Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Bacterial Reverse Mutation Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
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:
1 MATERIALS AND METHODS
1.2 Test Item
Designation in Test Facility: 17031402G
Date of Receipt: 14. Mar. 2017
Condition at Receipt Room temperature, in proper conditions
1.2.1 Specification.
Name Blendazol Red Blendwell
Batch no. E 328
Appearance Dark Red Powder
Composition 2-Naphtalenesulfonic acid, 7-amino-4-hydroxy-3,8-bis[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, triso dium salt
Purity 95 % by HPLC
Homogeneity homogeneous
Expiry date 17. Feb. 2019
Storage Room Temperature: (20 ± 5°C); Keep away from humidity
CAS No. 607724-47-0
EINECS-No. 612-028-6
Chemical Class not stated
Stability H2O: 96h; EtOH: unknown; acetone: unknown CH3CN: unknown; DMSO: unknown
Solubility H2O: to be determined*; EtOH: unknown; acetone: unknown CH3CN: unknown; DMSO: unknown


* Will be determined in another GLP-study at LAUS GmbH and will be stated in the final report, this
This information is not provided by the sponsor but determined at LAUS GmbH.


Method

Target gene:
hisD6610; hisD3052; hisG46; hisG428; uvrB; rfa; pKM101; pAQ1
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: All Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA97a: 4997D, TA98: 5011D, TA100: 4996D, TA102: 4982D, TA1535: 5012D)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Properly maintained: [yes/no]
- Periodically checked for Mycoplasma contamination: [yes/no]
- Periodically checked for karyotype stability: [yes/no)
- Periodically 'cleansed' against high spontaneous background: [yes/no]
Test concentrations with justification for top dose:
In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralized water, dimethyl sulfoxide
(DMSO) and ethanol.
Demin. water was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
On the day of the start of the experiment 1 and 2b, a stock solution containing 50 g/L (nominal concentration) of the test item in demin. water was prepared. The test item solution was not sterile filtrated before use.
The stock solution was used to prepare the geometric series of the concentrations to be tested.

The following nominal concentrations were prepared for the experiment 1:
5000 µg/plate, 1500 µg/plate, 500 µg/plate, 150 µg/plate and 50 µg/plate.

The following nominal concentrations were prepared for the experiment 2b:
5000 µg/plate, 2500 µg/plate, 1250 µg/plate, 625 µg/plate, 313 µg/plate and 156 µg/plate.
Vehicle / solvent:
- Vehicle(s): demineralized water batch: 20170309
- Justification for choice of vehicle:
In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralized water, dimethyl sulfoxide (DMSO) and ethanol.
Demin. water was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Controls
Untreated negative controls:
yes
Remarks:
demineralised water
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO), batch: 246245640
True negative controls:
yes
Remarks:
sterility control with Sterlised water and DMSO without colony
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
congo red
other: 4-Nitro-1,2-phenylene diamine, C6H7N3O2; CAS-No.: 99-56-9 and 2-amino-anthracene
Remarks:
Dimethylsulfoxide (DMSO), batch: 246245640, for the positive controls nitrophenylendia-mine, benzo-a-pyrene and 2-amino-anthracene Demineralised water, batch: 20170309 and 20170815 for the test item and for the positive control sodium azide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation;
7.2.3 Description of the Method
7.2.3.1 General preparation for the first experiment
Per bacteria strain and concentration, three plates with (S9 mix with hamster liver) and three plates without metabolic activation were used.
The test item solutions were prepared according to chapter 6.1.3
For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histi-dine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ±1 °C.
The applied volumes correspond to the following test item concentrations in the first ex-periment:
5000 µg/plate; 1500 µg/plate; 500 µg/plate; 150 µg/plate; 50 µg/plate.

7.2.3.2 General preparation for the second experiment
Per strain and dose, three plates with S9 mix containing hamster liver, three plates with S9 mix containing rat liver and three plates without S9 mix were used.
The applied volumes correspond to the following test item concentrations in the second experiment:
5000 µg/plate; 2500 µg/plate; 1250 µg/plate; 625 µg/plate; 313 µg/plate, 156 µg/plate.

DURATION
- Preincubation period: 30 min
- Exposure duration: 48 h
7.2.3.3 Pre-incubation method without shaking
The following materials were gently vortexed in a test tube and incubated at 37 °C for 30 minutes:
•100 µL test suspension at each dose level, solvent (negative control) or reference mutagen solution (positive control)
•500 µL S9 mix containing hamster liver (see chapter 6.4.23, for test with metabolic activation with S9 from Syrian hamster liver) or 500 µL S9 mix containing rat liver (see chapter 6.4.22, for test with metabolic activation with S9 from rat liver) or phos-phate buffer (for test without metabolic activation).
•100 µL bacteria suspension (see chapter 6.3.2, test system, culture of the strains)
After pre-incubation, 2000 µL overlay agar (top agar) was added, the tube was gently vor-texed and the mixture was poured onto the selective agar plate.
The plates were closed and left to harden for a few minutes, then inverted and placed in the dark incubator at 37°C.

