 31.6, 100.0, 316.0 and 1000.0* µg/mL

28 h: 31.6#, 100.0#, 316.0 and 1000.0* µg/mL

with S9-mix:

20 h: 31.6#, 100.0, 316.0 and 1000.0 µg/mL

28 h: 316.0# and 1000.0 µg/mL

* not evaluated because of high toxicity

# not used because higher concentrations were evaluated

The highest concentrations produced no relevant lowering of the mitotic index in the presence of metabolic activation and a distinct lowering of the mitotic index in the absence of metabolic activation. Microscopic visible precipitation of the test compound was observed at 10 µg/mL and above in the absence of S9-mix and at 100 µg/mL and above in the presence of S9-mix.

After treatment with the test compound there was no relevant increase in the number of polyploid cells as compared with the solvent controls.

There was an enhancement of the aberration rates inclusive and exclusive gaps 20 h and 28 h after the start of the treatment with the concentration of 316 µg/mL (relative mitotic index 19.4% and 44%, respectively) without S9-mix. In addition, an increased number of cells with break events was found in these groups. In the presence of S9-mix no relevant reproducible enhancement of metaphases over the range of the solvent control was found with any of the concentrations used.

The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

As the increase in aberration rates were only observed at 316 µg/mL without metabolic activation and in the presence of cytotoxicity and visible test substance precipitation, the relevance and reliability of this result is questionable.

Endpoint:
genetic toxicity in vitro, other
Remarks:
Gene mutation
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
Data predicted by OECD Toolbox v4.0. OECD Toolbox uses a valid estimation method; the substance was found to fall in the applicability domain of this method and results are considered relevant for risk assessment
Justification for type of information:
1. SOFTWARE
QSAR Toolbox

2. MODEL (incl. version number)
QSAR Toolbox 4.0
Database version: 4.0//3.4

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CAS#: 24610-00-2

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: gene mutation
see attachement

5. APPLICABILITY DOMAIN
see attachement

6. ADEQUACY OF THE RESULT
the substance lies well within the aplicability domain
Qualifier:
according to
Guideline:
other: REACH guidance on QSARs: Chapter R.6. QSARs and grouping of chemicals
GLP compliance:
no
Type of assay:
other: QSAR prediction
Key result
Species / strain:
not specified
Metabolic activation:
with and without
Genotoxicity:
negative
Additional information on results:
Prediction summary
Predicted endpoint: gene mutation, Gene mutation; No effect specified; No species specified; No duration specified; No guideline specified
Predicted value: Negative
Unit/scale: Gene mutation I
Data gap filling method: Read-across analysis

Calculation approach (OECD principle 2 - Unambiguous algorithm): takes the highest mode value from the nearest 5 neighbours
Active descriptor: log Kow (calculated)
Data usage: All values
Uncertainty of the prediction (OECD principle 4 - Uncertainty of the prediction):
The prediction is based on 36 values, 27 of them (75%) equal to predicted value
Prediction confidence is measured by the p-value: 1.14E-06
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
Based on the QSAR prediction results, the test substance has no mutagenic properties
Executive summary:

Prediction summary

Predicted endpoint: gene mutation, Gene mutation; No effect specified; No species specified; No duration specified; No guideline specified

Predicted value: Negative

Unit/scale: Gene mutation I

Data gap filling method: Read-across analysis

Calculation approach (OECD principle 2 - Unambiguous algorithm): takes the highest mode value from the nearest 5 neighbours

Active descriptor: log Kow (calculated): 5.94

Data usage: All values

Uncertainty of the prediction (OECD principle 4 - Uncertainty of the prediction):

The prediction is based on 36 values, 27 of them (75%) equal to predicted value

Prediction confidence is measured by the p-value: 1.14E-06

Analogue(s) selection

(OECD principle 3 - Applicability domain)

Database(s) used:

- Carcinogenic Potency Database (CPDB)

- Micronucleus OASIS

- Toxicity Japan MHLW

- Genotoxicity OASIS

- Transgenic Rodent Database

- ECVAM Genotoxicity & Carcinogenicity

- ECHA CHEM

- Bacterial mutagenicity ISSSTY

- Carcinogenicity&mutagenicity ISSCAN

- Micronucleus ISSMIC

Category boundaries (applicability domain):

- Active descriptor(s) range:

- log Kow: from 1.02 to 7.63 target chemical is in domain

- Response range:

- Gene mutation: from Negative to Positive

Profilers:

- DNA binding by OASIS v.1.4 (primary grouping) target chemical is in domain

- DNA binding by OECD (subcategorization) target chemical is in domain

- Eye irritation/corrosion Exclusion rules by BfR (subcategorization) target chemical is in domain

Additional data pruning:

none

Manually eliminated data points:

none

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
09 November 1998 to 14 December 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read across is based on the same physico-chemical properties, a close structural similarity and the same mechanism of action during use processes.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
- Source: Disperse Blue 291 Br
- Target: Disperse Red 184

3. ANALOGUE APPROACH JUSTIFICATION
see attachment in endpoint summary

4. DATA MATRIX
see attachment in endpoint summary
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: In vitro Mammalian Cell Gene Mutation Test EEC Directive 87/302, L133, pp. 61 - 63, March 1987
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
The compound was suspended in DMSO and tested at the following concentrations:
without S9-mix; 5.0, 7.5, 12.5, 25.0, 50.0, 75.0. 125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml (main mutation experiment)
50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)
with S9-mix: 5.0, 7.5, 12.5, 25.0, 50.0, 75.0, 125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml (main mutation experiment)
50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
ethylmethanesulphonate
Key result
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:
valid
Positive controls validity:
valid

Toxicity Data (Main mutation experiment)

 

Dose μg/ml

S9-mix

Extinction in microwell plates mean less blank values

Standard deviation

Relative survival*

Negative control

0.0

-

0.572

0.02

121.5

+

0.451

0.05

125.3

Solvent control

0.0

-

0.471

0.04

100.0

-

0.424

0.06

100.0

+

0.360

0.06

100.0

+

0.411

0.08

100.0

Positive control

1000.0

-

0.504

0.01

107.0

7.7

+

0.361

0.04

100.3

Test item

5.0

-

0.499

0.02

105.9

+

0.461

0.03

128.0

7.5

-

0.529

0.08

112.3

+

0.462

0.03

128.3

12.5

-

0.495

0.02

105.0

+

0.448

0.04

124.4

25.0

-

0.440

0.01

93.5

+

0.437

0.03

121.4

50.0

-

0.420

0.02

89.1 #

+

0.378

0.03

105.2 #

75.0

-

0.398

0.02

84.1 #

+

0.413

0.03

114.7 #

125.0

-

0.483

0.10

113.9 #

+

0.387

0.03

94.2 #

250.0

-

0.319

0.02

75.2 #§

+

0.418

0.05

101.8 #§

500.0

-

0.253

0.02

59.6 #§

+

0.325

0.05

79.1 #§

750.0

-

0.276

0.02

65.2 #§

+

0.256

0.05

62.3 #§

1285.0

-

0.226

0.03

63.2 #§

+

0.186

0.04

45.3 #§

# microscopical visible precipitation

§ macroscopical visible precipitation

* relative survival (mean value / mean value corresponding control x 100)

Solvent = DMSO

Positive control without S9-mix = EMS

Positive control with S9-mix = DMBA

 

Mutagenicity Data – Part 1 (Main experiment)

 

Dose μg/ml

S9-mix

Number of cells per flask

Factor* calculated

Cells* seeded

Cells*** survived

Seeded

Found

Mean

I / II

I

II

Negative control

0.0

-

403

158.0

164.5

161.3

0.40

342900

137203

+

401

101.0

98.0

99.5

0.25

337500

83744

Solvent control 1 (DMSO)

0.0

-

398

370.5

370.0

370.3

0.93

292350

271966

+

399

370.0

372.0

371.0

0.93

298800

277832

Solvent control 2 (DMSO)

0.0

-

400

313.0

323.5

318.3

0.80

291150

231646

+

402

201.5

200.5

201.0

0.50

287700

143850

Positive control (EMS)

1000.0

-

400

288.0

273.5

280.8

0.70

285000

200034

Positive control (DMBA)

7.7

+

398

281.0

299.0

290.0

0.73

276750

201652

Test item

5.0

-

399

258.5

261.0

259.8

0.65

291900

190028

+

400

368.0

386.5

377.3

0.94

258600

243892

7.5

-

401

188.5

191.5

190.0

0.47

291000

137880

+

401

284.0

274.0

279.0

0.70

326400

227096

12.5

-

399

278.0

277.0

277.5

0.70

307800

214071

+

394

271.0

278.5

274.8

0.70

331650

231271

25.0

-

399

348.0

362.5

355.3

0.89

294300

262030

+

402

361.5

380.0

370.8

0.92

276150

254683

50.0

-

403

320.0

310.5

315.3

0.78

288150

225408

+

402

292.5

296.0

294.3

0.73

280650

205426

75.0

-

399

357.0

338.5

347.8

0.87

294300

256498

+

405

276.5

272.5

274.5

0.68

299250

202825

125.0

-

400

381.0

338.0

359.5

0.90

294300

264502

+

399

340.0

351.0

345.5

0.87

269400

233277

250.0

-

402

368.0

353.0

360.5

0.90

288750

258941

+

401

450.5

449.0

449.8

1.12

258150

289534

500.0

-

403

322.0

314.0

318.0

0.79

279750

220748

+

402

302.0

314.5

308.3

0.77

288900

221526

750.0

-

402

271.0

299.5

285.3

0.71

349200

247784

+

399

313.0

314.0

313.5

0.79

336000

264000

1285.0

-

400

156.0

144.0

150.0

0.38

337500

126563

+

405

301.0

306.5

303.8

0.75

347250

260438

* Factor calculated: mean value / number of cells per flask seeded

** Cells seeded in 6-thioguanine (TG) containing medium

*** Cells survived after plating in (TG) containing medium (cells seeded x factor calculated)

 

Mutagenicity Data – Part 2 (Main experiment)

 

Dose μg/ml

S9-mix

Number of mutant colonies

Standard deviation

Mutation frequency

Stat. sig.

I

II

III

IV

V

Mean

Negative control

0.0

-

1

4

1

3

2

2.2

1.30

16.0

 

+

2

1

2

3

0

1.6

1.14

19.1

 

Solvent control 1 (DMSO)

0.0

-

3

3

1

2

1

2.0

1.00

7.4

 

+

1

1

1

1

3

1.4

0.89

5.0

 

Solvent control 2 (DMSO)

0.0

-

9

11

6

3

6

7.0

3.08

30.2

 

+

3

1

2

7

2

3.0

2.35

20.9

 

Positive control (EMS)

1000.0

-

166

147

161

156

148

155.8

8.20

777.9

*

Positive control (DMBA)

7.7

+

35

35

45

40

36

38.2

4.32

189.4

*

Test item

5.0

-

1

0

0

1

3

1.0

1.22

5.3

 

+

5

3

1

1

1

2.2

1.79

9.0

 

7.5

-

1

1

1

2

0

1.0

0.71

7.3

 

+

8

5

12

10

6

8.2

2.88

35.1

*

12.5

-

3

3

0

4

3

2.6

1.52

12.1

 

+

3

2

1

3

1

2.0

1.00

8.6

 

25.0

-

5

5

1

2

3

3.2

1.79

12.2

 

+

1

3

1

4

4

2.6

1.52

10.2

 

50.0

-

0

0

4

1

1

1.2

1.64

5.3

 

+

1

1

1

3

0

1.2

1.10

5.8

 

75.0

-

1

4

5

5

2

3.4

1.82

13.3

 

+

5

1

2

7

8

4.6

3.05

22.7

 

125.0

-

1

4

2

0

2

1.8

1.48

6.8

 

+

2

1

2

7

3

3.0

2.35

12.9

 

250.0

-

5

6

2

4

2

3.8

1.79

14.7

 

+

5

0

2

2

4

2.6

1.95

9.0

 

500.0

-

4

6

2

1

2

3.0

2.00

13.6

 

+

4

1

8

3

5

4.2

2.59

19.0

 

750.0

-

6

7

3

5

3

4.8

1.79

19.4

 

+

11

8

2

7

11

7.8

3.70

29.5

 

1285.0

-

1

6

5

5

3

4.0

2.00

31.6

 

+

1

7

2

2

3

3.0

2.35

11.5

 

Mutation frequency (mutant colonies per 1 million cells); mean value / cells surviving

* Statistical significant (p≤0.05) Mann-Whitney-U-Test

Conclusions:
The test substance did not induce gene mutation, i.e. was not mutagenic, in this HPRT-test with V79 Chinese hamster cells, either in the presence or in the absence of metabolic activation.
Executive summary:

The present study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 476 "Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Test". Adopted: July 21, 1997 and U.S. Environmental Protection Agency (EPA) Health Effect Test Guidelines, OPPTS 870.5300, In Vitro Mammalian Cell Gene Mutation Test, august 1998 and In vitro Mammalian Cell Gene Mutation Test EEC Directive 87/302, L133, pp. 61 - 63, March 1987. This study was conducted in compliance with the Principles of Good Laboratory Practice (GLP).

 

The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster in vitro.

Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix).

The compound was suspended in DMSO and tested at the following concentrations:

without S9-mix; 5.0, 7.5, 12.5, 25.0, 50.0, 75.0. 125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml (main mutation experiment)

50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)

With S9 -mix: 5.0, 7.5, 12.5, 25.0, 50.0, 75.0, 125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml (main mutaation experiment)

50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)

 

The concentration ranges were based on the results of preliminary tests for solubility and toxicity. The highest concentration showed slight toxic effects with and several toxic effects without metabolic activation.

In the presence of metabolic activation a significant increase of the mutation frequency was observed only at a concentration of 7.5 μg/ml. This effect was not dose-dependent and not three fold higher than the corresponding controls and therefore of no biological relevance.

Up to the highest investigated dose no further increase in mutant colony numbers was obtained in two independent experiments.

Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, thus indicating the sensitivity of the assay, and the efficacy of the S9-mix.

 

In conclusion, the test substance does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system under the experimental conditions described.

