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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Mar - 16 Apr 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted May 2008
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
his operon (S. typhimurium strains) and trp operon (E. coli strains)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
The dose-range finding test served as Experiment 1.
Experiment 1: 52, 164, 512, 1600 and 5000 µg/plate with and without metabolic activation
Experiment 2: 52, 164, 512, 1600 and 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Milli-Q water
- Justification for choice of solvent/vehicle: The test item was miscible in Milli-Q water at 25 °C and stable for at least 24 h at room temperature under normal laboratory light conditions, for at least 8 days in refrigerator (2-8°C) and for at least 3 weeks in the freezer (≤ -15°C), which was confirmed over the concentration range 1 to 60 mg/mL (project # 20144290 at the same testing facility).
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: -S9: 4-nitroquinoline N-oxide 10 µg/plate for WP2uvrA and ICR-191 2.5 µg/plate for TA1537 (Exp. 1); +S9: 2-aminoanthracene 1, 2.5 or 15 µg/plate for all strains
Remarks:
Each S9 batch was characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, Experiment 1); preincubation (Experiment 2)

DURATION
- Preincubation period: 30 ± 2 min (Experiment 2)
- Exposure duration: 48 ± 4 h (Experiment 1 and 2)

NUMBER OF REPLICATIONS: triplicates each in 2 independant experiments

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA1535, TA1537, TA98, TA100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item did not precipitate in the incubation medium at the start or at the end of the incubation period in any tester strain.

RANGE-FINDING/SCREENING STUDIES: Experiment 1 served as range-finding study.

HISTORICAL CONTROL DATA
- Positive historical control data: The positive control values were within the range of the historical control data (please refer to table 4 under "any other information on results incl. tables").
- Negative (solvent/vehicle) historical control data: The negative control values were within the range of the historical control data (please refer to table 4 under "any other information on results incl. tables").

Any other information on results incl. tables

Table 1: Results of dose-range finding study/Experiment 1 (direct plate assay)

Mean number of revertant colonies/3 replicate plates (± S.D.) with oneSalmonella typhimuriumand oneEscherichia colistrain.
Without S9-mix TA100 WP2uvrA
Positive control 1040± 42 1093 ± 98
Solvent control 123 ± 13 36 ± 8
1.7 123 ± 4 31 ± 7
5.4 132 ± 18 26 ± 6
17 117 ± 5 36 ± 5
52 105 ± 7 34 ± 4
164 124 ± 23 32 ± 8
512 123 ± 12 41 ± 8
1600 123 ± 27 44 ± 6
5000 121 ± 8 33 ± 6
With S9-mix TA100 WP2uvrA
Positive control 1513 ± 3 401 ± 118
Solvent control 141 ± 12 40 ± 4
1.7 128 ± 14 41 ± 8
5.4 122 ± 13 45 ± 4
17 108 ± 11 47 ± 7
52 110 ± 16 36 ± 6
164 114 ± 13 32 ± 6
512 103 ± 11 34 ± 9
1600 116 ± 10 36 ± 3
5000 91 ± 8 31 ± 6

Table 2: Results of Experiment 1 (direct plate assay)

Mean number of revertant colonies/3 replicate plates (± S.D.) with one Salmonella typhimurium and one Escherichia coli strain.
Without S9-mix TA1535  TA1537 Ta98
Positive control 1073 ± 97 1036 ± 50 1058 ± 40
Solvent control 8 ± 2 3 ± 2 15 ± 4
52 8 ± 1 6 ± 3 16 ± 1
164 12 ± 5 5 ± 5 20 ± 6
512 11 ± 4 4 ± 1 15 ± 6
1600 8 ± 7 6 ± 2 21 ± 2
5000 8 ± 4 7 ± 5 16 ± 6
With S9-mix TA1535  TA1537 Ta98
Positive control 349 ± 22 471 ± 37 1181 ± 40
Solvent control 10 ± 2 3 ± 2 20 ± 5
52 12 ± 2 11 ± 4 30 ± 11
164 12 ± 7 9 ± 9 26 ± 3
512 14 ± 2 7 ± 0 23 ± 8
1600 7 ± 7 4 ± 5 22 ± 3
5000 10 ± 1 5 ± 2 18 ± 2 

Table 3: Results of Experiment 2 (pre-incubation assay)

Mean number of revertant colonies/3 replicate plates (± S.D.) with one Salmonella typhimurium and one Escherichia coli strain.
Without S9-mix TA1535 TA1537 TA98 TA100 WP2uvrA
Positive control 1164 ± 79 145 ± 15 1147 ± 80 765 ± 93 217 ± 85
Solvent control 8 ± 4 3 ± 2 18 ± 2 109 ± 15 29 ± 6
52 12 ± 6 3 ± 1 11 ± 4 104 ± 14 32 ± 5
164 11 ± 4 6 ± 3 22 ± 19 105 ± 12 36 ± 8
512 15 ± 4 5 ± 5 17 ± 5 110 ± 17 31 ± 3
1600 17 ± 13 5 ± 2 16 ± 6 106 ± 17 34 ± 6
5000 4 ± 0 5 ± 2 15 ± 5 114 ± 9 39 ±8
With S9-mix          
Positive control 283 ± 20 217 ± 21 898 ± 85 1801 ± 423 477 ± 5
Solvent control 21 ± 9 12 ± 2 35 ± 6 110 ± 0 39 ± 8
52 20 ± 5 12 ± 4 32 ± 4 128 ± 14 38 ± 4
164 16 ± 2 18 ± 4 29 ± 2 106 ± 9 43 ± 5
512 19 ± 4 13 ± 4 34 ± 5 106 ± 20 39 ± 8
1600 14 ± 4 15 ± 4 29 ± 6 104 ± 2 46 ± 6
5000 21 ± 1 12 ± 3 27 ± 3  97 ± 13 38 ± 4

Table 4: Historical control data of the positive and negative control

  TA1535 TA1537 TA98 TA100 WP2uvrA
Positive control
S9-mix - + - + - + - + - +
Range 125 – 1248 73 – 1206 55 – 1353 54 – 1051 365 – 1995 250 – 1977 439 – 1848 408 - 2651 93 – 1951 111 - 1359
Mean 846 219 787 353 1406 887 901 1232 1094 437
SD 146 119 345 162 258 349 168 343 477 149
n 2348 2229 2003 2234 2200 2276 2335 2327 2021 2085
Negative control
S9-mix - + - + - + - + - +
Range 3 – 29 3 - 27 3 – 20 3 – 23 8 - 41 8 - 55 63 – 176 54 - 160 10 – 59 9 - 69
Mean 10 11 6 7 16 23 108 107 25 32
SD 3 4 2 3 5 7 19 20 7 8
n 2356 2336 2264 2235 2319 2360 2341 2336 2075 2078

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative