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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Thiourea did not induce gene mutation in bacterial cells (according to OECD No. 471).


Thiourea did not induce micronuclei in mammalian cells (according to OECD No. 487).


Thiourea did not induce gene mutation in mammalian cells (accoridng to OECD No. 476).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-04-20 - 2023-04-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
Batch: 021701
Retest: 2023-08-04
Purity: 100.1 %
Target gene:
HPRT-Gene
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix
Test concentrations with justification for top dose:
Experiment I without metabolic activation: 1.25, 2.5, 5, 7.5, 10 mM
Experiment I with metabolic activation: 1.25, 2.5, 5, 7.5, 10 mM
Experiment II with metabolic activation: 2, 4, 6, 8, 10 mM

No precipitation of the solved test item was noted in any experiment.
No growth inhibition (relative survival < 70%) was observed in any experiments.
Vehicle / solvent:
Culture Medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
Complete Culture Medium: MEM +
10 % FBS
100 U Penicillin / 100 µg/ml strepptomycin
2 mM L-glutamin
25 mM HEPES
2.5 µg/ml amphotericin B

Treatment Medium: MEM supplemented as above w/o FBS

Cells incubated at 37 °C and 5 % CO2
Statistics:
non-parametric Mann-Whitney test
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Conclusions:
In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Thiourea is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

The test substance Thiourea was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster.


The solved test substance was incubated with cells for 4 hours. The main experiments were performed independently with and without metabolic activation.


The concentrations used in the main experiments were selected based on data from the pre-experiments.


 


The test item was investigated at the following concentrations:


Main Experiment without metabolic activation:       1.25, 2.5, 5, 7.5, 10 mM


Main Experiment I with metabolic activation:               1.25, 2.5, 5, 7.5, 10 mM


Main Experiment II with metabolic activation:              2, 4, 6, 8, 10 mM


 


No precipitation of the solved test item was noted in any experiment.


No growth inhibition (relative survival < 70%) was observed in any experiments.


 


In the experiment without metabolic activation at concentrations 1.25 and 2.5 mM the Upper Control Limit of 47.4 was exceeded with mutant frequency values of 49.39 and 61.79, respectively. However, at higher concentrations up to the recommended maximum concentration of 10 mM no increase of mutant frequency values was observed. With 61.79 at 2.5 mM a statistical analysis displayed that the mutant frequency was significantly increased over those of the negative control. Nevertheless, the c² test for trend did not show a concentration-related increase of mutant frequencies. Therefore, no importance was attributed to both concentrations.


 


In the experiment I with metabolic activation at concentration 10 mM the Upper Control Limit of 51.6 was exceeded with mutant frequency values of 51.88. However, a statistical analysis displayed that the mutant frequency was significantly increased over those of the negative control. Nevertheless, the c² test for trend did show a concentration-related increase of mutant frequencies. Therefore, a confirmatory experiment with metabolic activation was performed.


 


In the experiment II with metabolic activation all mutant frequency values were within the historical control data. A statistical analysis displayed that there is no significant increase of mutant frequencies over those of the negative control. Furthermore, the c² test for trend did not show a concentration-related increase of mutant frequencies.


 


DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.


 


 

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-06-25 - 2022-03-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2016-07-29
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Batch: 021701
Purity: 100.1 %
Retest: 2023-08-04
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Homogenate
Test concentrations with justification for top dose:
Experiment I:
without and with metabolic activation: 0.625, 1.25, 2.5, 5, 7.5 and 10 mM
Experiment II:
without metabolic activation: 0.625, 1.25, 2.5, 5, 7.5 and 10 mM

The concentration of 10 mM was considered to be the highest test concentration used in this test
system following the recommendation of the corresponding OECD testing guideline 487.
Vehicle / solvent:
Culture medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
colchicine
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
Complete Culture Medium: MEM +
10 % FBS
100 U Penicillin / 100 µg/ml strepptomycin
2 mM L-glutamin
25 mM HEPES
2.5 µg/ml amphotericin B

Treatment Medium (short-term): MEM supplemented as above w/o FBS

After Treatment Medium / Treatment Medium (long-term exposure)
Complete culture medium with 10% FBS and 1.5 µg/mL cytochalasin B.

