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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:
2021-05-05 to 2021-06-14
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Agusut 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
26 June 2020
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Thiourea
EC Number:
200-543-5
EC Name:
Thiourea
Cas Number:
62-56-6
Molecular formula:
CH4N2S
IUPAC Name:
thiourea
Details on test material:
- Name of test material (as cited in study report): Thiourea
- Molecular formula (if other than submission substance): CH4N2S
- Molecular weight (if other than submission substance): 76.12 g/mol
- Substance type: organic; white, odorless solid
- Physical state: solid

Method

Target gene:
S. typhimurium: his;
E. coli: trp
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:
Type and composition of metabolic activation system: S9 mix
- source of S9:
S9 liver microsomal fraction was obtained from Trinova Biochem GmbH, Gießen, Germany - male Sprague Dawley rats were induced with phenobarbital/β-naphthoflavone
- method of preparation of S9 mix:
The S9 mix preparation was performed according to Ames et al. (1973).
1. 100 mM of ice-cold sodium-ortho-phosphate-buffer, pH 7.4, was added to the following pre-weighed sterilised reagents to give final concentrations in the S9 mix of:
8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP
2. This solution was mixed with the liver 9000 x g supernatant fluid in the following proportion:
co-factor solution 9.5 parts, liver preparation 0.5 parts (uring the experiment the S9 mix is stored on ice)
3. S9 Mix Substitution Buffer: the S9 mix substitution buffer was used in the study as a replacement for S9 mix, without metabolic activation (-S9). Phosphate-buffer (0.2 M) contains per litre of purified water:
0.2 M NaH2PO4 x H2O 120 mL, 0.2 M Na2HPO4 880 mL
4. The two solutions were mixed and the pH was adjusted to 7.4. Sterilisation was performed for 20 min at 121 °C in an autoclave.
5. This 0.2 M phosphate-buffer was mixed with 0.15 M KCl solution (sterile) in the following proportion:
0.2 M phosphate-buffer 9.5 parts, 0.15 M KCl solution 0.5 parts (this S9 mix substitution buffer was stored at 4 °C)
- volume of S9 mix and S9 in the final culture medium: 500 µL (or substitution buffer in case of -S9)
- quality controls of S9:
The following quality control determinations were performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-O-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)


References:
Ames, B.N., Durston, W.E., Yamasaki, E. and Lee, F.D. (1973), Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection, Proc. Natl. Acad. Sci. (USA) 70, 2281-2285
Test concentrations with justification for top dose:
5000 μg/plate was selected as the maximum concentration according to the concurrent guideline and pre-experiments for toxicity

The following concentrations of the test item were prepared and used in the experiments:
31.6, 100, 316, 1000, 2500 and 5000 μg/plate
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
Vehicle / solvent:
-solvent used: water (A. dest.)
-source: Eurofins Munich
-batch no.: 210506, 210326, 210520
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
A. dest
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine
Remarks:
+S9: 2-AA; 2-aminoanthracene for S. typhimurium: TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA
(pKM101)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE
- Cell density at seeding: approx. 10^9 cells/mL
- Test substance added in agar: plate incorporation test (experiment I) and the pre-incubation test (experiment II)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: experiment II: 60 min
- Exposure duration: 48 h (37 °C in the dark) after solidification

FOR GENE MUTATION:
- Method to enumerate numbers of viable and mutants cells:
The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH). If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA1535 and TA1537 were counted manually.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method:
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or “B”, respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

EVALUATION CRITERIA

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean
values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and E. coli WP2 uvrA (pKM101) the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher as compared to the reversion rate of the solvent control.


According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Rationale for test conditions:
the experimental design was based on the requirements of the concurrent guideline
Evaluation criteria:
see below
Statistics:
no statistical evaluation was performed

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
experiment I & II
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 examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
experiment I & II
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 examined
True negative controls validity:
not examined
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at a concentration of 5000 μg/plate (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental
conditions reported, Thiourea did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, Thiourea is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

The test item thiourea was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM101).
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:
31.6, 100, 316, 1000, 2500 and 5000 μg/plate
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). In experiment I toxic effects of the test item were noted in tester strain TA1537 at a concentration of 5000 μg/plate (with metabolic activation). No further toxic effects of the test item were observed. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Thiourea at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.