After incubation for 48 hours, the revertants were counted and the numbers for each plate were recorded.
In the evaluation of the study the test item and the positive control congo red (S9 mix with hamster liver) were compared with spontaneous revertants demineralised water with Min-imal Glucose Agar with 2% glucose. Test item (S9 mix with rat liver) was compared with spontaneous revertants demineralised water with Minimal Glucose Agar with 8% glucose.
Positive controls (S9 mix with rat liver) were compared with spontaneous revertants de-mineralised water resp. DMSO with Minimal Glucose Agar with 8% glucose


- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

Rationale for test conditions:
The test item was dissolved in demineralised water. A stock solution containing 50 g/L was prepared.
No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not reduced.
Evaluation criteria:
The colonies were counted visually, the numbers were recorded. A validated spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The increase factor f(I) of revertant induction (mean revertants divided by mean sponta-neous revertants) and the absolute number of revertants (“Rev. abs.”, mean revertants less mean spontaneous revertants) were also calculated.
A test item is considered to have mutagenic potential, if a significant, reproducible in-crease of revertant colonies per plate (increase factor  2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.
Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results
Statistics:
The determination of titre should give a number of at least 109 cells/mL, correlating to 100 colonies/plate after dilution.
The criterion was fulfilled, exact values are given in the table below.
Table 13.1 a Titre Values (colonies per plate)
Strain TA97a TA98 TA100 TA102 TA1535
Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Repl. 1 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001
Repl. 2 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001
Mean 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001
Stand. Dev. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Assessment ok ok ok ok ok ok ok ok ok ok
1001 colonies per plate means the bacteria growth was too strong for counting

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
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:
HISTORICAL CONTROL DATA
In the following table, the history of the spontaneous revertants and positive controls of the performed experiments with these strains up to 30. May 2017 is stated in comparison with the experiments performed within this study. Only experiments which were per-formed before finalisation of the study plan of this study were considered.
The following values result from the standard Ames method including the S9 mix only with S9 from rat liver. For the Prival & Mitchell method, no historical data are available.
Therefore, only the values of the second experiment without S9 mix and with S9 mix from rat liver are reported.

Table 16 a Historical Revertants
Strain TA97a TA98 TA100 TA102 TA1535
Induction - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9
Demin. water Mean 93 97 14 17 93 97 279 297 17 16
Min 60 63 6 8 62 66 85 67 6 7
Max 144 138 35 41 141 141 425 511 30 30
SD 19 16 5 5 15 15 66 77 6 5
Exp 2b n.t. 91 n.t. 36 n.t. 100 n.t. 348 n.t. 19
DMSO Mean 91 101 14 15 90 93 278 290 17 16
Min 58 70 7 8 60 63 79 80 8 6
Max 135 144 46 36 136 199 393 459 33 29
SD 19 17 6 5 16 19 62 67 6 6
Exp 2b n.t. 89 n.t. 39 n.t. 98 n.t. 295 n.t. 15
Positive Controls* Mean 549 499 403 78 508 721 1163 1240 250 117
Min 264 241 112 39 223 273 491 408 55 45
Max 1152 1181 793 237 984 1912 2331 6083 484 712
SD 174 146 140 40 153 296 459 682 89 81
Exp 2b n.t. 533 n.t. 260 n.t. 897 n.t. 1251 n.t. 157
* Different Positive Controls were used,



ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Any other information on results incl. tables

1.1     Determination of Titre

 Criterion: The determination of titre should give a number of at least 109cells/mL, correlating to 100 colonies/plate after dilution.

The criterion was fulfilled, exact values are given in the tables below.

Performance on plates with 2% MinGlu and for treatments with metabolic activation with S9 mix containing S9 from Syrian hamster liver:

Table14.1‑a   Titre Values (colonies per plate)

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Repl. 1

1001

1001

1001

1001

1001

1001

1001

1001

1001

1001

Repl. 2

1001

1001

1001

1001

1001

1001

1001

1001

1001

1001

Mean

1001

1001

1000

1001

1001

1001

1001

1001

1001

1001

Stand. Dev.

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

Assessment

ok

ok

ok

ok

ok

ok

ok

ok

ok

ok

1001 colonies per plate means the bacteria growth was too strong for counting.

Performance on plates with 8% MinGlu and for treatments with metabolic activation with S9 mix containing S9 from rat liver.

Table14.1‑b  Titre Values (colonies per plate)

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Repl. 1

1001

1001

1001

1001

1001

1001

1001

1001

1001

1001

Repl. 2

1001

1001

1001

1001

1001

1001

1001

1001

1001

1001

Mean

1001

1001

1000

1001

1001

1001

1001

1001

1001

1001

Stand. Dev.

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

Assessment

ok

ok

ok

ok

ok

ok

ok

ok

ok

ok

1001 colonies per plate means the bacteria growth was too strong for counting.

 

Applicant's summary and conclusion

Conclusions:
The test item Blendazol Red Blendwell showed no increase in the number of revertants in all bacteria strains in both experiments.
Based on the results of this study it is concluded that Blendazol Red Blendwell is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experi-mental conditions in the present study.
Executive summary:

The mutagenic potential of Blendazol Red Blendwell was determined using the Bacterial Reverse Mutation Test according to OECD 471 and EU B.13/14 according to Prival & Mitchell procedure,

The mutagenic Potentiel of Blendazol Red was checked by 3 experiments: The experiment 1 was valid. The experiment 2a was not valid, because the pre- incubation time was 20 minutes, instead of 30 minutes

1.Solubility and Toxicity

The test item was dissolved in demineralised water. A stock solution containing 50 g/L was prepared.

No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not reduced.

2. Mutagenicity

No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

 

Therefore, the test item is stated as not mutagenic under the test conditions.