The test substance is therefore considered to be non-mutagenic in this HPRT assay.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Plate incorporation test under oxidative conditions according to Maron and Ames (1983), with modifications, using sterile deionized water as the negative control.
All the experiments were performed in duplicate. The dye was tested under partially anaerobic (reducing) conditions, and the results were compared to the data obtained using the normal oxidative protocol. The assay under reductive conditions was performed according to Prival and Mitchell (1982).
The mutation spectra of CI Disperse Blue 291 were determined using the Salmonella TA7000 series described by Gee et al. (1994) using the microsuspension modification (Kado et al., 1983) under oxidative conditions. Overnight cultures of each strain (around 109 cells/ml) were concentrated 10 fold by centrifugation at 10,000g at 4 °C for 10 min and resuspended in 0.015 M sodium phosphate buffer. 50 µI of cell suspension, 50 µI of 0.015 M sodium phosphate buffer or S9 mix, and 7.5 µI of the test sample were added to a tube and incubated at 37 °C for 90 min without shaking. After incubation, 2 ml of molten agar was added, the mixture was poured onto a minimal agar plate, and the plates were incubated at 37 °C for 72 h. Colonies were counted using an automatic colony counter. When the number of colonies was less than ten, the counts were also confirmed by hand, using a light box with magnification.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Genotype and the type of mutation detected by each strain of Salmonella typhimurium

Strains of Salmonella. Genotype Type of mutation detected Reference
TA1535 HisG46, rfa, Abio, AuvrB Frameshift (mainly CG deletions) Maron and Ames (1983)
TA1537 HisC3076, rfa, Abio, AuvrB Frameshift (mainly CG deletions) Maron and Ames (1983)
TA1538 HisD3052, rfa, Abio, AuvrB Frameshift (mainly CG deletions) Maron and Ames (1983)
TA98 HisD3052, rfa, Abio, AuvrB, pKM101 Frameshift (mainly CG deletions) Maron and Ames (1983)
TA98 NR HisD3052, rfa, Abio, AuvrB, pKM101 nitroreductase enzyme activity deficient Frameshift (mainly CG deletions) Rosenkranz and Mermelstein (1983)
TA98 DNP6 HisD3052, rfa, Abio, AuvrB, pKM101 acetyltransferase enzyme activity deficient Frameshift (mainly CG deletions) Rosenkranz and Mermelstein (1983)
TA100 HisG46, rfa, Abio, AuvrB, pKM101 Missence (base pair substitutions) Maron and Ames (1983)
YG1021 HisD3052, rfa, Abio, AuvrB, pKM101, nitroreductase overproducing enzyme activity Frameshift (mainly CG deletions) Watanabe et al. (1989)
YG1024 HisD3052, rfa, Abio, AuvrB, pKM101, acetyltransferase overproducing enzyme activity Frameshift (mainly CG deletions) Watanabe et al. (1990)
YG1041 HisD3052, rfa, Abio, AuvrB, pKM101, acetyltransferase and nitroreductase overproducing enzyme activity Frameshift (mainly CG deletions) Hagiwara et al. (1993)
TA7001 IlisG1775, rfa, Abio, AuvrB, pKM101 T:A —> C:G Gee et al. (1994)
TA7002 IlisC9138, rfa, Abio, AuvrB, pKM101 T:A —> A:T Gee et al. (1994)
TA7003 HisG9074, rfa, Abio, AuvrB, pKM101 T:A —> G:C Gee et al. (1994)
TA7004 IlisG9133, rfa, Abio, AuvrB, pKM101 C:G —> T:A Gee et al. (1994)
TA7005 IlisG9130, rfa, Abio, AuvrB, pKM101 C:G —> A:T Gee et al. (1994)
TA7006 HisG9070, rfa, Abio, AuvrB, pKM101 C:G —> G:C Gee et al. (1994)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
other: TA98 NR
Additional strain / cell type characteristics:
nitroreductase deficient
Species / strain / cell type:
other: TA98 DNP6
Additional strain / cell type characteristics:
acetyltransferase deficient
Species / strain / cell type:
other: YG1021
Additional strain / cell type characteristics:
other: nitroreductase overproducing enzyme activity
Species / strain / cell type:
other: YG1024
Additional strain / cell type characteristics:
acetyltransferase proficient
Species / strain / cell type:
other: YG1041
Additional strain / cell type characteristics:
other: acetyltransferase and nitroreductase overproducing enzyme activity
Species / strain / cell type:
other: TA7001, TA7002, TA7003, TA7004, TA7005, TA7006
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
S. typhimurium TA 98
Metabolic activation:
with and without
Metabolic activation system:
Sprague Dawley rat liver S9 (MolTox, Boone, NC) induced with an Arochlor 1254 mix
Test concentrations with justification for top dose:
up to 500 µg/plate
TA 7000 stains: 250, 500, 1000 µg/plate
Vehicle / solvent:
sterile deionized water and DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Evaluation criteria:
Samples were considered positive when a significant ANOVA and dose response was obtained with the Bernstein model (Bernstein et al., 1982). For the TA7000 strain series, because of the low spontaneous mutation rate of some strains, we considered a response positive when the counts of the plates containing the sample were greater than the average of the negative controls plus 3 standard deviations. The results were expressed as number of reverants per µg of compound tested.
Statistics:
ANOVA
Species / strain:
other: TA1537, TA100, TA98, TA1538
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA98NR
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA98NR
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: YG1021, YG1024, YG1041
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA98DNP6
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA98DNP6
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
slight increase
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA7001, TA7002, TA7004, TA7005
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
TA7004, TA7005 at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA7003, TA7006
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
TA7006 at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA7000 series
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
under reductive cleavage conditions, the color of the tested dye did not change when using the modification of Prival and Mitchell (1982), and the mutagenicity remained the same as that seen with normal oxidative metabolism. This suggests that the azobond was not cleaved in the testing conditions.
Conclusions:
The study shows that the mitagenic effect of the test substance is due to bacterial nitroreductase and O-acetyltransferase activity
Executive summary:

Disperse Blue 291 was tested for mutagenic activity in the Salmonella assay with strains with different levels of nitroreductase andO-acetyltransferase (i.e., TA98DNP6, YG1024, and YG1041) as well as standard strains TA 1535, TA1537, TA1538, TA98 and TA100 and strains which provide more information on the base-pair substitution (TA 7001 to 7006). Disperse Blue 291 showed direct-acting mutagenic activity with all strains ofSalmonella typhimuriumtested, except forTA 1535.According to the classification of Claxton et al. (1991), the potency of this product can be considered moderate (10-100 revertants/µg). In the absence of S9, the nitroreduction is strongly related to the mutagenic activity, because the mutagenicity of Disperse Blue 291 was very low when tested with the strains lacking nitroreductase activity (TA98NR) and was increased with the nitroreductase overproducing strains, (YG1021 and YG1041). The same mutagenic pattern was observed for the acetyltransferase deficient and overproducing strains (TA98DNP6, YG1024, and YG1041) revealing also the importance of the acetyltransferase enzyme in the activation of Disperse Blue 291. Because of the remarkable increase in the response with the nitroreductase andO-acetyltransferase overproducing strain (YG1041), it is likely that the product of the nitroreductase is a substrate for theO-acetyltransferase.

In the presence of S9, the mutagenicity was slightly increased with TA98NR, TA98, YG1021, TA98DNP6, and YG1024 suggesting that, P450 enzymes also have a role in the activation of these compounds, besides the bacterial enzymes. This could be explained by the activation of other radicals of the molecule by the S9 enzymes, for example the —OCH3or the —N(CH2CH3)2of the CI Disperse Blue 291.

 

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
23 November 1998 to 15 January 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
chromosomal aberrations in cultured mammalian cells
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Source of cells: cell bank of "Genetic Toxicology", HMR Germany
Test organism: cell line V79 of Chinese hamster lung fibroblasts
Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
The test compound was suspended in DMSO and tested at the following concentrations:

First experiment with 3 h treatment time:
without S9-mix: 25, 50, 75, 125*, 250, 500*, 750 and 1285* μg/ml
with S9-mix: 25, 50, 75, 125*, 250, 500*, 750 and 1285* μg/ml

Second experiment with 20 h treatment time:
without S9-mix: 5, 10, 25*, 50, 75*. 125 and 250* μg/ml

Third experiment with 3 h treatment time:
with S9-mix: 1285* μg/ml

*= evaluated slides
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
Reference Compounds
Without metabolic activation
Name or number of compound (I.N.N. orU.S.A.N): ethyl methane sulfonate
Synonyms: EMS
Formula of the compound: C3HB03S
CAS-Register number: 62-50-0
Product number / Code: 820774
Supplier of reference compound: Dr. Theodor Schuchardt & Co., Germany
Batch number: 40606721
Certificate of analysis: certificated by the supplier, Analytical Department, Dr. Bolkart dated December 18th, 1995

With metabolic activation
Name or number of compound {I.N.N. orU.S.A.N): cyclophosphamide
Synonyms: Endoxan®
Formula of the compound: C7H15CI2N2P.H20
CAS-Register number: 50-18-0
Supplier of reference compound: ASTA Medica AG, Germany
Batch number: 603575 B
Certificate of analysis: certificated by the supplier, Quality Control, Dr. Weiss dated March 25th, 1998

Test groups
Treatment time 3h
Without S9-mix with S9-mix
Solvent control: 0.0μg/ml 0.0μg/ml
Positive control: EMS 1500.00μg/ml CPA 3.0μg/ml
Test group 1: 25μg/ml # 20μg/ml #
Test group 2: 50μg/ml # 50μg/ml #
Test group 3: 75μg/ml # 75μg/ml #
Test group 4: 125μg/ml * 125μg/ml *
Test group 5: 250μg/ml # 250μg/ml #
Test group 6: 500μg/ml * 500μg/ml *
Test group 7: 750μg/ml # 750μg/ml #
Test group 8: 1285μg/ml * 1285μg/ml *

Treatment time 20h
Without S9-mix
Solvent control: 0.0μg/ml
Positive control: EMS 400.0μg/ml
Test group 1: 5μg/ml #
Test group 2: 10μg/ml #
Test group 3: 25μg/ml *
Test group 4: 50μg/ml #
Test group 5: 75μg/ml *
Test group 6: 125μg/ml #
Test group 7: 250μg/ml *

*=evaluated slides
#= not used because higher concentrations were evaluated

Control groups:
Solvent controls: cultures treated with the solvent
Positive controls: without metabolic activation: EMS (ethyl methane sulfonate)
with metabolic activation: CPA (cyclophosphamide) = Endoxan®

Formulation of test compound: suspended in DMSO at appropriate concentrations immediately before use

Formulation of reference compounds
EMS dissolved in cell culture medium on the day of treatment, final concentration: 1.5 mg/ml (3 h treatment) final concentration: 0.4 mg/ml (20 h treatment)
CPA dissolved in cell culture medium on the day of treatment, final concentration in ceil culture medium: 3.0 μg/ml

Source of biological material: cell bank of "Genetic Toxicology", HMR Germany, ProTox

Test organism: cell line V79 of Chinese hamster lung fibroblasts

Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer

Experimental conditions in vitro: approx. 37 °C and approx. 4 % C02 in plastic flasks

Preparation and storage of a liver homogenate fraction (S9)
The S9 fraction was prepared by the testing facility according to Ames et. al (1975). Male Sprague Dawley rats (200-300 g), supplied by Harlan Winkelmann, Gartenstrasse 27, 33178 Borchen, Germany, received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) 5 days before killing. The livers were removed from at least 5-6 animals at approx. 0 to 4 °C using cold sterile solutions and glassware, and were then pooled and washed in approx. 150 mM KCl (approximately 1 ml/g wet liver). The washed livers were cut into small pieces and homogenized in three volumes of KCl. The homogenate was centrifuged at approx. 9000g for 10 minutes. The supernatant, the S9 fraction, was divided into small portions, rapidly frozen and stored at approx. - 80 °C for not longer than six months. The protein content was determined for every batch. Also for every batch of S9 an independent validation was performed with a minimum of two different mutagens, e.g., 2-aminoanthracene and dimethylbenzanthracene to confirm metabolic activation by microsomal enzymes.

Preparation of S9-mix
Sufficient S9 fraction was thawed to room temperature immediately before each test. An appropriate quantity of S9 fraction (batch no. 98/1, protein concentration 51.8 g/l) was mixed with S9 cofactor solution to yield a final protein concentration of 0.3 mg/ml in the cultures which was kept on ice until used. This preparation is termed S9-mix. The concentrations of the different cofactors of the S9-mix were:

8 mM MgCl2
33 mM KCI
5 mM glucose-6-phosphate
5 mM NADP
100 mM phosphate buffer pH 7.4

Cell culture
Large stocks of the mycoplasma-free V79 cell line are stored in liquid nitrogen in the cell bank of "Genetic Toxicology", thus permitting repeated use of the same ceil culture batch for numerous experiments. The identical characteristics of the cells ensure comparability of the experimental parameters.
Thawed stock cultures were kept at approx. 37 °C and approx. 4 % C02 in 175 cm2 plastic flasks. About 5 x 10 5 to 1 x 10 6 cells were seeded into each flask in 30 ml of MEM-medium supplement with approx. 10 % (v/v) FCS (fetal calf serum) containing approx. 2 mM L-glutamine and approx. 0.1 % (w/v) neomycinsulfate. The cells were subcultured twice a week.

Toxicity experiments and dose range finding
A preliminary toxicity test was undertaken in order to select appropriate dose levels for the cytogenetic assay. Cell cultures were subjected to the same treatment conditions as in the main experiment. Cytotoxic effects were determined by photometric measurement of V79 cell cultures grown in microwell plates and stained with crystal violet. The relative cell density in the microwell plates was nearly the same as in the Quadriperm® dishes.

The test included the following treatments:

Solvent control : the maximum final concentration of organic solvents was approx. 1 % (v/v).
Test compound : the highest dose level for the preliminary toxicity test was determined by the solubility of the test compound up to the maximum of 10 mM or 5000μg/ml.
Treatments were performed both in the presence and absence of S9 metabolic activation system using a duplicate cell culture at each test point.

Rationale for dose selection
The concentrations for the mutagenicity assay were based on the results of the toxicity experiment.
For non-toxic, freely soluble test compounds, the top dose is 10 mM or 5000μg/ml according to international testing guidelines.
For relatively insoluble test compounds, that are not toxic at concentrations lower than the insoluble concentration, the highest dose used should be a concentration above the limit of solubility in the final culture medium after the end of the treatment period. In the case of toxic effects, the highest dose level should reduce the survival rate to approximately 20 - 50 % and/or the mitotic index to approximately 50 % compared with the corresponding solvent control.
For toxic compounds additional concentrations may be included in the treatment series. According to the criteria described above, three adequately spaced dose levels extending over at least one decadic logarithm were evaluated. In the event of clearly positive results at the 3 hours treatment time, it is not necessary to perform an evaluation of the 20 h treatment time.
For each experimental point two cultures were used for each concentration.

Mutagenicity test
Two independent experiments were conducted. The first experiment with 3 hours treatment time of the test substance was performed in the presence and the absence of S9-mix. Cultured cells were seeded on-to slides (duplicate culture) then treated for either 3 hours (with and without S9-mix in the first experiment) or for 20 hours (without S9-mix in the second experiment). Colcemide was then added to arrest cell division and the chromosomes were stained and examined. In both assays, cells were sampled 20 hours after the start of treatment. For both assays, at least three dose levels were evaluated for chromosome aberrations. Where negative or equivocal results were obtained, cells were treated and also examined 20 hours after the start of treatment.

Before treatment, the pH values and osmolality of the treatment medium were determined. If necessary the pH was adjusted to pH 7.3 with NaOH or HCl. Any effects on the osmolality during the study were described in the study report.

