Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The reliability is rated 1 because the study followed the standard guideline of reference (OECD 471), which describes a procedure designed to evaluate this endpoint, the results were reviewed for reliability and assessed as valid, and the study was conducted under GLP condition.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Mono and bis and tris{tris[4-(mono and dimethylamino)phenyl]methylium} [12,21,30,32-tetrahydro-29H,31Hphthalocyanine- mono, bis and trisulfonato-k4N29,N30,N31,N32]cuprate.
IUPAC Name:
Mono and bis and tris{tris[4-(mono and dimethylamino)phenyl]methylium} [12,21,30,32-tetrahydro-29H,31Hphthalocyanine- mono, bis and trisulfonato-k4N29,N30,N31,N32]cuprate.
Details on test material:
- Name of test material (as cited in study report): Sepisol Fast Violet 2B
- Substance type: organic
- Physical state: dark violet powder
- Analytical purity: 98%
- Lot/batch No.: 423393
- Storage condition of test material: TA (20°C +/-2)

Method

Target gene:
histidine operon
and tryptophan operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0.5; 1.5; 5; 15; 50 µg/plate
Vehicle / solvent:
- solvent used: ethanol
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); and preincubation for the second assay with S9 mix if the first one was negative.

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 to 72 hours
- Expression time (cells in growth medium):number of revertant colonies per plate

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: 5 x 10 E 9

DETERMINATION OF CYTOTOXICITY
- Method: bacteriostatic activity

OTHER:
-Number of plates per assay: 3
Evaluation criteria:
R = (Number of revertants colonies wiith the test material) / (Number of revertant colonies without the test material)

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Bacterioastatic activity: Cytotoxic rate of 72% was observed with 50 µg/plate of test material. This rate stands below the tolerated threshold of 75%.
No cytotoxic effect observed for the other doses.

COMPARISON WITH HISTORICAL CONTROL DATA:
Rate of spontaneous revertants and positive controls (with and without S9 mix) fall within the range of observed historical values at the facility.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
For TA 1535, TA 98, TA 100 and E.coli, a significative decrease of the number of revertants colonies can be observed for the dose 50 µg/plate.This
finding must be correlated with the high toxicity measured at this concentration..

Any other information on results incl. tables

STRAIN

DOSE/PLATE

(µg)

R

Assay 1:

- S9 mix 10%

Assay 1:

+ S9 mix 10%

Assay 2:

- S9 mix 10%

Assay 2:

- S9 mix 10% and pre-incubation

TA 1535

50

0.31

0.53

0.15

0.43

15

1.08

0.80

0.85

1.04

5

1.42

0.83

1.19

1.00

1.5

0.88

0.85

0.85

1.04

0.5

1.08

0.75

1.04

1.07

 

TA 1537

50

0.60

2.52

0.42

2.42

15

1.75

2.22

2.58

1.13

5

2.25

1.22

2.67

1.48

1.5

125

0.96

1.42

1.00

0.5

0.85

1.00

1.33

0.94

 

TA 98

50

0.39

0.74

0.45

1.02

15

0.49

1.18

1.34

0.79

5

1.14

1.20

1.21

1.03

1.5

1.00

1.02

1.04

0.81

0.5

0.86

0.86

1.11

1.03

 

TA 100

50

0.30

0.38

0.36

0.66

15

0.66

0.51

0.63

0.87

5

0.95

0.85

0.83

0.90

1.5

1.06

1.03

0.96

1.01

0.5

1.01

1.00

1.04

0.91

 

E.Coli

50

0.18

0.56

0.55

0.10

15

0.50

1.25

0.73

0.34

5

1.01

0.92

0.99

0.91

1.5

0.72

0.98

0.92

1.15

0.5

0.97

1.07

1.06

1.32

R = (Number of revertants colonies wiith the test material) / (Number of revertant colonies without the test material)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions described, the test material Sepisol Fast Violet 2B did not show any mutagenic activity regarding 4 strains of
Salmonella typhimurium (TA 1535, TA 1537, TA 100 and TA 98) and regarding one strain of Escherichia Coli WP2 (uvr A-) (pKM 101) with and
without metabolic activation.

Executive summary:

The search for any mutagenic activity of Sepisol Fast Violet 2B, was studied by means of the Ames test (Salmonella his- and E.coli Trp- /microsome system) in compliance with the OECD Guideline 471 using the maximum tolerated concentration (standing below the threshold of 75%) recommended by OECD Guideline, i.e. 50μg/plate for this toxicity assay. Four lower dilutions chosen according to a geometrical (half-log) ratio were also tested.

Preparation of test material solution

The test material (Sepisol Fast Violet 2B) is diluted in ethanol so as to prepare a stock solution of 2 mg/mL.

Preliminary assay: Cytotoxicity

Different concentrations have been tested (0.5; 1; 5; 15; 25; 50; 100; 500; 1500 and 5000 µg/plate). 0.1 mL of a bacterial suspension from a culture containing 1 to 5 x 109bacteria/mL and 0.1 mL of the test item at the relevant concentration were successively added to 2 mL of top agar to which 10 % of 0.5 mM biotin histidine solution, maintained in a state of superfusion at 45°C, has been added. The content of each tube was agitated, then spread out in a Petri plate containing 20 mL of minimum agar and then incubated for 24 or 48 hours at 37 °C (n=3). Colonies are counted. A negative sample with pure solvent is run the same way.

 

Mutagenicity test

- Without metabolic activation:

The following technic was performed for Salmonella strains used in the test: 0.1 mL of a bacterial suspension containing 1 to 5 x 109bacteria/mL and 0.1 mL of the test item at the relevant concentration were successively added to 2 mL of top agar to which 10 % of 0.5 mM biotin histidine solution, maintained in a state of superfusion at 45°C, has been added. The content of each tube was agitated, then spread out in a Petri plate containing 20 mL of minimum agar.

For E.Coli the following technic was applied: 0.1 mL of a bacterial suspension containing 1 to 5 x 109bacteria/mL and 0.1 mL of the test item at the relevant concentration were successively added to 2 mL of top agar to which 5 % (v/v) of Nutrient broth n°2 and an extra 5µL of a L-Tryptophan solution at 2 mg/mL, maintained in a state of superfusion at 45°C, has been added. The content of each tube was agitated, then spread out in a Petri plate containing 20 mL of minimum agar.

Three plates were used per dose. The plates were then incubated at 37°C for 48 h approximately. Finally, colonies of revertants were counted for each plate.

- With metabolic activation :

Two techniques have been used:

- direct plate incorporation: Same technique as the one described above except that immediately before spreading in the

plates, 0.5 mL of the S9 mix metabolic activation system was added in soft agar.

- by pre-incubation: The bacterial mixture, the test material, and the S9 fraction is first incubated at 37 °C during at least

20 minutes before being added to the top agar.

If the first assay gives a positive response, the incorporation plate method has been used for the second S9 mix assay.

If the first assay gives a negative response, the pre-incubation method has been used for the the second S9 mix assay.

Solvent controls, positive controls were performed like in the mutagenicity assay without metabolic activation.

Bacterioastatic activity

Bacterioastatic activity: Cytotoxic rate of 72% was observed with 50 µg/plate of test material. This rate stands below the tolerated

threshold of 75%. No cytotoxic effect observed for the other doses.

For TA 1535, TA 98, TA 100 and E.coli, a significative decrease of the number of revertants colonies can be observed for the dose 50 µg/plate. This finding must be correlated with the high toxicity measured at this concentration.

Mutagenicity assays

The mutagenic activity of the test item was assessed by means of the Ames’s test in the four Salmonella typhimurium strains

TA1535, TA1537, TA98, TA100 and in the E. Coli WP2 (uvr A-) (pKM 101) tested either in presence or in absence of metabolic activation, in two independent assays. No mutagenic activity was found either with or without metabolic activation in any of the 5 strains. Values fall within the range of historical values observed at the facility.

For the Salmonella TA 1537 strain, an increase of the number of revertants at the 5; 15 and 50 µg/plate dose was observed. Nonetheless the mutagenic effet is only taken into account when the revertants' number is at least equal to 3 times the spontaneous revertant's rate for the TA 1537.

Conclusion

Under the experimental conditions described, the test material Sepisol Fast Violet 2B did not show any mutagenic activity regarding 4

strains of Salmonella typhimurium (TA 1535, TA 1537, TA 100 and TA 98) and Escherichia coli WP2 (uvr A-) (pKM 101) with and without metabolic activation, according to the OECD guideline 471.