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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Sept. 2016 - March 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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Test material form:
solid: crystalline
Details on test material:
- State of aggregation: Non-aggregated
- Particle size distribution: D50 = 12.2 µm
- Geometric standard deviation (GSD): 0.2
- Shape of particles: not assessed
- Surface area of particles: not determined
- Crystal structure: Crystalline, no further details known
- Coating: None
- Moisture content: 0.03%
- Residual solvent: Non detected
- Activation: Not applicable
- Stabilisation: With Phenothiazine (< 1000 ppm)
- Other:
Specific details on test material used for the study:
Designation in Test Facility: 16082904G
Date of Receipt: 29. Aug. 2016
Condition at Receipt: Room temperature, in proper conditions
Name: ROC-601
Batch no.: S23PSG0816
Appearance: White crystalline powder
Composition: 99.5% purity, see analytical certificate
Purity: 99.5% by LC-UV
Homogeneity: Uniform crystalline powder
Expiry date: Jul. 2017
Storage: Room Temperature (20 ±5 °C), keep away from light
CAS No.: 439661-46-8
EINECS-No.: Not yet assigned
Stability: H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility: H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
SMILES Code: C(=O)(C(=C)C)OCCCCCCOc1ccc(C=CC(=O)OC)cc1

Method

Target gene:
hisD6610, category frame shift, effect histidine deficiency: TA97a
hisD3052, category frame shift, effect histidine deficiency: TA98
hisG46, category base pair substitution, effect histidine deficiency: TA100, TA1535
hisG428, category base pair substitution, effect histidine deficiency: TA102
uvrB, category deletion, effect UV sensitivity, biotin deficiency: TA97a, TA98, TA100, TA1535
rfa, category deletion, effect lipopolysaccharide side chain deficiency: TA97a, TA98, TA100, TA102, TA1535
pKM101, category plasmid, effect ampicillin resistance: TA97a, TA98, TA100, TA102
pAQ1, category plasmid, effect tetracycline resistance: TA102
trpE, effect tryptophan deficiency: E.coli
uvrA, category deletion, effect UV sensitivity: E.coli
pKM101, Category plasmid, effect ampicillin resistance: E.coli
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 was obtained by Trinova Biochem. Gießen. Batch nos. 3652, 3597, 3568, produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intra-peritoneally.
Test concentrations with justification for top dose:
First Experiment
Concentrations tested: 5000 / 1500 / 500 / 150 / 50 µg/plate (5000 µg/plate is the max. conc. according to guideline)
Incubation time: 48 h at 37 ±1 °C
Tester strains TA97a, TA98, TA100, TA102, TA1535, E. coli WP2 (plate incorporation method)
Second Experiment
Concentrations tested: 5000 / 2500 / 1250 / 625 / 313 / 156 µg/plate (5000 µg/plate is the max. conc. according to guideline)
Incubation time: 48 h at 37 ±1 °C
Tester strains TA97a, TA98, TA100, TA102, TA1535, E. coli WP2 (pre-incubation method)
Vehicle / solvent:
DMSO 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. The test item solution was not sterile filtrated before use.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylene diamine
Remarks:
used for TA97a, TA98 and TA102, without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
used for TA100 and TA1535, without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
used for E.coli, without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Amino-anthracene
Remarks:
used for TA97a, TA100 and TA1535, with S9 metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Amino-anthracene
Remarks:
used for WP2, with S9 metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
used for TA98, with S9 metabolic activation
Details on test system and experimental conditions:
Preparation: 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. DMSO 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. The test item solution was not sterile filtrated before use. On the day of the start of the first and the second experiment, a stock solution containing 50 g/L of the test item in DMSO was prepared. The stock solution was used to prepare the geometric series of the concentrations to be tested.
The following nominal concentrations were prepared for the first experiment: 5000 µg/plate, 1500 µg/plate, 500 µg/plate, 150 µg/plate and 50 µg/plate.
The following nominal concentrations were prepared for the second experiment: 5000 µg/plate, 2500 µg/plate, 1250 µg/plate, 625 µg/plate, 313 µg/plate and 156 µg/plate.
Origin and Culture: Salmonella typhimurium and E. coli (all strains used) were obtained from TRINOVA BioChem (batch of the Salmonella typhimurium strains: TA97a: 4997D, TA98: 5011D, TA100: 4996D, TA102: 4982D, TA1535: 5012D and batch of E. coli strain: 4999D) and were stored as lyophilizates in the fridge at 2 - 8 °C. The lyophilizates were used to prepare permanent cultures which were filled into vials and stored at < -75 °C.
8 h before the start of each experiment, an aliquot of a permanent culture per strain to be used was taken from the deep freezer to inoculate a culture vessel containing nutrient broth. After incubation, overnight for 8 hours at 37 ±1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.
Chemicals
The purity of the chemicals which were used were either “analytical grade“ or “for microbiological purposes“. All solutions and media were sterilized, either by autoclaving (121 °C, 20 minutes) or by membrane filtration.
Composition is stated as nominal composition, exact weights differed by max. ± 10 %.
Nutrient Broth for Overnight Culture:
Nutrient broth Merck 5443: 2.8 g, H2O demineralised: ad 350 mL
Isotonic Sodium Chloride Solution for Dilution Purposes
Sodium chloride: 0.9 g, H2O demineralised: ad 100 mL
Vogel-Bonner-Medium 20-fold
Magnesium sulphate (MgSO4*7H2O): 4.0 g, Citric acid mono hydrate (MR 210.14 g/mol): 40.0 g, Potassium phosphate, dibasic (anhydrous) (K2HPO4): 200.0 g, Sodium ammonium phosphate, monobasic, tetra hydrate (Na(NH4)HPO4*4H2O): 70.0 g, H2O demineralised: ad 1000.0 mL
Glucose Solution 40%
Glucose monohydrate: 440.0 g, H2O demineralised: ad 1000.0 mL
Minimal Glucose Agar
Vogel-Bonner-Solution 20-fold: 500.0 mL, Glucose solution 40%: 500.0 mL H2O demineralised: 9000.0 mL, Agar: 150.0 g
Biotin Agar
Minimal-Glucose-Agar. 80 °C: 500.0 mL, Biotin solution 0.5 mM: 3.0 mL
Histidine-Biotin-Agar
Biotin-Agar, 80 °C: 350.0 mL, Histidine solution 0.5%: 3.5 mL
Ampicillin-Agar
Histidine-biotin agar, 80 °C: 200.0 mL, Ampicillin solution 0.8%: 0.6 mL
Ampicillin-Tetracycline Plates
Ampicillin agar, 80 °C : 50.0 mL, Tetracycline solution 0.8% : 0.01 mL
Nutrient Agar Plates
Nutrient broth Merck 5443: 0.8 g, Sodium chloride (NaCl): 0.5 g, Agar: 1.52 g, H2O demineralised: 100 mL
Basis for Top-Agar and Maximal-Soft-Agar
Agar: 6 g, Sodium chloride (NaCl): 5 g, H2O demineralised: ad 1000 mL
Histidine-Biotin-Solution 0.5 mM/0.5 mM (Use: Top Agar)
D-Biotin (MR 244.3 g/mol): 12.2 mg, L-Histidine* HCl*1H2O (MR 209.7 g/mol): 10.5 mg, H2O demineralised 90 °C: ad 100.0 mL
To 100 mL basis, 10 mL histidine-biotin-solution 0.5 mM/0.5 mM were added.
Histidine-Biotin-Solution 5 mM/ 0.5 mM (Use: Maximal-Soft-Agar)
D-Biotin (MR 244.3 g/mol): 12.2 mg, L-Histidine* HCl*1H2O (MR 209.7 g/mol): 105 mg, H2O demineralised 90 oC: ad 100.0 mL
To 100 mL basis, 10 mL histidine-biotin-solution 5 mM/0.5 mM were added.
Tryptophan-solution 0.5 mM (Use: Top Agar for E. coli)
L-Tryptophan (MR 204.2 g/mol): 10.2 mg, H2O demin.: ad 100.0 ml
To 100 mL basis, 10 mL tryptophan-solution 0.5 mM were added.
Tryptophan-solution 5mM (Use: Maximal-Soft-Agar for E. coli)