Two-day old, exponentially growing stock cultures which were over 50 % confluent were trypsinised and a single cell suspension (culture) was prepared. The trypsin concentration was approx. 0.25 % (v/v) in Ca-Mg-free salt solution. Two slides were placed in Quadripem dishes which were then seeded with cells to yield 2-3 x 10 4 cells/slide. Thus for each dose level and treatment time, duplicate cultures were used. The Quadriperm® dishes contained 5 ml MEM with approx. 10 % (v/v) FCS.

After 48 h, the medium was replaced with one containing approx. 10 % (v/v) FCS and the test compound, or positive control, or solvent and in the presence of metabolic activation additionally 2 % ( v/v) S9-mix.
For the 3 hour treatment time, the medium was replaced by normal medium following two rinses. In the repeat experiment the cells were exposed to the treatment medium without S9-mix for 20 h.

18 h after the start of the treatment, colcemide was added (approx. 0.05μg/ml/culture medium) to the cultures to arrest mitosis and 2 h later (20 h after the start of treatment) metaphase spreads were prepared as follows:

The cultures were made hypotonic by adding about 4 ml of approx. 0.075 M potassium chloride solution at around 37 °C. The cells were then incubated for 20 minutes at approx. 37 °C. The next step was the addition of 1.5 ml fixative.

Then the liquid was replaced by 5 ml fixative (methanol: glacial acetic acid, 3:1). After 10 minutes the procedure was repeated. After at least another 10 minutes, the slides were taken out and airdried for 24 hours. The chromosomes were stained as follows:

staining for 10 minutes in approx. 2 % (w/v) orcein solution
rinsing 3 times in distilled water
rinsing twice in acetone
brief rinsing in acetone/xylene
2 minutes in acetone/xylene
5 minutes in xylene
10 minutes in xylene
embedding in Enteilan® or Corbit®

Duplicate cultures were prepared from each experimental group.

For both treatment times the solvent and the positive controls were prepared 20 h after treatment in the same way.
Evaluation criteria:
Analysis of metaphases
The slides were coded and 25 - 100 metaphases per experimental group and cell culture were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed. Only metaphases with 22 ±2 chromosomes are included in the analysis. The metaphases were examined for the following aberrations: chromatid gap, chromosome gap, chromatid break, chromosome break, minute, double minute, chromatid deletion, chromosome deletion, chromatid exchanges including intrachanges, chromosome exchanges including intrachanges, dicentrics, pulverization and ring formation. Furthermore the incidence of polyploid metaphases was determined in 1000 cells of each cell culture.
Additionally a mitotic index was determined by counting the number of cells undergoing mitosis in a total of 1000 cells. The mitotic index is given in per cent.
After the metaphases had been evaluated, the code was broken. The values for the control group were compared with the results from the dose groups and the positive control at each sampling time.

Criteria for a valid assay
The assay was considered valid if the following criteria are met:
the solvent control data were within the laboratory's normal control range for the spontaneous mutant frequency
the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range

Criteria for a positive response
The evaluation of the results was performed as follows:
The test compound is classified as mutagenic if it induces a statistically significant increase in the aberration rate (without gaps) with one or more of the concentrations tested as compared with the solvent controls.
The test compound is classified as mutagenic if there is a concentration-related increase in the aberration rate (without gaps).
The test compound is classified as non-mutagenic if the tests are negative both with and without metabolic activation.
Statistics:
Statistics
The Biometry of the results was performed with a one-sided Fisher - Exact test.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Solubility and preliminary toxicity testing
The test substance was suspended in DMSO.
Evaluation of the solubility of that suspension in cell culture medium showed that 1285 μg/ml was the highest practicable concentration and produced precipitate.

Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 1285μg/ml and a range of lower dose levels down to 10μg/ml.

Following treatment for 3 hours, several toxicity was observed at 100μg/ml and above. Survival declined in a dose-related manner reaching 35.3 % in the absence of metabolic activation, respectively 55.4 % in the presence of S9-mix of the solvent control value at a dose level of 750μg/ml.
After 20 hours treatment survival was also dose-dependent reduced reaching 17.5 % of the solvent control value at a concentration of 500μg/ml.
On account of the precipitation of the test substance the extinction values at higher dose levels were slightly increased at both treatment times.

Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test compound solutions did not have any effect on these parameters.

Mutagenicity test
In the main experiments cytotoxicity was also evaluated by treatment of cells seeded in microwell plates. Survival was reduced in a dose-related manner reaching 39.8 % of the solvent control value without S9-mix at the 20 hours treatment time at the highest dose, 250μg/ml and 42.0 % respectively 54.7 % in the absence respectively in the presence of S9-mix at the 3 hours treatment time at a concentration of 750μg/ml.
In accordance to the preliminary experiment the extinction values at the dose level of 1285μg/ml were increased on account of the precipitation of the test substance in the wells.
In the main and in the repeat experiments the mitotic index was reduced (indication of toxicity) after treatment with the highest dose levels.
After treatment with the test compound there was no relevant increase in the number of polyploid cells as compared with the solvent controls.
There was an enhancement of the aberration rates at the 3 h treatment time with 1285μg/ml with S9-mix, but only in one culture. These data were found significantly enhanced in the Fisher's exact-test.
Because of the different results between the duplicate cultures a third independent experiment with two slides and duplicate cultures was performed additionally. In this experiment the enhancement of the aberration rate was not reproduced.

The test compound was assessed for its mutagenic potential in vitro in the chromosome aberration test in two independent experiments.
No relevant reproducible enhancement of metaphases with aberrations over the range of the solvent control was found with any of the concentrations used, either with or without metabolic activation by S9-mix. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

MAIN EXPERIMENT TOXICITY TABLES

 

Table 2a (3 hours treatment time without S9-mix)

 

Dose

μg/ml

S9-mix

Extinction in microwell plates mean less blank values

Standard deviation

Relative survival*

Solvent control

0

-

0.485

0.03

100.0

+

0.402

0.03

100.0

Test item

25

-

0.427

0.01

88.1

+

0.376

0.03

93.9

50

-

0.402

0.03

82.9 #

+

0.338

0.02

84.0 #

75

-

0.378

0.03

78.0 #

+

0.340

0.02

84.6 #

125

-

0.343

0.08

70.6 #

+

0.308

0.02

78.8 #

250

-

0.291

0.04

60.0 #

+

0.277

0.02

69.0 #

500

-

0.236

0.02

48.6 #§

+

0.250

0.02

62.2 #§

750

-

0.204

0.03

42.0 #§

+

0.220

0.02

54.7 #§

1285

-

0.318

0.02

65.6 #§

+

0.268

0.02

66.7 #§

 

Table 2b (20 hours treatment time without S9-mix)

 

Doseμg/ml

S9-mix

Extinction in microwell plates mean less blank values

Standard deviation

Relative survival*

Solvent control

0

-

0.489

0.04

100.0

Test item

5

-

0.480

0.06

98.1

 

10

-

0.461

0.02

94.3

 

25

-

0.421

0.02

86.0

 

50

-

0.378

0.02

77.4 #

 

75

-

0.328

0.02

67.1 #

 

125

-

0.248

0.03

50.7 #

 

250

-

0.195

0.03

39.8 #

 

* = relative survival (mean value/mean value corresponding control x 100)

Solvent control = DMSO

# = microscopical visible precipitation of the test compounds

§= macroscopical visible precipitation of the test compounds

 

TABLE OF THE MITOTIC INDEX

First experiment

Doseμg/ml

S9-Mix

Treatment time (h)

Mitotic index

Mean

Relative mitotic index percent*

Test group

1

2

Solvent control DMSO

0.0

-

3

5.1

5.7

5.9

100.0

Test item

125.0

-

3

4.5

4.3

4.4

74.8

500.0

-

3

2.8

4.3

3.6

61.0

1285.0

-

3

4.6

3.2

3.9

66.1

Positive control EMS

1500.0

-

3

5.7

3.3

4.5

76.3

 

Solvent control DMSO

0.0

+

3

7.0

8.2

7.6

100.0

Test item

125.0

+

3

7.3

7.2

7.3

96.1

500.0

+

3

5.4

6.5

6.0

79.0

1

1285.0

+

3

4.3

5.0

4.7

61.8

Positive control CPA

3.0

+

3

4.1

3.8

4.0

52.6

 

Second experiment

Doseμg/ml

S9-mix

Treatment time (h)

Mitotic index

Mean

Relative mitotic index percent*

Test group

1

2

Solvent control DMSO

0.0

-

20

5.0

5.4

5.2

100.0

Test item

25.0

-

20

5.6

4.2

4.9

94.2

75.0

-

20

4.9

4.7

4.8

92.3

250.0

-

20

2.3

2.9

2.6

50.0

Positive control EMS

400.0

-

20

3.1

2.9

3.0

57.7

 

Third experiment

Doseμg/ml

S9-mix

Treatment time (h)

Mitotic index

Mean

Relative mitotic index percent*

Test group

1

2

Solvent control DMSO

0.0

+

3

8.4

9.1

8.8

100.0

Test item

1285.0

+

3

4.0

6.6

 

 

 

 

 

4.8

5.2

5.2

59.1

Positive control CPA

3.0

+

3

5.4

3.2

4.3

48.9

* The mitotic index was determined in 1000 cells

 

TABLE OF THE NUMBER OF POLYPLOID CELLS

First experiment

Doseμg//ml

S9-mix

Treatment time (h)

Polyploid cells*

Mean

Test group

1

2

Solvent control DMSO

0.0

-

3

2

1

1.5

Test item

125.0

-

3

0

3

1.5

500.0

-

3

0

2

1.0

1285.0

-

3

0

0

0.0

Positive control EMS

1500.0

-

3

1

1

1.0

 

 

 

 

 

 

 

Solvent control DMSO

0.0

+

3

4

1

2.5

Test item

125.0

+

3

0

1

0.5

500.0

+

3

2

0

1.0

1285.0

+

3

0

4

2.0

Positive control CPA

3.0

+

3

0

1

0.5

 

Second experiment

Doseμg/ml

S9-mix

Treatment time (h)

Polyploid cells*

Mean

Test group

1

2

Solvent control DMSO

0.0

-

20

0

4

2.0

Test item

25.0

-

20

6

6

6.0

75.0

-

20

66

2

4.0

250.0

-

20

0

1

0.5

Positive control EMS

400.0

-

20

0

1

0.5

 

Third experiment

Doseμg/ml

S9-mix

Treatment time (h)

Polyploid cells*

Mean

Test group

1

2

Solvent control DMSO

0.0

+

3

3

2

2.5

Test item

1285.0

+

3

1

7

 

 

 

 

5

6

4.8

Positive control CPA

3.0

+

3

2

0

1.0

* The number of polyploid cells was determined in 1000 cells

Conclusions:
The test substance was not mutagenic in this chromosome aberration test system in vitro with cells of the V79 Chinese hamster cell line under the conditions described in this report.
Executive summary:

The present study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 473 "Genetic Toxicology:In Vitro Mammalian Chromosome Aberration Test". Adopted: July 21st, 1997 and U.S. EPA: OPPTS 870.5375 Health Effects Test Guidelines In Vitro Mammalian Chromosome Aberration Test, August 1998 and EEC Directive 92/69, L 383 A, Annex B. 10, p. 148 -150. This study was conducted in compliance with the Principles of Good Laboratory Practice (GLP).

 

In this study the potential of the test substance to induce chromosome aberrations was investigated in V 79 cells of the Chinese hamster lung in vitro. For each experiment duplicate cultures were used for each concentration.

 

The test compound was suspended in DMSO and tested at the following concentrations:

- First experiment with 3 h treatment time:

without S9-mix: 25, 50, 75,125*, 250, 500*, 750 and 1285*μg/ml

with S9-mix:   25, 50, 75, 125*, 250, 500*, 750 and 1285*μg/ml

- Second experiment with 20 h treatment time:

without S9-mix: 5, 10, 25*, 50, 75*. 125 and 250*μg/ml

- Third experiment with 3 h treatment time:

with S9-mix: 1285*μg/ml

*= evaluated slides

 

The concentration ranges were based on the results of preliminary testing for solubility and toxicity. The highest concentration produced a distinct lowering of the mitotic index.

 

At concentrations of 500 μg/ml and above macroscopic visible precipitation of the test substance onto the slides was observed. Microscopically precipitation of the test compound was observed at 50 μg/ml and above.

 

There was an enhancement of the aberration rates at the 3 h treatment time with 1285 μg/ml with S9-mix, but only in one culture. These data were found significantly enhanced in the Fisher's exact-test.

Because of the different results between the duplicate cultures a third independent experiment with two slides and duplicate cultures was performed. In this experiment the enhancement of the aberration rate was not reproduced.

Without S9-mix up to the highest investigated dose the test compound induced no significant increase in the number of chromosome aberrations.

 

Appropriate reference mutagens used as positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay, and the efficacy of the S9-mix.

 

In conclusion, the test substance does not induce chromosome mutations (=aberrations) in V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described. It is therefore considered to be non-mutagenic in this chromosome aberration assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
09 November 1998 to 14 December 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: In vitro Mammalian Cell Gene Mutation Test EEC Directive 87/302, L133, pp. 61 - 63, March 1987
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase) locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
The compound was suspended in DMSO and tested at the following concentrations:
without S9-mix; 5.0, 7.5, 12.5, 25.0, 50.0, 75.0. 125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml (main mutation experiment)
50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)
with S9-mix: 5.0, 7.5, 12.5, 25.0, 50.0, 75.0, 125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml (main mutation experiment)
50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
Reference Compounds

Without metabolic activation
Name or number of compound (I.N.N. orU.SAN): ethyl methane sulfonate
Synonyms: EMS
Formula of the compound: C3Ha03S
CAS-Register number: 62-50-0
Product number / Code: 820774
Supplier of reference compound: Dr. Theodor Schuchardt & Co. Chemische Fabrik, Germany
Batch number: 40606721
Certificate of analysis: certificated by the supplier, Analytical Department, Dr. Bolkart dated December 18th, 1995

With metabolic activation
Name or number of compound (I.N.N. orU.S.A.N): 9,10-dimethyl-1,2-benzanthracene
Synonyms: DMBA
Chemical name: 7,12-dimethylbenz[a]anthracene
CAS-Register number: 57-97-6
Product number / Code: 21,626-7
Supplier of reference compound: Fa. Aldhch – Chemie, Germany
Batch number: TL 1825LL
Certificate of analysis: certificated by the supplier, Aldrich chemical company, David Swessel, dated April 3rd, 1996

Test system

Test groups
with and without metabolic activation: 5.0, 7.5, 12.5, 25.0, 50.0, 75.0, 125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml (main mutation experiment)
50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)

Control groups
negative controls: untreated control & cultures treated with the solvent

positive controls:
without metabolic activation: EMS (Ethyl methane sulfonate)
with metabolic activation: DMBA (9,10-dimethyl-1,2-benzanthracene)

Formulation of test compounds: suspended in DMSO at appropriate concentrations immediately before use.

Formulation of reference compounds: EMS dissolved in cell culture medium on the day of treatment, final concentration: 1.0 mg/ml = 8 mM.
DMBA dissolved in DMSO and frozen in small portions. Aliquot thawed on the day of treatment, final concentration in cell culture medium: 7.7 μg/ml = 30 μM

Source of biological material: cell bank of "Genetic Toxicology", HMR Deutschland GmbH, ProTox

Test organism: cell line V79 of Chinese hamster lung fibroblasts

Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer

Experimental conditions in vitro: approx. 37 °C and approx. 4 % CO2 in plastic flasks
Observations and Measurements

Preparation and storage of a liver homogenate fraction (S9)
The S9 fraction was prepared by the testing facility according to Ames et. al (1975). Male Sprague Dawley rats (200-300 g), supplied by Harlan Winkelmann, Gartenstrasse 27, 33178 Borchen, Germany, received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) 5 days before killing. The livers were removed from at least 5-6 animals at approx. 0 to 4 °C using cold sterile solutions and glassware, and were then pooled and washed in approx. 150 mM KCI (approximately 1 ml/g wet liver). The washed livers were cut into small pieces and homogenized in three volumes of KCI. The homogenate was cenfrifuged at approx. 9000g for 10 minutes. The supernatant, the S9 fraction, was divided into small portions, rapidly frozen and stored at approx. - 80 °C for not longer than six months. The protein content was determined for every batch. Also for every batch of S9 an independent validation was performed with a minimum of two different mutagens, e.g., 2-aminoanthracene and dimethylbenzanthracene to confirm metabolic activation by microsomal enzymes.

Preparation of S9-mix
Sufficient S9 fraction was thawed to room temperature immediately before each test. An appropriate quantity of S9 fraction (batch no. 98/1 for both mutation experiments, protein concentration 51.8 g/l) was mixed with S9 cofactor solution to yield a final protein concentration of 0.3 mg/ml in the cultures which was kept on ice until used. This preparation is termed S9-mix. The concentrations of the different components of the S9-mix were:
8 mM MgCI3
33 mM KCI
5 mM glucose-6-phosphate
5 mM NADP
100 mM phosphate buffer pH 7.4

Cell culture
Large stocks of the mycoplasma-free V79 cell line are stored in liquid nitrogen in the cell bank of "Genetic Toxicology", thus permitting repeated use of the same cell culture batch for numerous experiments. The identical characteristics of the cells ensure comparability of the experimental parameters.
Thawed stock cultures were kept at approx. 37 °C and approx. 4 % C03 in 175 cm2 plastic flasks. About 5 x 105 to 1 x 106 cells were seeded into each flask in 30 ml of MEM-medium supplement with approx. 10 % (v/v) FCS (fetal calf serum) containing approx. 2 mM L-glutamine and approx. 0.1 % (w/v) neomycinsulfate. The cells were subcultured twice a week.
For the selection of mutants the medium was supplemented with approx. 11 μg/ml thioguanine.

Toxicity experiments and dose range finding
A preliminary toxicity test was undertaken in order to select appropriate dose levels for the mutation assay. In this test a wide range of dose levels of test compound was used. Cell cultures were subjected to the same treatment conditions as in mutation assays, and the survival of the cells was subsequently determined.

The test included the following treatments:
Solvent control : the maximum final concentration of organic solvents will not exceed approx. 1 % (v/v).
Test compound : the highest dose level for the preliminary toxicity test was determined by the solubility of the test compound up to the maximum of 10 mM or 5000 μg/ml.
Treatments were performed both in the presence and absence of S9 metabolic activation system using a single cell culture at each test point.

Test procedure
In preliminary toxicity experiments approximately 4500 cells were seeded in each well of a microliter plate, allowed to attach overnight and then exposed to the test and control compound for four hours.
For each concentration at least 6 wells were used. Approx. 24 hours after treatment, the cells were fixed and stained with crystal violet.
Survival was determined by measurement of the crystal violet extinction.

In the main mutation experiments the cultures for assessing toxicity were prepared and treated with the test compound in the same way as for the preliminary experiment. 24 hours after seeding of approx. 4500 cells per well in a microtiter plate, the medium was replaced with serum-reduced (5 % v/v) medium containing the test compound to which either buffer or S9-mix was added as appropriate. After 4 hours the treatment medium was replaced with normal medium after rinsing twice with this. The cultures were stained with crystal violet and survival was determined after an incubation period of approx. 24 hours.

Rationale for dose selection
For non-toxic, freely soluble test compounds, the top dose should be 10 mM or 5000 ug/ml according to international testing guidelines.
For non-toxic, poorly soluble test compounds, the top dose should be the highest evaluable dose.
For toxic compounds the percentage survival relative to the solvent control should be calculated for each treatment. The dose level which resulted in a predicted survival of less than 30 % should be chosen as the highest dose level. At least eight respectively seven lower dose levels should be also included in each experiment.

Mutagenicity test
Two independent mutation tests were performed.
Two-day old, exponentially growing cultures which were more than 50 % confluent were trypsinated and a single cell suspension was prepared. The trypsin concentration was approx. 0.25 % (v/v) in Ca-Mg-free salt solution. The Ca-Mg-free salt solution was prepared as follows (per liter): NaCI 6.8 g; Ka 0.4 g; glucose 1 g; NaHC03 2.2 g; phenol red 5 mg; trypsin 2.5 g.
Subsequently the cells were replated to determine the mutation frequency and plating efficiency.

The treatment schedule of the mutagenicity test is described below:

Day 1: Subculturing of an exponentially growing culture
a) Approx. 4500 cells in each well of a microtiter plate for determination of the plating efficiency.
b) 6x 105 - 1 x 106 cells in 175 cm2 flasks with 30 ml medium for the mutagenicity test, one flask per experimental point.

Day 2: Treatment of a) and b) with the test compound in the presence and absence of S9-mix (final protein concentration: approx. 0.3 mg/ml) for 4 hours.

Day 3: Fixation and staining of the cells in a) for the determination of the plating efficiency.

Day 5: Subculturing of b) in 175 cm2 flasks

Day 9: Subculturing of b) in five 75 cm1 flasks with culture medium containing 6-thioguanine:
Mutant selection (about 300 000 cells/flask);
subculturing of b) in two 25 cm2 flasks for plating efficiency (about 400 cells per flask)

Day 16: Fixation and staining of colonies of b) - from subcultures seeded on day 9.

All incubations were carried out at approx. 37 °C and 4 % CO2.
Staining was performed with approx. 10 % (v/v) methylene blue in approx. 0.01 % (w/v) KOH solution.
Only colonies with more than 50 cells were counted.
Evaluation criteria:
Evaluation of data

Criteria for a valid assay
The assay is considered valid if the following criteria are met:
the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range
the plating efficacy for the solvent control was greater than 50 %

Criteria for a positive response
The test compound is classified as mutagenic if:
it reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment
there is a reproducible dose-related increase in the mutation frequency. Such an evaluation may be considered independently from the number induced mutants
survival of the responding dose group is at least 30 %
However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.
Statistics:
The biometry of the results for the test compound is performed off-line with the MANN-WHITNEY-U-TEST
Key result
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:
valid
Positive controls validity:
valid
Additional information on results:
Solubility and toxicity
The test substance was suspended in DMSO.
Evaluation of the solubility of that suspension in cell culture medium showed that 1285.0 μg/ml was the highest practicable concentration and produced precipitate.
Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 1285.0 μg/ml and a range of lower dose levels down to 10.0 pg/ml.
Following treatment in the absence of S9 metabolic activation, several toxicity was observed. Survival declined in a dose-related manner reaching 23.0 % of the solvent control value at the concentration of 750 μg/ml.
In the presence of S9 metabolic activation survival declined steeply up to the concentration of 500.0 μg/ml where survival was reduced to 64.8 % of the solvent control value.
Based on these results 1285 μg/ml was selected as the maximum dose level for the main mutation experiments in both the absence and in the presence of S9-mix. Ten lower concentrations down to 5 μg/ml were included in the main mutation experiment and seven lower concentrations down to 50 μg/ml in the repeat mutation experiment.

Mutagenicity
Experimental design
Two independent mutation assays to examine resistance to 6-thioguanine were performed.
In the absence and in the presence of S9 metabolic activation dose levels of 5.0, 7.5, 12.5, 25.0, 50.0, 75.0,125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml were used in the main mutation experiment, For the repeat mutation experiment a dose range of 50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml was selected in the absence and in the presence of S9-mix.
Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test compound solutions did not have any effect on these parameters.

Survival after treatment
In the absence of S9 metabolic activation in both mutation experiments a dose-related decrease in survival was observed reaching 53.2 % respectively 39.4 % of the solvent control value in the microtiter plates at the highest dose level tested, 1285.0 μg/ml.
In the presence of S9 metabolic activation survival decreased in a dose-related manner reaching approximately 45.3 % respectively 53.8 % of the solvent control value in the microtiter plates after treatment at the highest dose level, 1285.0 μg/ml.

Mutation results
The test compound was assessed for its mutagenic potential in vitro in the HPRT-test in two independent experiments without metabolic activation and two independent experiments with metabolic activation.
In the presence of metabolic activation a statistically significant increase of the mutation frequency was observed only at a concentration of 7.5 μg/ml. This effect was not dose-dependent and not three fold higher than the corresponding controls and therefore of no biological relevance.
No relevant reproducible increases in the mutant colonies or mutant frequency over the range of the solvent control was found with any of the concentrations used, either with or without metabolic activation by S9-mix.
The sensitivity of the test system and efficacy of the S9-mix was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

Toxicity Data (Main mutation experiment)

 

Dose μg/ml

S9-mix

Extinction in microwell plates mean less blank values

Standard deviation

Relative survival*

Negative control

0.0

-

0.572

0.02

121.5

+

0.451

0.05

125.3

Solvent control

0.0

-

0.471

0.04

100.0

-

0.424

0.06

100.0

+

0.360

0.06

100.0

+

0.411

0.08

100.0

Positive control

1000.0

-

0.504

0.01

107.0

7.7

+

0.361

0.04

100.3

Test item

5.0

-

0.499

0.02

105.9

+

0.461

0.03

128.0

7.5

-

0.529

0.08

112.3

+

0.462

0.03

128.3

12.5

-

0.495

0.02

105.0

+

0.448

0.04

124.4

25.0

-

0.440

0.01

93.5

+

0.437

0.03

121.4

50.0

-

0.420

0.02

89.1 #

+

0.378

0.03

105.2 #

75.0

-

0.398

0.02

84.1 #

+

0.413

0.03

114.7 #

125.0

-

0.483

0.10

113.9 #

+

0.387

0.03

94.2 #

250.0

-

0.319

0.02

75.2 #§

+

0.418

0.05

101.8 #§

500.0

-

0.253

0.02

59.6 #§

+

0.325

0.05

79.1 #§

750.0

-

0.276

0.02

65.2 #§

+

0.256

0.05

62.3 #§

1285.0

-

0.226

0.03

63.2 #§

+

0.186

0.04

45.3 #§

# microscopical visible precipitation

§ macroscopical visible precipitation

* relative survival (mean value / mean value corresponding control x 100)

Solvent = DMSO

Positive control without S9-mix = EMS

Positive control with S9-mix = DMBA

 

Mutagenicity Data – Part 1 (Main experiment)

 

Dose μg/ml

S9-mix

Number of cells per flask

Factor* calculated

Cells* seeded

Cells*** survived

Seeded

Found

Mean

I / II

I

II

Negative control

0.0

-

403

158.0

164.5

161.3

0.40

342900

137203

+

401

101.0

98.0

99.5

0.25

337500

83744

Solvent control 1 (DMSO)

0.0

-

398

370.5

370.0

370.3

0.93

292350

271966

+

399

370.0

372.0

371.0

0.93

298800

277832

Solvent control 2 (DMSO)

0.0

-

400

313.0

323.5

318.3

0.80

291150

231646

+

402

201.5

200.5

201.0

0.50

287700

143850

Positive control (EMS)

1000.0

-

400

288.0

273.5

280.8

0.70

285000

200034

Positive control (DMBA)

7.7

+

398

281.0

299.0

290.0

0.73

276750

201652

Test item

5.0

-

399

258.5

261.0

259.8

0.65

291900

190028

+

400

368.0

386.5

377.3

0.94

258600

243892

7.5

-

401

188.5

191.5

190.0

0.47

291000

137880

+

401

284.0

274.0

279.0

0.70

326400

227096

12.5

-

399

278.0

277.0

277.5

0.70

307800

214071

+

394

271.0

278.5

274.8

0.70

331650

231271

25.0

-

399

348.0

362.5

355.3

0.89

294300

262030

+

402

361.5

380.0

370.8

0.92

276150

254683

50.0

-

403

320.0

310.5

315.3

0.78

288150

225408

+

402

292.5

296.0

294.3

0.73

280650

205426

75.0

-

399

357.0

338.5

347.8

0.87

294300

256498

+

405

276.5

272.5

274.5

0.68

299250

202825

125.0

-

400

381.0

338.0

359.5

0.90

294300

264502

+

399

340.0

351.0

345.5

0.87

269400

233277

250.0

-

402

368.0

353.0

360.5

0.90

288750

258941

+

401

450.5

449.0

449.8

1.12

258150

289534

500.0

-

403

322.0

314.0

318.0

0.79

279750

220748

+

402

302.0

314.5

308.3

0.77

288900

221526

750.0

-

402

271.0

299.5

285.3

0.71

349200

247784

+

399

313.0

314.0

313.5

0.79

336000

264000

1285.0

-

400

156.0

144.0

150.0

0.38

337500

126563

+

405

301.0

306.5

303.8

0.75

347250

260438

* Factor calculated: mean value / number of cells per flask seeded

** Cells seeded in 6-thioguanine (TG) containing medium

*** Cells survived after plating in (TG) containing medium (cells seeded x factor calculated)

 

Mutagenicity Data – Part 2 (Main experiment)

 

Dose μg/ml

S9-mix

Number of mutant colonies

Standard deviation

Mutation frequency

Stat. sig.

I

II

III

IV

V

Mean

Negative control

0.0

-

1

4

1

3

2

2.2

1.30

16.0

 

+

2

1

2

3

0

1.6

1.14

19.1

 

Solvent control 1 (DMSO)

0.0

-

3

3

1

2

1

2.0

1.00

7.4

 

+

1

1

1

1

3

1.4

0.89

5.0

 

Solvent control 2 (DMSO)

0.0

-

9

11

6

3

6

7.0

3.08

30.2

 

+

3

1

2

7

2

3.0

2.35

20.9

 

Positive control (EMS)

1000.0

-

166

147

161

156

148

155.8

8.20

777.9

*

Positive control (DMBA)

7.7

+

35

35

45

40

36

38.2

4.32

189.4

*

Test item

5.0

-

1

0

0

1

3

1.0

1.22

5.3

 

+

5

3

1

1

1

2.2

1.79

9.0

 

7.5

-

1

1

1

2

0

1.0

0.71

7.3

 

+

8

5

12

10

6

8.2

2.88

35.1

*

12.5

-

3

3

0

4

3

2.6

1.52

12.1

 

+

3

2

1

3

1

2.0

1.00

8.6

 

25.0

-

5

5

1

2

3

3.2

1.79

12.2

 

+

1

3

1

4

4

2.6

1.52

10.2

 

50.0

-

0

0

4

1

1

1.2

1.64

5.3

 

+

1

1

1

3

0

1.2

1.10

5.8

 

75.0

-

1

4

5

5

2

3.4

1.82

13.3

 

+

5

1

2

7

8

4.6

3.05

22.7

 

125.0

-

1

4

2

0

2

1.8

1.48

6.8

 

+

2

1

2

7

3

3.0

2.35

12.9

 

250.0

-

5

6

2

4

2

3.8

1.79

14.7

 

+

5

0

2

2

4

2.6

1.95

9.0

 

500.0

-

4

6

2

1

2

3.0

2.00

13.6

 

+

4

1

8

3

5

4.2

2.59

19.0

 

750.0

-

6

7

3

5

3

4.8

1.79

19.4

 

+

11

8

2

7

11

7.8

3.70

29.5

 

1285.0

-

1

6

5

5

3

4.0

2.00

31.6

 

+

1

7

2

2

3

3.0

2.35

11.5

 

Mutation frequency (mutant colonies per 1 million cells); mean value / cells surviving

* Statistical significant (p≤0.05) Mann-Whitney-U-Test

Conclusions:
The test substance did not induce gene mutation, i.e. was not mutagenic, in this HPRT-test with V79 Chinese hamster cells, either in the presence or in the absence of metabolic activation.
Executive summary:

The present study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 476 "Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Test". Adopted: July 21, 1997 and U.S. Environmental Protection Agency (EPA) Health Effect Test Guidelines, OPPTS 870.5300, In Vitro Mammalian Cell Gene Mutation Test, august 1998 and In vitro Mammalian Cell Gene Mutation Test EEC Directive 87/302, L133, pp. 61 - 63, March 1987. This study was conducted in compliance with the Principles of Good Laboratory Practice (GLP).

 

The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster in vitro.

Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix).

The compound was suspended in DMSO and tested at the following concentrations:

without S9-mix; 5.0, 7.5, 12.5, 25.0, 50.0, 75.0. 125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml (main mutation experiment)

50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)

With S9 -mix: 5.0, 7.5, 12.5, 25.0, 50.0, 75.0, 125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml (main mutation experiment)

50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)

 

The concentration ranges were based on the results of preliminary tests for solubility and toxicity. The highest concentration showed slight toxic effects with and several toxic effects without metabolic activation.

In the presence of metabolic activation a significant increase of the mutation frequency was observed only at a concentration of 7.5 μg/ml. This effect was not dose-dependent and not three fold higher than the corresponding controls and therefore of no biological relevance.

Up to the highest investigated dose no further increase in mutant colony numbers was obtained in two independent experiments.

Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, thus indicating the sensitivity of the assay, and the efficacy of the S9-mix.

 

In conclusion, the test substance does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system under the experimental conditions described.

The test substance is therefore considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Read across with structural analogues showed no induction of DNA repair (as measured by unscheduled DNA synthesis) in rat liver nor of micronuclei in the polychromatic erythrocytes of treated rats.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
26 October to 18 November 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read across is based on the same physico-chemical properties, a close structural similarity and the same mechanism of action during use processes.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
- Source: Disperse Red 82
- Target: Disperse Red 184

3. ANALOGUE APPROACH JUSTIFICATION
see attachment in endpoint summary

4. DATA MATRIX
see attachment in endpoint summary
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
OECD Guideline For Testing Of Chemicals, 474 Genetic Toxicology, Mammalian Erythrocyte Micronucleus Test, Adopted 21st, July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
EEC Directive 92/69, L 383 A, Annex B. 12., p. 154 -156
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
U.S. EPA: OPPTS 870.5395 Health Effects Test Guidelines; Mammalian Erythrocyte Micronucleus Test, August 1998
Deviations:
no
Route of administration:
oral: gavage
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
All animals survived after treatment. No signs of toxicity were observed.
The bone marrow smears were examined for the occurrence of micronuclei in red blood cells.
The incidence of micronudeated polychromatic erythrocytes in the dose group of C. I. Disperse Red 82 was within the normal range of the negative control groups. No statistically significant increase of micronudeated polychromatic erythrocytes was observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and was not less than 20% of the control values.
Cyclophosphamide (Endoxan®) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system.

Summary tables and statistics

Test compound: C.I. Disperse Red 82

Sex

Dose

mg/kg b.w.

Killing time

Number

Poly/Ery

Poly/Ery

SD

Poly with MN

Poly with MN

[%]

Poly with MN

SD

of animals

Poly counted

Mean

Mean

Mean

Mean

Mean

Male

Male

Male

0 – Control

2000

50 – Endoxan

24 h

24 h

24 h

5

5

5

2000

2000

2000

0.48

0.48

0.42

0.07

0.04

0.04

1.6

2.2

60.6

0.08

0.11

3.03

0.03

0.11

0.69

Female

Female

Female

0 –Control

2000

50 – Endoxan

24 h

24 h

24 h

5

5

5

2000

2000

2000

0.54

0.53

0.44

0.05

0.06

0.05

1.2

2.4

53.8

0.06

0.12

2.69

0.07

0.06

0.59

 

Sex

Dose

mg/kg b.w.

Killing time

Number

Poly/Ery

Poly/Ery

SD

Poly with MN

Poly with MN

[%]

Poly with MN

SD

Mut I.

of animals

Poly counted

Mean

Mean

Mean

Mean

Mean

Pooled

Pooled

Pooled

0 – Control

2000

50 – Endoxan

24 h

24 h

24 h

10

10

10

2000

2000

2000

0.51

0.51

0.43

0.07

0.06

0.04

1.40

2.30

57.20*

0.1

0.1

2.9

0.05

0.09

0.63

1.0

1.6

40.9

Mut I. = Mutagenic index

Control = Vehicle Tylose HEC 4000 (0.5% w/v)

* = significantly different from control (p < 0.05)

 

Table of individual date

Test compound: C.I. Disperse Red 82

Group

Animal number

Sex

Dose

mg/kg b.w.

Poly/200 Ery

Poly

with MN

Poly/Ery

Poly

with MN [%]

1

1

1

1

1

1

1

1

1

1

1

2

3

4

5

6

7

8

9

10

Male

Male

Male

Male

Male

Female

Female

Female

Female

Female

0 – Control

0 – Control

0 – Control

0 – Control

0 – Control

0 – Control

0 – Control

0 – Control

0 – Control

0 – Control

114

78

97

102

91

111

99

123

97

113

2

2

1

2

1

0

2

1

0

3

0.57

0.39

0.49

0.51

0.46

0.56

0.50

0.62

0.49

0.57

0.10

0.10

0.05

0.10

0.05

0.00

0.10

0.05

0.00

0.15

2

2

2

2

2

2

2

2

2

2

11

12

13

14

15

16

17

18

19

20

Male

Male

Male

Male

Male

Female

Female

Female

Female

Female

2000

2000

2000

2000

2000

2000

2000

2000

2000

2000

89

99

109

91

90

100

107

91

122

113

2

2

0

6

1

2

3

4

2

1

0.45

0.50

0.55

0.46

0.45

0.50

0.54

0.46

0.61

0.57

0.10

0.10

0.00

0.30

0.05

0.10

0.15

0.20

0.10

0.05

3

3

3

3

3

3

3

3

3

3

21

22

23

24

25

26

27

28

29

30

Male

Male

Male

Male

Male

Female

Female

Female

Female

Female

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

73

96

83

79

84

80

82

79

100

95

79

71

56

49

48

58

361

33

60

57

0.37

0.48

0.42

0.40

0.42

0.40

0.41

0.40

0.50

0.48

3.95

3.55

2.80

2.45

2.40

2.90

3.05

1.65

3.00

2.85

 

Historical control values

 

Based on OECD 474, adopted 1997:

 

Micronucleated PCE / 2000 PCE

PCE / Ery (200 Ery counted)

Vehicle

Sex

N

Mean

STD

Min

Med

Max

N

Mean

STD

Min

Med

Max

DMA / PEG / citrate buffer

Female

5

2.4

0.89

1

3

3

5

0.43

0.06

0.37

0.42

0.51

Male

5

3.8

2.49

2

2

7

5

0.45

0.07

0.35

0.45

0.52

Deionized water

Female

25

1.88

1.36

0

2

6

25

0.50

0.06

0.41

0.51

0.66

Male

25

1.64

1.19

0

1

5

25

0.45

0.07

0.33

0.46

0.59

Sesame oil

Female

5

2

1.58

0

2

4

5

0.50

0.08

0.40

0.51

0.60

Male

5

3

1.58

1

3

5

5

0.48

0.10

0.34

0.47

0.59

 

Total

Female

35

1.97

1.32

0

2

6

35

0.49

0.07

0.37

0.50

0.66

Male

35

2.14

1.65

0

2

7

35

0.46

0.07

0.33

0.46

0.59

 

Based on OECD 474, adopted 1983:

 

Micronucleated PCE / 2000 PCE

PCE / Ery (200 Ery counted)

Vehicle

Sex

N

Mean

STD

Min

Med

Max

N

Mean

STD

Min

Med

Max

NaCl 0.9% +

1 N sodium hydroxid

Female

5

1.2

2.17

0

0

5

5

1.12

0.13

0.94

1.11

1.28

Male

5

0.6

0.55

0

1

1

5

1.00

0.10

0.87

1.01

1.11

Beriate buffer

Female

5

1.0

1.0

0

1

2

5

1.48

0.32

1.17

1.31

1.85

Male

5

1.0

1.0

0

1

2

5

1.09

0.56

0.65

0.85

2.01

DMSO

Female

5

0.2

0.45

0

0

1

5

0.98

0.15

0.79

1.00

1.14

Male

5

1.4

0.89

0

2

2

5

0.89

0.17

0.63

0.88

1.06

Dextrose 5%

Female

5

1.4

0.89

0

2

2

5

0.68

0.05

0.62

0.68

0.74

Male

5

1.4

0.55

1

1

2

5

0.96

0.24

0.70

0.96

1.26

Eluat Tecegen S

Female

5

1.4

1.52

0

1

3

5

1.08

0.07

1.01

1.08

1.19

Male

5

0.8

0.84

0

1

2

5

0.83

0.18

0.55

0.91

1.01

Deionized water

Female

55

0.87

0.90

0

1

3

55

0.92

0.17

0.57

0.91

1.36

Male

55

0.95

0.89

0

1

4

55

0.87

0.21

0.50

0.89

1.39

Lecithin emulsion

Female

5

1.0

0.71

0

1

2

5

0.80

0.014

0.69

0.73

1.02

Male

5

1.4

1.52

0

1

3

5

0.91

0.12

0.80

0.89

1.12

Methyl cellulose 1%

Female

15

1.2

1.08

0

1

3

15

0.89

0.20

0.61

0.84

1.35

Male

15

1.27

0.96

0

1

3

15

0.78

0.20

0.41

0.78

1.10

NaCl 0.9%

Female

15

1.27

1.10

0

1

4

15

1.01

0.21

0.62

0.94

1.33

Male

15

1.73

1.22

0

2

4

15

0.85

0.11

0.64

0.85

1.03

Insulin-placebo

Female

5

1.4

1.67

0

1

4

5

1.52

0.37

0.90

1.62

1.88

Male

5

2.0

1.41

1

1

4

5

2.10

0.34

1.53

2.24

2.35

Sesame oil

Female

50

1.36

1.10

0

1

4

50

0.93

0.19

0.52

0.93

1.25

Male

50

1.32

1.04

0

1

4

50

0.80

0.13

0.55

0.79

1.14

Starch mucilage

Female

120

1.13

1.02

0

1

4

120

0.97

0.18

0.53

0.99

1.38

Male

120

1.43

1.16

0

1

5

120

0.87

0.19

0.48

0.89

1.38

Tylose 0.1%

Female

10

0.9

0.99

0

1

3

10

0.93

0.20

0.67

0.89

1.24

Male

10

1

0..94

0

1

2

10

0.89

0.20

0.60

0.95

1.11

H2O + Na2CO3(2.24% w/v)

Female

5

0.6

0.55

0

1

1

5

0.65

0.25

0.39

0.57

1.04

Male

5

1.2

0.84

0

1

2

5

1.02

0.32

0.62

1.15

1.40

 

Total

Female

340

1.147

1.07

0

1

5

340

0.94

0.23

0.39

0.93

1.88

Male

340

1.31

1.09

0

1

6

340

0.87

0.25

0.41

0.85

2.35

PCE: Polychromatic Erythrocytes

NCE: Normochromatic Erythrocytes

Ery: Total Erythrocytes (PCE + NCE)

Conclusions:
The results lead to the conclusion that C. I. Disperse Red 82 did not lead to a substantial increase of micronudeated polychromatic erythrocytes and is not mutagenic in the micronucleus test under the conditions described in this report.
Executive summary:

The test compound was suspended in Tylose HEC 4000 (0.5% w/v) and was given twice at an interval of 24 hours as an orally dose of 2000 mg per kg body weight to male and female mice, based on the results of a previous dose range finding assay (see preliminary study).

According to the test procedure the animals were killed 24 hours after administration.

 

Endoxan® was used as positive control substance and was administered once orally at a dose of 50 mg per kg body weight.

 

The number of polychromatic erythrocytes containing micronuclei was not increased.

The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment and was not less than 20% of the control value.

