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Toxicological information

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:
Experiment start: 21 August 2019; experiment end: 12 September 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
(4R)-2-oxo-1,3-oxazolidine-4-carboxylic acid
EC Number:
849-166-3
Cas Number:
83841-00-3
Molecular formula:
C4H5NO4
IUPAC Name:
(4R)-2-oxo-1,3-oxazolidine-4-carboxylic acid
Test material form:
solid

Method

Target gene:
Histidine-dependent auxotrophic mutants of S. typhimurium and a tryptophan-dependent mutant of E. coli were used in the test.
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
The S9 Microsomal fraction (Sprague-Dawley) was purchased from Moltox and stored at approximately -196 °C in a liquid nitrogen freezer; Lot No. 4123 was used in this study and the protein level was adjusted to 20 mg/mL. For a 10% S9-mix, an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approximately 10% (v/v) in the S9-mix. The S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium orthophosphate buffer (100 mM, pH 7.4) and was prepared using sterilized co-factors immediately prior to use and maintained on ice for the duration of the test.
Test concentrations with justification for top dose:
Plate incorporation test (experiment 1): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Pre-incubation test (experiment 2): 15, 50, 150, 500, 1500 and 5000 μg/plate
Vehicle / solvent:
Sterile distilled water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
Without S9 mix: 4-nitroquinoline-N-oxide, 9-aminoacridine, N-ethyl-N-nitro-N-nitrosoguanidine
With S9 mix: 2-aminoanthracene, benzo(a)pyrene
Details on test system and experimental conditions:
Bacterial cultures were prepared from frozen stocks by incubating for 10 hours at 37 °C in a shaking incubator. The bacteria were obtained from Trinova Biochem GmbH on 27 June 2017 and British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.
Media: Top agar was prepared using 0.6% w/v Bacto agar (lot number 8255817 07/2023) and 0.5% w/v sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot number 51673 09/2019).
Pre-cultures: A culture of each of the bacterial strains was prepared by inoculating nutrient broth with the appropriate coded stock culture and incubated, with shaking, for approximately 10 hours at 37 ± 3 °C. The bacterial cell count for each culture was determined by viable count analysis on nutrient agar plates on the day of test and was as follows
TA100 - 3.1 and 4.0E09/mL (experiment 1 and 2)
TA1535 - 1.3 and 3.5E09/mL (experiment 1 and 2)
WP2 uvrA pKM101 - 2.4 and 4.4E09/mL (experiment 1 and 2)
TA98 - 2.2 and 2.2E09/mL (experiment 1 and 2)
TA1537 - 1.7 and 2.409/mL (experiment 1 and 2)
Experiment 1: Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Conditions experiment 1, without S9 mix: 0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlaid onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
Conditions experiment 1, with S9 mix: The procedure was the same as described previously (see 3.5.2.2) except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.
Incubation and scoring, experiment 1: All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Sporadic manual counts were performed due to spreading colonies which prevented an accurate automated count.
Experiment 2: The concentration range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 μg/plate.
Conditions experiment 2, without S9 mix: 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 30 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
Conditions experiment 2, with S9 mix: The procedure was the same as described previously (see 3.5.3.2) except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 30 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.
Incubation and scoring experiment 2: All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
Rationale for test conditions:
The maximum concentration in experiment 1 was 5000 μg/plate (the maximum recommended concentration). In experiment 2, six test item concentrations per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic concentration levels and the maximum recommended concentration following the change in test methodology from plate incorporation to pre-incubation. No toxicity was observed up to the maximum concentration, with and without S9 mix. No test substance precipitate was observed on the plates at any of the concentrations tested with our without S9 mix in experiments 1 and 2.
Evaluation criteria:
If exposure to a test item produces a reproducible increase, in one or more concentration, in mean revertant colony numbers of at least twice that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship in at least one strain with or without metabolic activation system, it will be considered to exhibit mutagenic activity in this test system (Mortelmans and Zeiger 2000). No statistical analysis was performed.
If exposure to a test item does not produce an increase in mean revertant colony numbers, it will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis was performed.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used will usually be Dunnett’s test followed, if appropriate, by trend analysis (Mahon et al, 1989). Biological significance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below twice those of the concurrent vehicle controls (as described above) will not be considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director will use his/her scientific judgment.
Statistics:
No statistical analysis was performed.

Results and discussion

Test resultsopen allclose all
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
True 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:
valid
True 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:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
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:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Table: summary of results of plate incorporation test

















































































































































































































































































































































































































































































































































 Number of revertants (mean) +/- SD
 Base-pair substitution strainsFrameshift strains
Concentration TA100TA1535WP2uvrApKM101TA98TA1537
Per PlateWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mix
Solvent Control1071441018146114293877
(Water)106142781121082021128
 146140101112012437251416
1.5 µg1221361412117136109115
 1451358101151063020157
 11914111141031431720104
5 µg1111431011941431721149
 11212310141021422732114
 13512515141171301830108
15 µg10315110141171351518125
 110147138112136223067
 12212211161201421920154
50 µg1151171219136130914173
 1211167812815517121510
 10313110111321282813137
150 µg13011313101281332128139
 12913417231691142022312
 105148129121142202879
500 µg1041471212125141201655
 114126712144124162078
 10712412131371131114610
1500 µg10312512151311531522519
 9710110141321352722912
 11816312111311182528102
5000 µg10611579117153232557
 116114813121121194946
 11313717131291383120711
Positive controlsENNG ENNG ENNG 4NQO 9AA 
Concentration 3 µg 5 µg 0.5 µg 0.2 µg 80 µg 
 Without S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mix
 486 367 910 126 281 
 439 546 719 168 380 
 408 664 792 228 305 
Positive controls 2AA 2AA 2AA BP 2AA
Concentration  1 µg 2 µg 10 µg 5 µg 2 µg
  1639 295 1143 147 155
  1876 252 1115 130 160
  1869 268 1096 190 179

Table: summary of results of pre-incubation test



































































































































































































































































































































































































































































 Number of revertants (mean) +/- SD
 Base-pair substitution strainsFrameshift strains
Concentration TA100TA1535WP2uvrApKM101TA98TA1537
Per PlateWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mix
Solvent Control108114911957727311211
(Water)129136191312111818291216
 134119108841482926914
15 µg1171319121009421441716
 109111499810427291416
 1271451568711032341713
50 µg109102912911192825129
 114136112013612431311814
 14112913101401042130117
150 µg1111207590116173698
 10710151017014626381714
 10112812116816130291713
500 µg10912918710614423281910
 11213117910614515261012
 1141337810714627451015
1500 µg1321341291038722431414
 11712613101061121935910
 12011511810812418241516
5000 µg971301112105124253999
 11813381211212522301614
 113129151910712720361211
Positive controlsENNG ENNG ENNG 4NQO 9AA 
Concentration 3 µg 5 µg 0.5 µg 0.2 µg 80 µg 
 Without S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mixWithout S9 mixWith S9 mix
 716 2235 632 205 148 
 637 2753 603 195 197 
 657 2187 687 209 144 
Positive controls 2AA 2AA 2AA BP 2AA
Concentration  1 µg 2 µg 10 µg 5 µg 2 µg
  1491 322 1035 102 175
  1311 350 1067 154 191
  1283 313 1124 121 222

Applicant's summary and conclusion

Conclusions:
The substance did not induce gene mutations by base pair changes or frameshifts in the genome of S. typhimurium and E. coli strains, and was considered to be non-mutagenic in the bacterial reverse mutation assay.
Executive summary:

The mutagenic potential of the substance was studied under GLP to OECD TG 471 with histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2uvrApKM101. The bacteria were exposed to the substance diluted in sterile distilled water. Two independent mutation experiments were performed in the presence and absence of liver preparations (S9-mix) from rats treated with phenobarbital and β-naphthoflavone. Experiment 1 was a standard plate incorporation assay and experiment 2 a pre-incubation test. The maximum concentration of the test item in experiment 1 was 5000 µg/plate, the standard limit concentration recommended in the regulatory guideline that this assay follows. This maximum concentration was also selected for experiment 2. There was no toxicity, evident as a reduction in the number of revertants (below an induction factor of 0.5) or a reduction in the background lawn, in any of the five tester strains either with or without S9 mix following exposure to the substance in either the plate incubation or pre-incubation experiments. No test item precipitate was observed on the plates at any of the concentrations tested in either the presence or absence of metabolic activation (S9-mix) in experiments 1 and 2. The vehicle (sterile distilled water) control plates gave counts of revertant colonies generally within the normal range. There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any concentration of the test item, either with or without metabolic activation (S9-mix) in experiments 1 and 2. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.