L-Tryptophan (MR 204.2 g/mol): 102.1 mg, H2O demin.: ad 100.0 ml
To 100 mL basis, 10 mL tryptophan-solution 5mM were added
Phosphate Buffer
Sodium di-hydrogen phosphate monohydrate NaH2PO4*H2O: 0.184 g, Di-sodium hydrogen phosphate dihydrate Na2HPO4 * 2H2O: 1.722 g, H2O demineralised: ad 100.0 mL, The pH of the solution is adjusted to 7.4.
Salt Solution for S9-Mix
Potassium chloride (KCl): 1.23 g, Magnesium chloride hexahydrate MgCl2*6H2O: 0.814 g, H2O demineralised: ad 10.0 mL
NADP-Solution for S9-Mix, 0.1 M
NADP disodium salt (MR = 787.4 g/mol): 787.4 mg, H2O demineralised: 10 mL
Glucose-6-Phosphate (G6P) Solution for S9-Mix, 1 M
Glucose-6-phosphate (MR = 340.13 g/mol): 3000.5 mg, H2O demineralised: ad 10 mL
S9-Mix
Phosphate buffer: 22.5 mL, 0.1 M NADP-solution: 1.0 mL, 1 M G6P-solution: 0.125 mL, Salt solution: 0.5 mL, Rat liver S9: 1.0 mL
S9
S9 was obtained by Trinova Biochem, Gießen. Batch nos.: 3652, 3597, 3568 (produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intra¬peritoneally).
Test Vessels: All vessels used are made of glass or sterilizable plastic. They were sterilized before use by autoclaving.
The following vessels were used: Schott-bottles, glass vials, and culture flasks for solutions and media, Plastic petri plates, test tubes for top-agar-bacteria-substance mix. Standard laboratory material (glassware) and equipment was also used.
Performance of the Study
Culture of Bacteria: Eight hours before the start of each experiment, one vial permanent culture of each strain was taken from the deep freezer and an aliquot was put into a culture flask containing nutrient broth. After incubation for eight hours at 37 ±1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.
Conduct of Experiment
Preparations: In the days before each test (exact production dates are documented in the raw data), the media and solutions were prepared. On the day of the test, the bacteria cultures were checked for growth. The incubation chambers were heated to 37 ±1 °C. The water bath was turned to 43 ±1 °C. The table surface was disinfected. The S9 mix was freshly prepared and stored at 0 °C.
First Experiment
Date of treatment 27. Sep. 2016
Concentrations tested 5000 / 1500 / 500 / 150 / 50 µg/plate
Incubation time 48 h
Incubation temperature 37 ±1 °C
Tester strains TA97a, TA98, TA100, TA102, TA1535, E. coli WP2
Method plate incorporation method
Second Experiment
Date of treatment 05. Oct. 2016
Concentrations tested 5000 / 2500 / 1250 / 625 / 313 / 156 µg/plate
Incubation time 48 h
Incubation temperature 37 ±1 °C
Tester strains TA97a, TA98, TA100, TA102, TA1535, E. coli WP2
Method pre-incubation method
Description of the Method
General preparation: Per strain and dose, three plates with and three plates without S9 mix were used. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine-biotin-solution 0.5 mM per 100 mL basis was added and the bottle was placed in the water bath at 43 ±1 °C.
Plate incorporation method: The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
100 µL test solution at each dose level, 100 µL solvent (negative control) or reference mutagen solution (positive control), except for MMS  2 µL were applied directly
500 µL S9 mix (for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
100 µL bacteria suspension (test system, culture of the strains)
2000 µL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.
Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ±1 °C for 20 minutes:
100 µL test solution at each dose level, 100 µL solvent (negative control) or reference mutagen solution (positive control), except for MMS  2 µL were applied directly
500 µL S9 mix (for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
100 µL bacteria suspension (test system, culture of the strains)
After pre-incubation, 2000 µL overlay agar (top agar) was added, the tube was gently vortexed and the mixture was poured onto the selective agar plate. The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.
Genotype Confirmation: Genotype confirmation is performed for each batch of lyophilized bacteria before stock culture preparation.
Histidine requirement: Each Salmonella typhimurium strain was streaked on a biotin and a histidine-biotin-plate, using a sterilized wire loop. The plates were incubated for 24 hours at 37 ±1 °C.
Tryptophan requirement: The strain Escherichia coli WP2 was streaked on a tryptophan-plate and a plate without tryptophan, using a sterilized wire loop. The plates were incubated for 24 hours at 37 ± 1 °C.
Ampicillin-resistance (pKM 101): The Salmonella typhimurium strains and the Escherichia coli WP2 were streaked on ampicillin agar. TA1535 was taken as control strain, since it is not ampicillin resistant. The plates were incubated for 24 hours at 37 ±1 °C.
UV-sensitivity (uvrB): Two plates were streaked with the five strains (four strains Salmonella typhimurium and one strain Escherichia coli WP2, and one half of the plate was covered with aluminium foil so that one half of each streak was protected against light. Then, the plates with the strains were irradiated. Incubation for 24 hours at 37 ±1 °C followed.
Crystal violet sensitivity (deep rough): For each Salmonella typhimurium strain, two plates were used. 0.1 mL of bacteria suspension were mixed with 2 mL Top-Agar and poured on nutrient agar. Sterile paper discs ( 9 mm), each soaked with crystal violet solution (0.1%) were placed into the middle of each plate, followed by incubation for 24 hours at 37 ±1 °C.
Spontaneous Revertants: Three replicates, with/without S9, for each solvent which was used in the test; incubation for 48 hours at 37 ±1 °C.
Determination of Titre: The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. Incubation for 48 hours at 37 ±1 °C followed. It should give a density of 109 cells/mL (at the least), two replicates with and without metabolic activation.
Toxicity Control: Performed in experiment 1 only, analogously to the titre control with the maximum dose of test item with and without S9 on maximal-soft agar, two replicates with and without metabolic activation; incubation for 48 hours at 37 ±1 °C.
Sterility Control: Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar, 4 replicates.
Solubility: Plates were checked for precipitation of test item at the end of the incubation by visual inspection.
Positive Controls: Using diagnostic mutagens, three replicates were prepared. The stock solutions of the substances were diluted to effect an application volume of 0.1 mL/plate for the following substances: 4-nitro-1,2-phenylene diamine, sodium azide, 2-amino-anthracene and benzo-a-pyrene. The substance methyl methanesulfonate was used directly.
Evaluation criteria:
Evaluation: The colonies were counted visually and 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 mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Only in second experiment at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
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
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:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2
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:
In the following table, the nominal concentrations and the real concentrations (based on weights used) in the experiments are compared.
First Experiment
Nominal concentrations 5000 µg/plate - Real concentrations 5028 µg/plate
Nominal concentrations 1500 µg/plate - Real concentrations 1508 µg/plate
Nominal concentrations 500 µg/plate - Real concentrations 503 µg/plate
Nominal concentrations 150 µg/plate - Real concentrations 151 µg/plate
Nominal concentrations 50 µg/plate - Real concentrations50 µg/plate

Second Experiment
Nominal concentrations 5000 µg/plate - Real concentrations 5001 µg/plate
Nominal concentrations 2500 µg/plate - Real concentrations 2501 µg/plate
Nominal concentrations 1250 µg/plate - Real concentrations 1250 µg/plate
Nominal concentrations 625 µg/plate - Real concentrations 625 µg/plate
Nominal concentrations 313 µg/plate - Real concentrations 313 µg/plate
Nominal concentrations 156 µg/plate - Real concentrations 156 µg/plate

Any other information on results incl. tables

Findings

The detailed data of the two experiments are listed in the annex to the full study report.

Confirmation of genotype is performe d for each batch of lyophilized bacteria before stock culture preparation. The last performance showed no abnormalities.

First Experiment:

Confirmation of the Criteria and Validity

All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory.All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.

Solubility and Toxicity

In the first experiment, the test item showed no precipitates on the plates in all tested concentrations.

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

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.

To verify this result, a further experiment was performed.

Survey of the Findings

The mean revertant values of the three replicates are presented in the following table: 

Strain

TA97a

TA98

TA100

TA102

TA1535

E. coli

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin.

water

Mean

87

88

19

19

106

113

387

383

24

23

95

87

sd

21.1

20.4

3.6

2.1

18.0

18.5

76.6

18.0

1.0

5.6

8.1

9.5

DMSO

Mean

73

105

12

17

105

110

356

357

26

24

89

101

sd

1.5

39.0

1.0

4.0

15.3

9.2

6.9

40.3

3.5

6.4

16.3

11.0

Positive
Controls

Mean

351

388

296

61

647

1176

760

1088

365

145

1001

467

sd

175.5

90.6

69.3

9.8

16.2

259.6

28.8

350.9

96.0

23.7

0.0

44.1

f(I)

4.81

3.70

24.67

3.59

6.10

10.69

2.13

3.05

15.21

6.04

11.25

4.62

5000 µg/plate

Mean

88

112

12

17

139

130

315

368

23

27

158

127

sd

24.1

5.1

2.1

0.6

12.1

17.8

20.1

6.9

3.1

4.4

19.7

36.3

f(I)

1.21

1.07

1.00

1.00

1.32

1.18

0.88

1.03

0.88

1.13

1.78

1.26

1500 µg/plate

Mean

106

128

12

18

116

114

349

301

22

18

152

117

sd

11.1

14.5

1.5

2.1

4.9

10.6

12.9

68.6

2.9

6.1

25.1

32.5

f(I)

1.45

1.22

1.00

1.06

1.10

1.04

0.98

0.84

0.85

0.75

1.71

1.16

500 µg/plate

Mean

104

110

15

16

119

125

317

297

26

20

97

101

sd

7.0

33.4

1.2

3.8

5.0

17.2

26.6

46.4

1.5

3.5

17.0

20.0

f(I)

1.42

1.05

1.25

0.94

1.13

1.14

0.89

0.83

1.00

0.83

1.09

1.00

150 µg/plate

Mean

100

101

15

13

109

121

307

281

21

20

100

132

sd

5.5

20.5

4.0

3.2

19.4

5.0

30.6

52.2

3.0

4.6

10.6

42.0

f(I)

1.37

0.96

1.25

0.76

1.04

1.10

0.86

0.79

0.81

0.83

1.12

1.31

50 µg/plate

Mean

97

113

10

12

111

122

341

344

24

25

145

111

sd

10.1

5.8

2.3

3.0

28.0

5.5

25.7

24.3

3.1

1.7

11.0

21.7

f(I)

1.33

1.08

0.83

0.71

1.06

1.11

0.96

0.96

0.92

1.04

1.63

1.10

f(I) = increase factor (mean revertants divided by mean spontaneous revertants)

Second Experiment:

Confirmation of the Criteria and Validity

All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.

Solubility and Toxicity: In the second experiment, the test item showed no precipitates on the plates in all tested concentrations. Signs of toxicity towards the bacteria strain TA97a could be observed in the highest concentration (5000 µg/plate), only. The bacterial background lawn was visible and not affected, but the number of revertant colonies was reduced. Towards the other bacteria strains no signs of toxicity were observed.

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.

Survey of the Findings

The mean revertant values of the three replicates are presented in the following table: 

Strain

TA97a

TA98

TA100

TA102

TA1535

E. coli

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin.

water

Mean

108

91

9

14

100

96

264

331

19

12

99

99

sd

7.0

8.7

1.5

4.0

2.0

9.2

13.9

22.0

2.9

1.5

3.2

22.5

DMSO

Mean

91

89

13

10

89

98

257

309

13

14

93

87

sd

14.4

4.9

3.0

1.2

6.0

12.5

6.1

37.8

1.2

2.1

10.1

14.6

Positive
Controls

Mean

665

448

372

79

444

480

769

1525

291

64

1001

396

sd

40.5

150.3

44.0

6.2

40.6

48.0

10.1

113.5

74.4

14.0

0.0

77.9

f(I)

7.31

5.03

28.62

7.90

4.44

4.90

2.99

4.94

15.32

4.57

10.76

4.55

5000 µg/plate

Mean

3

15

13

13

78

80

323

409

21

17

114

77

sd

0.6

4.5

1.7

1.0

6.5

5.5

34.0

48.2

2.1

1.5

6.5

1.7

f(I)

0.03

0.17

1.00

1.30

0.88

0.82

1.26

1.32

1.62

1.21

1.23

0.89

2500 µg/plate

Mean

88

85

12

12

95

83

384

413

19

17

84

114

sd

6.6

11.6

4.0

1.2

17.6

11.0

58.1

52.2

5.9

5.3

11.5

3.5

f(I)

0.97

0.96

0.92

1.20

1.07

0.85

1.49

1.34

1.46

1.21

0.90

1.31

1250 µg/plate

Mean

88

86

14

16

81

101

373

436

15

16

91

95

sd

9.2

10.7

3.6

4.9

10.1

11.5

10.1

68.4

3.2

5.5

4.0

3.5

f(I)

0.97

0.97

1.08

1.60

0.91

1.03

1.45

1.41

1.15

1.14

0.98

1.09

625 µg/plate

Mean

80

94

15

11

79

97

413

407

17

20

83

91

sd

13.5

5.7

4.7

2.1

4.9

18.1

23.4

51.6

0.6

2.9

9.1

24.4

f(I)

0.88

1.06

1.15

1.10

0.89

0.99

1.61

1.32

1.31

1.43

0.89

1.05

312 µg/plate

Mean

99

82

15

19

81

113

399

387

16

18

107

128

sd

12.2

10.1

1.2

0.6

13.2

15.4

40.5

50.6

4.6

1.7

9.6

20.6

f(I)

1.09

0.92

1.15

1.90

0.91

1.15

1.55

1.25

1.23

1.29

1.15

1.47

156 µg/plate

Mean

85

102

15

16

82

104

373

401

13

13

103

111

sd

12.2

9.9

2.1

5.7

13.1

12.9

19.7

44.6

2.5

3.1

12.7

4.2

f(I)

0.93

1.15

1.15

1.60

0.92

1.06

1.45

1.30

1.00

0.93

1.11

1.28

f(I) = increase factor (mean revertants divided by mean spontaneous revertants)

 

Mutagenicity of Test Item: The test itemROC-601 showed no increase in the number of revertants in all bacteria strains in both experiments. All negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that ROC-601 is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 and also not mutagenic in the Escherichia coli strain WP2 in the absence and presence of metabolic activation under the experimental conditions in the present study.

Acceptability of Study, Discussion: In all experiments, no precipitation of the test item was observed at any of the tested concentrations up to 5000 µg/plate. In the first experiment, the test item caused no cytotoxicity towards all bacteria strains. In the second experiment, the test item caused cytotoxicity towards the bacteria strain TA97a in the highest concentrations (5000 µg/plate), only.

The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 109 bacteria/mL.

All of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. All numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory and were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens. Since all criteria for acceptability have been met, the study is considered valid.

Deviations from the Study Plan: The following deviation of the study plan was observed: For the positive control MMS, 2 µL were applied directly, instead 100 µL. This can be seen as uncritical, because the procedure was correct. The deviation was assessed and signed by the study director on 09. Feb. 2017.

Applicant's summary and conclusion

Conclusions:
ROC-601 is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102, TA1535 and Escherichia coli strain WP2 in the absence and presence of metabolic activation under the experimental conditions in this study.
Executive summary:

Findings and results from the study “Determination of the mutagenic potential ofROC-601with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14” were as follows:

The test item ROC-601 was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535) and one strain of Escherichia coli (WP2). The test was performed in two experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

In the first experiment, ROC-601 (dissolved in DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102, TA1535 and E. coli WP2 using the plate incorporation method.ROC-601 showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item ROC-601 showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed that none of the tested concentrations induced a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

On the base of the first experiment, ROC-601 was tested in a second experiment up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in all bacteria strain using the pre-incubation method. ROC-601 showed no precipitates on the plates at any of the concentrations. The number of revertants was reduced at the highest concentration (5000 µg/plate) at the bacteria strain TA97a, only. At the other strains, no relevant decrease in the number of revertants was observed. The test item ROC-601 showed no signs of toxicity towards the bacteria strains TA98, TA100, TA102, TA1535 and Escherichia coli (WP2) in both the absence and presence of metabolic activation.

The results of this experiments showed that the test item ROC-601 caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item ROC-601 did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

Based on the results of this study it is concluded that ROC-601 is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102, TA1535 and Escherichia coli strain WP2 in the absence and presence of metabolic activation under the experimental conditions in this study.