 

Endoxan® induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

 

Under the conditions of the present study the results indicate that the test compound is not mutagenic in the micronucleus test.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
26 October to 18 November 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
OECD Guideline For Testing Of Chemicals, 474 Genetic Toxicology, Mammalian Erythrocyte Micronucleus Test, Adopted 21st, July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
EEC Directive 92/69, L 383 A, Annex B. 12., p. 154 -156
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
U.S. EPA: OPPTS 870.5395 Health Effects Test Guidelines; Mammalian Erythrocyte Micronucleus Test, August 1998
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
Solubility: suspension in Tylose HEC 4000 (0.5% w/v)
Stability and homogeneity in the vehicle: confirmed over 5 hours in Tylose HEC 4000 (0.5% w/v), signed October 26th, 1998
Concentration of stock suspension: 200 mg/ml
Species:
mouse
Strain:
NMRI
Details on species / strain selection:
Species of animals: mouse
Strain of animals: HsdWin:NMRI

Micronuclei are small secondary nuclear structures resulting from either chromosomal breakage or malfunction of the spindle apparatus of the cell which regulates the distribution of chromosomes during mitosis. The micronucleus test originally described by Schmid is a suitable in vivo method for investigating the clastogenic potential of substances and their ability to affect the mitotic spindle mechanism. Micronuclei rarely occur in normal dividing cells. The mouse has been chosen for this study since it provides a convenient in vivo mammalian model.
Sex:
male/female
Details on test animals and environmental conditions:
Species of animals: mouse
Strain of animals: HsdWin:NMRI
Origin (supplier) of animals: Harlan Winkelmann GmbH, Gartenstrasse 27, 33178 Borchen
Animal identification: fur marking with KMnO4 and cage numbering
Body weight at start of study
male animals
mean = 33.5 g (=100%)
min = 32.0 g (- 4.5 %)
max = 36.0 g (+ 7.5 %)
n =15

female animals
mean = 27.1 g (=100%)
min = 24.0 g (-11.4%)
max = 29.0 g (+ 7.0 %)
n =15

Age at the start of study male/female animals approximately 7 weeks
Randomization procedure randomization schemes 98.0890 and 98.0891

Animal maintenance: in fully air-conditioned rooms in makrolon cages type 3 (five animals per cage) on soft wood granulate
Room temperature: 22 ± 3 °C
Relative humidity: 50 ± 20 %
Lighting times: 12 hours daily
Acclimatization: 5 days under study conditions
Food: rat/mice diet ssniff® R/M-H (V 1534), ad libitum ssniff® GmbH, Postbox 2039, 59480 Soest
Water: tap water in plastic bottles, ad libitum
Route of administration:
oral: gavage
Vehicle:
Vehicle used: Tylose HEC 4000 (0.5% w/v)
Solubility in the vehicle: Suspension in Tylose HEC 4000 (0.5% w/v)
Stability and homogeneity in the vehicle: confirmed over 5 hours in Tylose HEC 4000 (0.5% w/v)
Concentration of test item in vehicle: 200 mg/ml
Amount of vehicle: 10 ml/kg body weight
Details on exposure:
Formulation of test compound: On the days of administration the test substance was suspended in Tylose HEC 4000 (0.5% w/v) at the appropriate concentration. A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.
Formulation of reference Compound: CPA dissolved in distilled water on the second day of experiment; final concentration: 0.5 % (w/v)
Duration of treatment / exposure:
48 hours
Frequency of treatment:
Twice at an interval of 24 hours
Post exposure period:
24 hours after last administration.
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per dose group (5 male/5 female).
Control animals:
yes
yes, concurrent vehicle
Positive control(s):
Name or number of compound (I.N.N, or U.S.A.N): cyclophosphamide
Synonyms: Endoxan®
Formula of the compound: C7H15CI2N2P-H2O
CAS-Register number: 50-18-0
Supplier of reference compound: ASTA Medica AG, Weismüllerstr. 45, D-60314 Frankfurt, Germany
Batch number: 603575B
Certificate of analysis: certified by the supplier, Quality Control, Dr. Milsmann dated January 10th, 1996
Tissues and cell types examined:
polychromatic erythrocytes
Details of tissue and slide preparation:
Test procedure
The test substance was administered twice at an interval of 24 hours orally by gavage to the test animals at a dose of 2000 mg per kg body weight. The vehicle, Tylose HEC 4000 (0.5% w/v), was administered in the same way to the negative control groups. The study included a concurrent positive control using Endoxan®, which was administered once orally by gavage at a dose of 50 mg per kg body weight.
Following dosing, the animals were examined regularly for mortality and clinical signs of toxicity.

Preparation and staining
Animals were killed by carbon dioxide asphyxiation 24 hours after dosing. For each animal, about 3 ml fetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.

Staining was performed as follows:
- 5 minutes in methanol
- 5 minutes in May-Grünwald's solution
- brief rinsing twice in distilled water
- 1 0 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan®

Evaluation
2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. In addition, the ratio of polychromatic erythrocytes to 200 total erythrocytes was determined. Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.
Evaluation criteria:
Both biological and statistical significances were considered together for evaluation purposes.
A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
A one-sided Wilcoxon-Test was evaluated to check the validity of the study. The study was considered as valid in case the proportion of polychromatic erythrocytes with micronuclei in the positive control was significantly higher than in the negative control (p=0.05).
If the validity of the study had been shown the following sequential test procedure for the examination of the mutagenicity was applied: Based on a monotone-dose relationship one-sided Wilcoxon tests were performed starting with the highest dose group. These test were performed with a multiple level of significance of 5%.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
All animals survived after treatment. No signs of toxicity were observed.
The bone marrow smears were examined for the occurrence of micronuclei in red blood cells.
The incidence of micronudeated polychromatic erythrocytes in the dose group of C. I. Disperse Red 82 was within the normal range of the negative control groups. No statistically significant increase of micronudeated polychromatic erythrocytes was observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and was not less than 20% of the control values.
Cyclophosphamide (Endoxan®) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system.

Summary tables and statistics

Test compound: C.I. Disperse Red 82

Sex

Dose

mg/kg b.w.

Killing time

Number

Poly/Ery

Poly/Ery

SD

Poly with MN

Poly with MN

[%]

Poly with MN

SD

of animals

Poly counted

Mean

Mean

Mean

Mean

Mean

Male

Male

Male

0 – Control

2000

50 – Endoxan

24 h

24 h

24 h

5

5

5

2000

2000

2000

0.48

0.48

0.42

0.07

0.04

0.04

1.6

2.2

60.6

0.08

0.11

3.03

0.03

0.11

0.69

Female

Female

Female

0 –Control

2000

50 – Endoxan

24 h

24 h

24 h

5

5

5

2000

2000

2000

0.54

0.53

0.44

0.05

0.06

0.05

1.2

2.4

53.8

0.06

0.12

2.69

0.07

0.06

0.59

 

Sex

Dose

mg/kg b.w.

Killing time

Number

Poly/Ery

Poly/Ery

SD

Poly with MN

Poly with MN

[%]

Poly with MN

SD

Mut I.

of animals

Poly counted

Mean

Mean

Mean

Mean

Mean

Pooled

Pooled

Pooled

0 – Control

2000

50 – Endoxan

24 h

24 h

24 h

10

10

10

2000

2000

2000

0.51

0.51

0.43

0.07

0.06

0.04

1.40

2.30

57.20*

0.1

0.1

2.9

0.05

0.09

0.63

1.0

1.6

40.9

Mut I. = Mutagenic index

Control = Vehicle Tylose HEC 4000 (0.5% w/v)

* = significantly different from control (p < 0.05)

 

Table of individual date

Test compound: C.I. Disperse Red 82

Group

Animal number

Sex

Dose

mg/kg b.w.

Poly/200 Ery

Poly

with MN

Poly/Ery

Poly

with MN [%]

1

1

1

1

1

1

1

1

1

1

1

2

3

4

5

6

7

8

9

10

Male

Male

Male

Male

Male

Female

Female

Female

Female

Female

0 – Control

0 – Control

0 – Control

0 – Control

0 – Control

0 – Control

0 – Control

0 – Control

0 – Control

0 – Control

114

78

97

102

91

111

99

123

97

113

2

2

1

2

1

0

2

1

0

3

0.57

0.39

0.49

0.51

0.46

0.56

0.50

0.62

0.49

0.57

0.10

0.10

0.05

0.10

0.05

0.00

0.10

0.05

0.00

0.15

2

2

2

2

2

2

2

2

2

2

11

12

13

14

15

16

17

18

19

20

Male

Male

Male

Male

Male

Female

Female

Female

Female

Female

2000

2000

2000

2000

2000

2000

2000

2000

2000

2000

89

99

109

91

90

100

107

91

122

113

2

2

0

6

1

2

3

4

2

1

0.45

0.50

0.55

0.46

0.45

0.50

0.54

0.46

0.61

0.57

0.10

0.10

0.00

0.30

0.05

0.10

0.15

0.20

0.10

0.05

3

3

3

3

3

3

3

3

3

3

21

22

23

24

25

26

27

28

29

30

Male

Male

Male

Male

Male

Female

Female

Female

Female

Female

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

50 – Endoxan

73

96

83

79

84

80

82

79

100

95

79

71

56

49

48

58

361

33

60

57

0.37

0.48

0.42

0.40

0.42

0.40

0.41

0.40

0.50

0.48

3.95

3.55

2.80

2.45

2.40

2.90

3.05

1.65

3.00

2.85

 

Historical control values

 

Based on OECD 474, adopted 1997:

 

Micronucleated PCE / 2000 PCE

PCE / Ery (200 Ery counted)

Vehicle

Sex

N

Mean

STD

Min

Med

Max

N

Mean

STD

Min

Med

Max

DMA / PEG / citrate buffer

Female

5

2.4

0.89

1

3

3

5

0.43

0.06

0.37

0.42

0.51

Male

5

3.8

2.49

2

2

7

5

0.45

0.07

0.35

0.45

0.52

Deionized water

Female

25

1.88

1.36

0

2

6

25

0.50

0.06

0.41

0.51

0.66

Male

25

1.64

1.19

0

1

5

25

0.45

0.07

0.33

0.46

0.59

Sesame oil

Female

5

2

1.58

0

2

4

5

0.50

0.08

0.40

0.51

0.60

Male

5

3

1.58

1

3

5

5

0.48

0.10

0.34

0.47

0.59

 

Total

Female

35

1.97

1.32

0

2

6

35

0.49

0.07

0.37

0.50

0.66

Male

35

2.14

1.65

0

2

7

35

0.46

0.07

0.33

0.46

0.59

 

Based on OECD 474, adopted 1983:

 

Micronucleated PCE / 2000 PCE

PCE / Ery (200 Ery counted)

Vehicle

Sex

N

Mean

STD

Min

Med

Max

N

Mean

STD

Min

Med

Max

NaCl 0.9% +

1 N sodium hydroxid

Female

5

1.2

2.17

0

0

5

5

1.12

0.13

0.94

1.11

1.28

Male

5

0.6

0.55

0

1

1

5

1.00

0.10

0.87

1.01

1.11

Beriate buffer

Female

5

1.0

1.0

0

1

2

5

1.48

0.32

1.17

1.31

1.85

Male

5

1.0

1.0

0

1

2

5

1.09

0.56

0.65

0.85

2.01

DMSO

Female

5

0.2

0.45

0

0

1

5

0.98

0.15

0.79

1.00

1.14

Male

5

1.4

0.89

0

2

2

5

0.89

0.17

0.63

0.88

1.06

Dextrose 5%

Female

5

1.4

0.89

0

2

2

5

0.68

0.05

0.62

0.68

0.74

Male

5

1.4

0.55

1

1

2

5

0.96

0.24

0.70

0.96

1.26

Eluat Tecegen S

Female

5

1.4

1.52

0

1

3

5

1.08

0.07

1.01

1.08

1.19

Male

5

0.8

0.84

0

1

2

5

0.83

0.18

0.55

0.91

1.01

Deionized water

Female

55

0.87

0.90

0

1

3

55

0.92

0.17

0.57

0.91

1.36

Male

55

0.95

0.89

0

1

4

55

0.87

0.21

0.50

0.89

1.39

Lecithin emulsion

Female

5

1.0

0.71

0

1

2

5

0.80

0.014

0.69

0.73

1.02

Male

5

1.4

1.52

0

1

3

5

0.91

0.12

0.80

0.89

1.12

Methyl cellulose 1%

Female

15

1.2

1.08

0

1

3

15

0.89

0.20

0.61

0.84

1.35

Male

15

1.27

0.96

0

1

3

15

0.78

0.20

0.41

0.78

1.10

NaCl 0.9%

Female

15

1.27

1.10

0

1

4

15

1.01

0.21

0.62

0.94

1.33

Male

15

1.73

1.22

0

2

4

15

0.85

0.11

0.64

0.85

1.03

Insulin-placebo

Female

5

1.4

1.67

0

1

4

5

1.52

0.37

0.90

1.62

1.88

Male

5

2.0

1.41

1

1

4

5

2.10

0.34

1.53

2.24

2.35

Sesame oil

Female

50

1.36

1.10

0

1

4

50

0.93

0.19

0.52

0.93

1.25

Male

50

1.32

1.04

0

1

4

50

0.80

0.13

0.55

0.79

1.14

Starch mucilage

Female

120

1.13

1.02

0

1

4

120

0.97

0.18

0.53

0.99

1.38

Male

120

1.43

1.16

0

1

5

120

0.87

0.19

0.48

0.89

1.38

Tylose 0.1%

Female

10

0.9

0.99

0

1

3

10

0.93

0.20

0.67

0.89

1.24

Male

10

1

0..94

0

1

2

10

0.89

0.20

0.60

0.95

1.11

H2O + Na2CO3(2.24% w/v)

Female

5

0.6

0.55

0

1

1

5

0.65

0.25

0.39

0.57

1.04

Male

5

1.2

0.84

0

1

2

5

1.02

0.32

0.62

1.15

1.40

 

Total

Female

340

1.147

1.07

0

1

5

340

0.94

0.23

0.39

0.93

1.88

Male

340

1.31

1.09

0

1

6

340

0.87

0.25

0.41

0.85

2.35

PCE: Polychromatic Erythrocytes

NCE: Normochromatic Erythrocytes

Ery: Total Erythrocytes (PCE + NCE)

Conclusions:
The results lead to the conclusion that C. I. Disperse Red 82 did not lead to a substantial increase of micronudeated polychromatic erythrocytes and is not mutagenic in the micronucleus test under the conditions described in this report.
Executive summary:

The micronucleus test was carried out with C. I. Disperse Red 82. The test compound was suspended in Tylose HEC 4000 (0.5% w/v) and was given twice at an interval of 24 hours as an orally dose of 2000 mg per kg body weight to male and female mice, based on the results of a previous dose range finding assay (see preliminary study).

According to the test procedure the animals were killed 24 hours after administration.

 

Endoxan® was used as positive control substance and was administered once orally at a dose of 50 mg per kg body weight.

 

The number of polychromatic erythrocytes containing micronuclei was not increased.

The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with C. I. Disperse Red 82 and was not less than 20% of the control value.

 

Endoxan® induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

 

Under the conditions of the present study the results indicate that C. I. Disperse Red 82 is not mutagenic in the micronucleus test.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
Two doses only of the test compound were applied: Only two test compound concentrations were required for scoring as the test substance dosed at a limit concentration is not cytotoxic in the liver. A third dose was therefore considered unnecessary.
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: ICI Barriered Animal Breeding Uni (BABU), Alderley Park, Macclesfield
- Age at study initiation:
- Weight at study initiation: 180-280 g
- Assigned to test groups randomly: yes - according to the order in which they are removed from the stock cage
- Fasting period before study:
- Housing: - pre dosing: up to 5 per cage
- for dosing: up to three per cage in a fume cupboard for a total period not exceeding 24 hours
- Diet (e.g. ad libitum): Porton Combined Diet, Special Diets Services Ltd, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: Since the animals were eventually moved in a fume cupboard, acclimatisation was not considered appropriate


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 21
- Humidity (%): 40 - 60
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: test substance in insoluble in water
- Concentration of test material in vehicle: 125 and 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg body weight
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing suspensions of the test substance were prepared in corn oil.


