Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Sepisol fast Blue 85219
- Substance type: organic salt - UVCB
- Appearance/Physical state: dark blue powder
- Stability under test conditions: stable
- Storage condition of test material:room temperature
Specific details on test material used for the study:
- Batch N° 310551

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:
50; 150; 500; 1500 and 5000 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

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

STAIN (for cytogenetic assays): Methyl violet

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
Key result
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
Key result
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 50,5%, 23,5 % and 13,5% were observed with respectively 5000, 1500 and 500 µg/plate of test material.Those rates stand below the tolerated threshold of 75%. No cytotoxic effect observed for the following doses: 150 et 50 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
the rates 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:
A significative decrease of the number of revertants colonies is observed for the dose 5000 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

5000

0,26

0,31

0,08

0,13

1500

0,89

0,81

0,35

0,55

500

1,04

0,94

0,42

0,58

150

0,93

1,03

0,73

0,90

50

1,07

1,22

0,73

0,97

 

TA 1537

5000

0,16

0,42

0,20

0,19

1500

0,58

0,85

0,47

0,68

500

0,74

0,58

0,4

0,61

150

1,37

0,73

0,47

0,74

50

0,95

1,04

0,73

0,87

 

TA 98

5000

0,53

1,4

0,44

0,55

1500

0,74

0,84

0,46

0,77

500

0,60

0,81

0,90

1,08

150

0,83

1,21

0,90

1,18

50

0,96

1,26

0,76

1,23

 

TA 100

5000

0,05

0,06

0,06

0,22

1500

0,12

0,16

0,19

0,24

500

0,13

0,45

0,36

0,36

150

0,32

0,86

0,35

0,88

50

0,65

0,98

0,77

0,91

 

E.Coli

5000

0,14

0,33

0,13

0,22

1500

0,26

0,25

0,31

0,43

500

0,30

0,66

0,51

0,89

150

0,44

0,66

0,75

1,01

50

0,62

1,42

0,82

1,09

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

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions described, the test material Sepisol Fast Blue 85219 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 actvation.

Executive summary:

The search for any mutagenic activity of Sepisol Fast Blue 85219, 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 concentration recommended by OECD Guideline, i.e. 5000 μg/plate for the toxicity assay. Four lower dilutions chosen according to a geometrical (half-log) ratio were also tested. In the main assay, the maximum dose was chosen in accordance with the recommendations of OECD Guideline, i.e. 5000μg/plate.

Preparation of test material solution

The test material (Sepisol Fast Blue 85219) is diluted in ethanol so as to prepare a stock solution of 100 mg/L.

Preliminary assay: Cytotoxicity

Five concentrations have been tested (50; 150; 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 each one of the 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 treatment. 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. The first method (plate incorporation) was the same 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. The other method: Pre-incubation, in which the mixture of bacteria, the test material, and the S9 fractioin is first incubated at 37 °C during at least 20 minutes before to be added to the top agar.

For the first essay, incorporation plate method have been used. The pre-incubation method have beee used for the second assay with S9 mix.

Solvent controls, positive controls and assay for the control of media sterility were performed like in the mutagenicity assay without metabolic activation.

Bacterioastatic activity

Cytotoxic rate of 50,5%, 23,5 % and 13,5% were observed with respectively 5000, 1500 and 500 µg/plate of test material. Those rates stand below the tolerated threshold of 75%. No cytotoxic effect observed for the following doses: 150 et 50 µg/plate.

A significative decrease of the number of revertants colonies is observed for the dose 5000 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.

Conclusion

Under the experimental conditions described, the test material Sepisol Fast Blue 85219 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 actvation.