Duration of treatment / exposure:
single dose
Frequency of treatment:
once

Post exposure period:
4 and 12 h after treatment

Dose / conc.:
1 250 mg/kg bw/day (nominal)
Remarks:
in corn oil
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
in corn oil
No. of animals per sex per dose:
5 males/dose/time point in treatment group
4 males/group/time point in vehicle and positive control group - only 1 animal/group/time point scored

Control animals:
yes, concurrent vehicle
Positive control(s):
2-acetylaminofluorene [2AAF] at twelve hours and N-nitrosodimethylamine [NDMA] or 6-p-dimethylaminophenylazobenzthiazole [6BT] at four hours
Tissues and cell types examined:
Hepatocytes

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on acute oral toxicity study in rats, in which the acute MTD for the test substance was >5000 mg/kg bw and a range finding study in 5 males at 2000 mg/kg bw

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Single oral dose by oral gavage. Slides from the animals were subsequently analysed. Of the two positive and two vehicle control animals in each experiment, only one of each was scored for induction of UDS

DETAILS OF SLIDE PREPARATION: Preparation of hepatocytes were made 4 or 12 h after dosage. Hepatocytes were prepared from treated animals by a two stage collagenase perfusion technique. Hepatocyte cultures were prepared by allowing cells to attach to plastic cover slips. Medium was removed from the dishes and replaced with fresh medium containing [3H] thymidine. After 4 h incubation at 37 °C within a 5% CO2/95% air (v/v) atmosphere, the medium was removed, the cells washed three times with medium containing unlabelled thymidine and the cultures incubated overnight with the same medium. Cultures were fixed and coverslips mounted onto microscope slides. Slides were coated with photographic emulsion and left for 14 d at 4 °C in the dark. The emulsion was developed, fixed and the cell nuclei and cytoplasm stained with Meyers haemalum and eosin Y phloxine.
Slides were examined microscopically for signs of undue cytotoxicity to enable selection of those to be examined for UDS.


METHOD OF ANALYSIS: Prior to microscopic assessment, all slides were furnished with code numbers, so that the counting was blind. The following counts were made:
- at least 25 but normally 50 cells/slide of two slides for each animal. The third slide was not normaly read, unles a total of 10 cells could not be obtained from the
first two slides examined.
- The nuclear count (the number of silver grains over the nucleus) and the cytoplasmic count (the number of grains in an adjacent, nuclear sized, most heavily labelled area of cytoplasm) were measured using an automated image analyser and the data captured directly into a computer.
- The mean net grain count (nuclear count - cytoplasmic count), the mean nuclear count and cytoplasmic counts and the percent of cells in repair (net grain count of 5 or greater) to be calculated.
Evaluation criteria:
EVALUATION CRITERIA
Criteria for a positive response:
- The criteria outlined in the ASTM Guideline are adopted, based on the mean net group grain count and the percentage of cells in repair. A treated group showing mean net nuclear grain counts of 5 or greater and 20% or greater of the cells in repair is considered to be a positive response. A compound can only be assigned as an unequivocal genotoxin in this assay if such a response is reproduced.

Criteria for a negative response:
- A negative response is obtained where the mean net nuclear grain count of the treated cultures is less than 0, and the percentage of cells in repair is also less than 20.


VALIDATION CRITERIA
Negative Controls
Cytoplasmic counts of less than 40 are considered acceptable. Mean net nuclear grain counts for the negative controls should be less than zero.

Positive Controls
Mean net grain counts for the positive control in each experiment shall be sufficient to be regarded as an unequivocal positive response; mean net nuclear grain value of 5 or greater, with at least 20% of the cells examined in repair (ie with 5 or more net grains).
Statistics:
difference between treated and control cultures
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
No significant adverse reactions to treatment
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: No adverse effects at 5000 mg/kg bw in acute oral or 2000 mg/kg body weight for four days in range finding study

RESULTS OF DEFINITIVE STUDY
- Animal Toxicity: No significant signs of acute toxic effects were observed.

Others:
Test substance caused no significant increases over the vehicle control in mean net nuclear grain count, nor in percentage of cells in repair, at either dose level (1250 or 2000 mg/kg bw) or time point (4 or 14 h) investigated. Hepatocytes from test substance treated animals had mean net nuclear grain values of less than zero. These data therefore provided no evidence for induction of UDS by test substance.

None

Conclusions:
Under the test conditions, the test substance did not induce DNA repair in rat liver in vivo up to a limit dose of 2000 mg/kg bw
Executive summary:

CI Disperse Blue 291 was tested for the ability to induce unscheduled DNA synthesis (UDS) in an in vivo rat hepatocyte assay. Male Fischer 344 rats were treated with a single oral dose of CI Disperse Blue 291 by gavage at 1250, or 2000 mg/kg body weight. The highest test dose, 2000 mg/kg was the limit test dose for a non-toxic test agent in this assay. Animals were killed and hepatocytes prepared four hours and twelve hours following administration of the chemical. Two independent experiments were carried out for each time point.

Hepatocytes from treated rats were exposed to [³H]-thymidine and the amount of radioactivity incorporated into the nucleus [N] and an equal area of cytoplasm [C] determined by autoradiography. The cytoplasmic grain count was subtracted from that of the nucleus. The value obtained, the mean net nuclear grain count [N-C], is an index of UDS activity. In the respective testing laboratory, no negative control animal has shown a mean net nuclear grain count greater than zero. An [N-C] of more than zero in a treated animal is therefore considered indicative of a UDS response.

Each experiment was validated by concurrent control treatments of rats with corn oil, the solvent for CI Disperse Blue 291 and with the carcinogens 2-acetylaminofluorene [2AAF] at twelve hours and N-nitrosodimethylamine [NDMA] or 6-p-dimethylaminophenylazobenzthiazole [6BT] at four hours. Solvent treated rats gave rise to mean net grain counts of less than zero, whilst hepatocytes from 2AAF, 6BT or NDMA treated animals had mean net nuclear grain counts of greater than +5. These data showed that background levels of UDS were normal and that the tester animals were responsive to known carcinogens requiring metabolic activation for genotoxic activity.

Hepatocytes from CI Disperse Blue 291 treated animals were assessed for UDS at two dose levels of 1250 and 2000 mg/kg body weight. Treatments with CI Disperse Blue 291 in no case resulted in a mean net grain count greater than zero, at either time point.

It is concluded that, when tested up to a limit dose of 2000 mg/kg body weight, the test sample of CI Disperse Blue 291 did not induce DNA repair (as measured by unscheduled DNA Synthesis) in hepatocytes from rats treated in vivo.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read across is based on the same physico-chemical properties, a close structural similarity and the same mechanism of action during use processes.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
- Source: Disperse Blue 291 Br
- Target: Disperse Red 184

3. ANALOGUE APPROACH JUSTIFICATION
see attachment in endpoint summary

4. DATA MATRIX
see attachment in endpoint summary
Reason / purpose:
read-across source
Principles of method if other than guideline:
Two doses only of the test compound were applied: Only two test compound concentrations were required for scoring as the test substance dosed at a limit concentration is not cytotoxic in the liver. A third dose was therefore considered unnecessary.
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Fischer 344
Route of administration:
oral: gavage
Dose / conc.:
1 250 mg/kg bw/day (nominal)
Remarks:
in corn oil
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
in corn oil
Control animals:
yes, concurrent vehicle
Positive control(s):
2-acetylaminofluorene [2AAF] at twelve hours and N-nitrosodimethylamine [NDMA] or 6-p-dimethylaminophenylazobenzthiazole [6BT] at four hours
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
No significant adverse reactions to treatment
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: No adverse effects at 5000 mg/kg bw in acute oral or 2000 mg/kg body weight for four days in range finding study

RESULTS OF DEFINITIVE STUDY
- Animal Toxicity: No significant signs of acute toxic effects were observed.

Others:
Test substance caused no significant increases over the vehicle control in mean net nuclear grain count, nor in percentage of cells in repair, at either dose level (1250 or 2000 mg/kg bw) or time point (4 or 14 h) investigated. Hepatocytes from test substance treated animals had mean net nuclear grain values of less than zero. These data therefore provided no evidence for induction of UDS by test substance.

None

Conclusions:
Under the test conditions, the test substance did not induce DNA repair in rat liver in vivo up to a limit dose of 2000 mg/kg bw
Executive summary:

CI Disperse Blue 291 was tested for the ability to induce unscheduled DNA synthesis (UDS) in an in vivo rat hepatocyte assay. Male Fischer 344 rats were treated with a single oral dose of CI Disperse Blue 291 by gavage at 1250, or 2000 mg/kg body weight. The highest test dose, 2000 mg/kg was the limit test dose for a non-toxic test agent in this assay. Animals were killed and hepatocytes prepared four hours and twelve hours following administration of the chemical. Two independent experiments were carried out for each time point.

Hepatocytes from treated rats were exposed to [³H]-thymidine and the amount of radioactivity incorporated into the nucleus [N] and an equal area of cytoplasm [C] determined by autoradiography. The cytoplasmic grain count was subtracted from that of the nucleus. The value obtained, the mean net nuclear grain count [N-C], is an index of UDS activity. In the respective testing laboratory, no negative control animal has shown a mean net nuclear grain count greater than zero. An [N-C] of more than zero in a treated animal is therefore considered indicative of a UDS response.

Each experiment was validated by concurrent control treatments of rats with corn oil, the solvent for CI Disperse Blue 291 and with the carcinogens 2-acetylaminofluorene [2AAF] at twelve hours and N-nitrosodimethylamine [NDMA] or 6-p-dimethylaminophenylazobenzthiazole [6BT] at four hours. Solvent treated rats gave rise to mean net grain counts of less than zero, whilst hepatocytes from 2AAF, 6BT or NDMA treated animals had mean net nuclear grain counts of greater than +5. These data showed that background levels of UDS were normal and that the tester animals were responsive to known carcinogens requiring metabolic activation for genotoxic activity.

Hepatocytes from CI Disperse Blue 291 treated animals were assessed for UDS at two dose levels of 1250 and 2000 mg/kg body weight. Treatments with CI Disperse Blue 291 in no case resulted in a mean net grain count greater than zero, at either time point.

It is concluded that, when tested up to a limit dose of 2000 mg/kg body weight, the test sample of CI Disperse Blue 291 did not induce DNA repair (as measured by unscheduled DNA Synthesis) in hepatocytes from rats treated in vivo.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

The test item Disperse Red 184 was tested positivein the Bacteria Reverse Mutation Assay (Ames test) in Salmonella strains with and without metabolic activation. In the OECD QSAR Toolbox, the prediction for gene mutation was negative. In the chromosome aberration test in V79 cells,Disperse Red 184was negative with metabolic activation and showed a positive response only at a concentration which showed distinct cytotoxicity and visible precipitation. The prediction by the OECD QSAR Toolbox was clearly negative. A close structural analogue (Structural Analogue 02), was also tested positive in the Ames Test, but negative in the in-vitro mutation assay in mammalian cells (HPRT) and an in-vitro chromosome aberration test in V79 cells. Another structural analogue (Disperse Blue 291) was tested positive in an Ames test with nitroreductase and O‑acetyltransferase positive Salmonella typhimurium strains, but negative with nitroreductase and O‑acetyltransferase negative strains andin a test for unscheduled DNA synthesis. Another structural analogue (Disperse Red 82) was tested negative in an in-vivo mouse micronucleus test.

This positive effect in the bacterial mutation assay is a bacteria-specific effect due to bacterial nitro-reductases, which are highly effective in these bacterial strains, but not in mammalian cells.

This effect was also in-depth investigated in another structurally similar dye Disperse Blue 367and in several pharmaceuticals (see below).

Disperse Blue 367was tested positive in a standard Ames test, but negative in the following tests:

Study Type

Metabolic activation

Result

Mitotic recombination assay withSaccharomyces cerevisae

Rat liver S9-mix

Negative

Point mutation assay withSaccharomyces cerevisae

Rat liver S9-mix

Negative

HPRT assay with V79 hamster cells

Rat liver S9-mix

Negative

UDS assay with rat hepatocytes

Metabolic competent hepatocytes were used

Negative

Micronucleus assay in vivo (mouse)

In vivo assay

Negative

The nitroreductase family comprises a group of flavin mononucleotide (FMN)- or flavin adenine dinucleotide (FAD) -dependent enzymes that are able to metabolize nitroaromatic and nitroheterocyclic derivatives (nitrosubstituted compounds) using the reducing power of nicotinamide adenine dinucleotide (NAD(P)H). These enzymes can be found in bacterial species and, to a lesser extent, in eukaryotes. The nitroreductase proteins play a central role in the activation of nitro-compounds. Type I nitroreductases can transfer two electrons from NAD(P)H to form the nitroso and hydroxylamino intermediates and finally the amino group. Type II nitroreductases transfer a single electron to the nitro group, forming a nitro anion radical, which in the presence of oxygen generates the superoxide anion in a futile redox cycle, regenerating the nitro group [de Oliveira et al. 2010].

The positive effect in the bacterial reverse mutation test (Ames) was clearly related to a bacteria-specific metabolism of the test substance, as it is well-known for aromatic nitro compounds to be positive in the Ames assay resulting from metabolism by the bacteria-specific enzyme nitro-reductase [Tweats et al. 2012]. This could be also be proved to be true in studies with Disperse Blue 291, which was tested for mutagenic activity in the Salmonella assay with strains with different levels of nitroreductase and O-acetyltransferase[Umbuzeiro et al. 2005]. In this study,Disperse Blue 291 showed mutagenic activity with all standard strains of Salmonella typhimurium tested (TA1537, TA1538, TA98 and TA100), except for TA1535.In nitroreductase and O-acetyltransferasenegative strains (TA98NR, TA98DNP6) not mutagenic activity was observed in the absence of S9, whereas themutagenic activity was increased with the nitroreductaseand/or O‑acetyltransferaseoverproducing strains, (YG1021, YG1024 and YG1041) This shows the importance of the bacterial acetyltransferase enzyme in the activation of Disperse Blue 291. Because of the remarkable increase in the response with the nitroreductase and O‑acetyltransferase overproducing strain (YG1041), it is assumed that the product of the nitroreductaseis a substrate for the O-acetyltransferase. As there was a very slight increase in mutagenicity with TA98NR, TA98, YG1021, TA98DNP6, and YG1024 in the presence of S9, it was assumed that P450 enzymes have also a role in the activation ofDisperse Blue 291, besides the bacterial enzymes. This could however not proven true in studies withDisperse Orange 288 in mammalian cells (HPRT, CA assay) or in in-vivo studies with Disperse Blue 291 (UDS) or Disperse Red 82 (MNT).

It has also been demonstrated in various other publications that this mutagenic activity is a bacteria-specific effect and that these Ames positive nitro-substances are not mutagenic in mammalian assays.

That the reduction of these nitro-compounds to mutagenic metabolites is a bacteria-specific effect is demonstrated in the following by means of the two compounds AMP397 and fexinidazole.

  • AMP397 is a drug candidate developed for the oral treatment of epilepsy. The molecule contains an aromatic nitro group, which obviously is a structural alert for mutagenicity. The chemical was mutagenic in Salmonellastrains TA97a, TA98 and TA100, all without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR. Accordingly, the ICH standard battery mouse lymphomatkand mouse bone marrow micronucleus tests were negative, although a weak high toxicity-associated genotoxic activity was seen in a micronucleus test inV79 cells [Suter et al. 2002].The amino derivative of AMP397 was not mutagenic in wild type TA98 and TA100. To exclude that a potentially mutagenic metaboliteis released by intestinal bacteria, a MutaTMMouse study was done in colon and liver with five daily treatments at the MTD, and sampling of 3, 7 and 21 days post-treatment. No evidence of a mutagenic potential was found in colon and liver. Likewise, a comet assay did not detect any genotoxic activity in jejunum and liver of rats, after single treatment with a roughly six times higher dose than the transgenic study, which reflects the higher exposure observed in mice. In addition, a radioactive DNA binding assay in the liver of mice and rats did not find any evidence for DNA binding. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. It was hypothesized that the positive Ames test was due to activation by bacterial nitro-reductase, as practically all mammalian assays including fourin vivoassays were negative, and no evidence for activation by mammalian nitro-reductase or other enzymes were seen. Furthermore, no evidence for excretion of metabolites mutagenic for intestinal cells by intestinal bacteria was found.
  • Fexinidazolewas in pre-clinical development as a broad-spectrum antiprotozoal drug by the Hoechst AG in the 1970s-1980s, but its clinical development was not pursued. Fexinidazole was rediscovered by the Drugs for Neglected Diseases initiative (DNDi) as drug candidate to cure the parasitic disease human African trypanomiasis (HAT), also known as sleeping sickness. The genotoxicity profile of fexinidazole, a 2-substituted 5-nitroimidazole, and its two active metabolites, the sulfoxide and sulfone derivatives were investigated [Tweats et al. 2012]. All the three compounds are mutagenic in the Salmonella/Ames test; however, mutagenicity is either attenuated or lost in Ames Salmonella strains that lack one or more nitroreductase(s). It is known that these enzymes can nitroreduce compounds with low redox potentials, whereas their mammalian cell counterparts cannot, under normal conditions. Fexinidazole and its metabolites have low redox potentials and all mammalian cell assays to detect genetic toxicity, conducted for this study either in vitro (micronucleus test in human lymphocytes) or in vivo (ex vivo unscheduled DNA synthesis in rats; bone marrow micronucleus test in mice), were negative.

Based on these data and the common mechanism between the reduction of these nitro-compounds, which is widely explored in literature [de Oliveira et al. 2010], it is concluded, that the mutagenic effects observed in the Ames test withDisperse Red 184is a bacteria specific effect and not relevant to mammalians.

Disperse Red 184and its structural analogues were not genotoxic in the in-vitro mammalian cell mutagenicity (HPRT assay) and chromosome aberration test and the in-vivo UDS and MNT test. Therefore, a direct genotoxic effect as well as a metabolisation towards genotoxic structures by mammalian species can be excluded.

References

De Oliveira IM, Bonatto D, Pega Henriques JA. Nitroreductases: Enzymes with Environmental Biotechnological and Clinical Importance. InCurrent Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology; Mendez-Vilas, A., Ed.; Formatex: Badajoz, Spain, 2010:1008–1019.

Suter W, Hartmann A, Poetter F, Sagelsdorff P, Hoffmann P, Martus HJ. Genotoxicity assessment of the antiepileptic drug AMP397, an Ames-positive aromatic nitro compound. Mutat Res. 2002 Jul 25;518(2):181-94.

Umbuzeiro GA, Freeman H, Warren SH, Kummrow F, Claxton LD. Mutagenicity evaluation of the commercial product CI Disperse Blue 291 using dierent protocols of the Salmonella assay. Food and Chemical Toxicology 2005;43:49–56.

Tweats D, Bourdin Trunz B, Torreele E. Genotoxicity profile of fexinidazole--a drug candidate in clinical development for human African trypanomiasis (sleeping sickness). Mutagenesis. 2012 Sep;27(5):523-32.

Additional information

Bacteria reverse mutation

The mutagenic activity of Disperse Red 184,was conducted according to ETAD Test Method 005 and was designed to assess the mutagenic potential of the test material using a bacterial/microsome test system . The study was based on the in vitro technique described by Ames and his co-workers and Garner et al in which mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium to various concentrations of the test compound.

The strains used in this assay were all mutants derived from Salmonella typhimurium and were those recommended for general screening: TA 1535, TA 100, TA 1537, TA 98.

In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test compound to the tester organisms. No cytotoxicity was observed at the tested concentrations of 4 to 2500 µg/plate.

Concentrations of 4, 20, 100, 500, and 2500 µg/plate were hence used for the main assay.

The test substance was negative in TA 1535 without metabolic activation and weakly positive in TA 100 and TA 1537 without metabolic activation. A stronger positive reaction was observed in TA 98 without metabolic activation and in all strains with metabolic activation.

Disperse Blue 291 (commercial product: 30 to 50% dye content) was tested for mutagenic activity in the Salmonella assay with strains with different levels of nitroreductase andO-acetyltransferase (i.e., TA98DNP6, YG1024, and YG1041) as well as standard strains TA 1535, TA1537, TA1538, TA98 and TA100 and strains which provide more information on the base-pair substitution (TA 7001 to 7006). Disperse Blue 291 showed direct-acting mutagenic activity with all strains of Salmonella typhimuriumtested, except for TA 1535.According to the classification of Claxton et al. (1991), the potency of this product can be considered moderate (10-100 revertants/µg). In the absence of S9, the nitroreduction is strongly related to the mutagenic activity, because the mutagenicity of Disperse Blue 291 was very low when tested with the strains lacking nitroreductase activity (TA98NR) and was increased with the nitroreductase overproducing strains, (YG1021 and YG1041). The same mutagenic pattern was observed for the acetyltransferase deficient and overproducing strains (TA98DNP6, YG1024, and YG1041) revealing also the importance of the acetyltransferase enzyme in the activation of Disperse Blue 291. Because of the remarkable increase in the response with the nitroreductase andO-acetyltransferase overproducing strain (YG1041), it is likely that the product of the nitroreductase is a substrate for theO-acetyltransferase.

In the presence of S9, the mutagenicity was slightly increased with TA98NR, TA98, YG1021, TA98DNP6, and YG1024 suggesting that, P450 enzymes also have a role in the activation of these compounds, besides the bacterial enzymes. This could be explained by the activation of other radicals of the molecule by the S9 enzymes, for example the —OCH3or the —N(CH2CH3)2 of Disperse Blue 291, which however proved not to happen in studied with mammalian cells or in-vivo studies, as shown in the negative results with these studies.

Mammalian cell gene mutation

The study was performed to investigate the potential of the test item, Structural Analogue 02, to induce gene mutations at the HPRT locus in V 79 cells of the Chinese hamster in vitro. Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix). The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster in vitro. Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix). The compound was suspended in DMSO and tested at concentrations of 5, 7.5, 12.5, 25, 50, 75, 125, 250, 500, 750 and 1285 µg/mL in the main mutation experiment and 50, 75, 125, 250, 500, 750, 1000 and 1285 µg/mL in the repeat mutation experiment.

The concentration ranges were based on the results of preliminary tests for solubility and toxicity. The highest concentration showed slight toxic effects with and several toxic effects without metabolic activation.

In the presence of metabolic activation, a significant increase of the mutation frequency was observed only at a concentration of 7.5 µg/mL. This effect was not dose-dependent and not three-fold higher than the corresponding controls and therefore of no biological relevance. Up to the highest investigated dose no further increase in mutant colony numbers was obtained in two independent experiments.

In conclusion, the test substance does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system under the experimental conditions described. The test substance is therefore considered to be non-mutagenic in this HPRT assay.

Chromosome aberration

In this study the potential of Disperse Red 184 to induce chromosome aberrations was investigated in V 79 cells of the Chinese hamster lung in vitro. For each experiment two cell cultures were used. Disperse Red 184 was suspended in DMSO. Evaluation of the solubility of that suspension in cell culture medium showed that 1000 µg/mL was the highest practicable concentration and produced precipitate. Accordingly, a preliminary toxicity study was carried out using a maximum concentration of 1000 µg/mL and a range of lower dose levels down to 10 µg/mL. Following treatment in the absence of S9 metabolic activation, severe toxicity was observed at 500 µg/mL and above. Survival declined in a dose-related manner reaching 29.2 % of the solvent control value at the highest dose level, 1000 µg/mL. In the presence of metabolic activation (S9-mix) there was only a slight indication of toxicity up to the limit of solubility. Hence, the test compound was suspended in DMSO at the following concentrations:

without S9-mix:

20 h: 3.16#, 10.0#, 31.6, 100.0, 316.0 and 1000.0* µg/mL

28 h: 31.6#, 100.0#, 316.0 and 1000.0* µg/mL

with S9-mix:

20 h: 31.6#, 100.0, 316.0 and 1000.0 µg/mL

28 h: 316.0# and 1000.0 µg/mL

* not evaluated because of high toxicity

# not used because higher concentrations were evaluated

The highest concentrations produced no relevant lowering of the mitotic index in the presence of metabolic activation and a distinct lowering of the mitotic index in the absence of metabolic activation. Microscopic visible precipitation of the test compound was observed at 10 µg/mL and above in the absence of S9-mix and at 100 µg/mL and above in the presence of S9-mix. After treatment with the test compound there was no relevant increase in the number of polyploid cells as compared with the solvent controls.

There was an enhancement of the aberration rates inclusive and exclusive gaps 20 h and 28 h after the start of the treatment with the concentration of 316 µg/mL (relative mitotic index 19.4% and 44%, respectively) without S9-mix. In addition, an increased number of cells with break events was found in these groups. In the presence of S9-mix no relevant reproducible enhancement of metaphases over the range of the solvent control was found with any of the concentrations used. As the increase in aberration rates were only observed at 316 µg/mL without metabolic activation and in the presence of cytotoxicity and visible test substance precipitation, the relevance and reliability of this result is questionable.

The potential of Structural Analogue 02 to induce chromosome aberrations was investigated in V 79 cells of the Chinese hamster lung in vitro. For each experiment duplicate cultures were used for each concentration. The test item was suspended in DMSO. Evaluation of the solubility of that suspension in cell culture medium showed that 1285 µg/mL was the highest practicable concentration and produced precipitate. Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 1285 µg/mL and a range of lower dose levels down to 10 µg/mL. Following treatment for 3 hours, severe toxicity was observed at 100 µg/mL and above. Survival declined in a dose-related manner reaching 35.3 % in the absence of metabolic activation and 55.4 % in the presence of S9-mix of the solvent control value at a dose level of 750 µg/mL. After 20 hours treatment survival was also dose-dependently reduced reaching 17.5 % of the solvent control value at a concentration of 500 µg/mL. Hence, the test compound was suspended in DMSO at the following concentrations:

- First experiment with 3 h treatment time:

without S9-mix: 25, 50, 75,125*, 250, 500*, 750 and 1285*µg/mL

with S9-mix:   25, 50, 75, 125*, 250, 500*, 750 and 1285*µg/mL

- Second experiment with 20 h treatment time:

without S9-mix: 5, 10, 25*, 50, 75*. 125 and 250*µg/mL

- Third experiment with 3 h treatment time:

with S9-mix: 1285*µg/mL

*= evaluated slides

In the main experiments cytotoxicity was also evaluated by treatment of cells seeded in microwell plates. The highest concentration produced a distinct lowering of the mitotic index. At concentrations of 500 µg/mL and above macroscopic visible precipitation of the test substance onto the slides was observed. Microscopically precipitation of the test compound was observed at 50 µg/mL and above.

After treatment with the test compound there was no relevant increase in the number of polyploid cells as compared with the solvent controls. There was an enhancement of the aberration rates at the 3 h treatment time with 1285 µg/mL with S9-mix, but only in one culture. Because of the different results between the duplicate cultures a third independent experiment with two slides and duplicate cultures was performed additionally. In this experiment the enhancement of the aberration rate was not reproduced. No relevant reproducible enhancement of metaphases with aberrations over the range of the solvent control was found with any of the concentrations used, either with or without metabolic activation by S9-mix.

In conclusion, the test item was not clastogenic in this chromosome aberration test system in vitro with cells of the V79 Chinese hamster cell line under the conditions described in this report.

Unscheduled DNA Synthesis

Disperse Blue 291 (96%purity)was tested for the ability to induce unscheduled DNA synthesis (UDS) in an in vivo rat hepatocyte assay. Male Fischer 344 rats were treated with a single oral dose of CI Disperse Blue 291 by gavage at 1250, or 2000 mg/kg body weight. The highest test dose, 2000 mg/kg was the limit test dose for a non-toxic test agent in this assay. Animals were killed and hepatocytes prepared four hours and twelve hours following administration of the chemical. Two independent experiments were carried out for each time point. Hepatocytes from treated rats were exposed to [³H]-thymidine and the amount of radioactivity incorporated into the nucleus [N] and an equal area of cytoplasm [C] determined by autoradiography. The cytoplasmic grain count was subtracted from that of the nucleus. The value obtained, the mean net nuclear grain count [N-C], is an index of UDS activity. In the respective testing laboratory, no negative control animal has shown a mean net nuclear grain count greater than zero. An [N-C] of more than zero in a treated animal is therefore considered indicative of a UDS response. Each experiment was validated by concurrent control treatments of rats with corn oil, the solvent for Disperse Blue 291 and with the carcinogens 2-acetylaminofluorene [2AAF] at twelve hours and N-nitrosodimethylamine [NDMA] or 6-p-dimethylaminophenylazobenzthiazole [6BT] at four hours. Solvent treated rats gave rise to mean net grain counts of less than zero, whilst hepatocytes from 2AAF, 6BT or NDMA treated animals had mean net nuclear grain counts of greater than +5. These data showed that background levels of UDS were normal and that the tester animals were responsive to known carcinogens requiring metabolic activation for genotoxic activity. Hepatocytes from Disperse Blue 291 treated animals were assessed for UDS at two dose levels of 1250 and 2000 mg/kg body weight. Treatments with Disperse Blue 291 in no case resulted in a mean net grain count greater than zero, at either time point. It is concluded that, when tested up to a limit dose of 2000 mg/kg body weight, the test sample of Disperse Blue 291 did not induce DNA repair (as measured by unscheduled DNA Synthesis) in hepatocytes from rats treated in vivo.

Mammalian erythrocyte micronucleus test

A study was performed to assess the potential of Disperse Red 82 (94%purity) to produce damage to chromosomes or the mitotic apparatus of mice when administered by the oral route. The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 474 “Genetic Toxicology: Micronucleus Test”. The test compound was suspended in Tylose HEC 4000 (0.5% w/v) and was given twice at an interval of 24 hours as an orally dose of 2000 mg per kg body weight to 5 male and 5 female mice, based on the results of a previous dose range finding assay. According to the test procedure the animals were killed 24 hours after administration. Endoxan® was used as positive control substance and was administered once orally at a dose of 50 mg per kg body weight.  

The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with Disperse Red 82 and was not less than 20% of the control value. Endoxan® induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that Disperse Red 82 is not clastogenic or aneugenic in the micronucleus test.

Justification for classification or non-classification

Based on the results of in vitro and in vivo testing, no classification for genotoxicity is required for the test substance according to CLP (EC 1272/2008) criteria.