Cells incubated at 37 °C and 5 % CO2
Statistics:
non-parametric chi² test
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
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
Conclusions:
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item Thiourea did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells.
Therefore, Thiourea is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Qualifier:
no guideline followed
Principles of method if other than guideline:
Seventy-five chemicals, carcinogens and noncarcinogens, were tested in the SHE (Syrian hamster embryo) micronucleus test in vitro.
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
no data
Species / strain / cell type:
mammalian cell line, other: syrian hamster embryo
Details on mammalian cell type (if applicable):
no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
not specified
Metabolic activation system:
no data
Test concentrations with justification for top dose:
no data
Vehicle / solvent:
no data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
no data
Evaluation criteria:
no data
Statistics:
no data
Species / strain:
mammalian cell line, other: Syrian hamster embryo cells
Metabolic activation:
not specified
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
no data

no data

Conclusions:
Interpretation of results: positive
In this in vitro SHE (Syrian hamster embryo) micronucleus test Thiourea is considered to be clastogenic.
Executive summary:

Thiourea was tested in a SHE (Syrian hamster embryo) micronucleus test in vitro in three independent experiments. After an incubation period of 18 h 6 times 2000 cells were evaluated per experiment. In this study thiourea is considered to be clastogenic.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Qualifier:
no guideline followed
Principles of method if other than guideline:
The methods include the detection of unscheduled DNA synthesis (UDS) with either autoradiography (5 laboratories) or liquid scintillation counting (2 laboratories) and the assessment of DNA single-strand breaks with the alkaline elution assay (1 laboratory)
GLP compliance:
not specified
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Target gene:
no data
Species / strain / cell type:
hepatocytes: rat
Details on mammalian cell type (if applicable):
no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
not specified
Metabolic activation system:
no data
Test concentrations with justification for top dose:
A concentration range of 0.064 to 10000 µg/ml was tested.
Vehicle / solvent:
no data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
no data
Evaluation criteria:
no data
Statistics:
no data
Key result
Species / strain:
hepatocytes: rat
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
no data

no data

Conclusions:
Interpretation of results: negative
Thiourea did not induce of unscheduled DNA synthesis and DNA single-strand breaks.
Executive summary:

Thiourea did not induce unscheduled DNA synthesis (UDS) and DNA single-strand breaks in primary rat hepatocytes at concentrations of 0.064 to 10000 µg/ml.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Qualifier:
no guideline followed
Principles of method if other than guideline:
SOS/umu test according to Oda et al. 1985
GLP compliance:
not specified
Type of assay:
SOS/umu assay
Target gene:
umu
Species / strain / cell type:
S. typhimurium TA 1535 pSK1002
Details on mammalian cell type (if applicable):
n.a.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
1670 µg per plate as highest test concentration
Vehicle / solvent:
no data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
no data
Details on test system and experimental conditions:
no data
Evaluation criteria:
no data
Statistics:
no data
Species / strain:
S. typhimurium TA 1535 pSK1002
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
no data

no data

Conclusions:
Interpretation of results: negative
In this study thiourea is not considered to have mutagenic potential in an umu assay.
Executive summary:

The Umu Chromotest test system is a method to evaluate genotoxic activities of a wide variety of environmental carcinogens and mutagens (Oda et al., 1985). In the present study the abilities of 151 chemicals to induce umu gene expression in Salmonella typhimurium TA1535/pSK1002 was examined. The highest concentration of 1670 µg thiourea per plate did not result in gentoxic effects with and without metabolic activation (S9-mix). Therefore, in this study thiourea is not considered to have mutagenic potential.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
gene comprising the his-operon
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
Details on mammalian cell type (if applicable):
n.a.
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli, other: WP2uvrA-, W3110/pol A+, p3478/pol A-
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0, 0.3, 1.0, 3.3, 10, 33.3, 100, 333.3 µg
Vehicle / solvent:
water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with metabolic activation: 2-anthramine and DMN; without metabolic activation: Sodium azide, 9-Aminoacridine, MMS
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: no
- Exposure duration: 48 h


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
no data
Statistics:
no data
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: weak to moderate cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
not specified
Key result
Species / strain:
E. coli, other: E.coli: W3110/pol A+, p3478/pol A-
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: weak to moderate cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: weak to moderate cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: weak to moderate cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: weak to moderate cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: weak to moderate cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: weak to moderate cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
not specified
Additional information on results:
The test substance exhibited weak to moderate toxicity for the bacteria at high concentration levels
Conclusions:
Interpretation of results: negative
In this study Thiourea is not considered to be genotoxic.
Executive summary:

Thiourea was tested in the concentrations 0, 0.3, 1, 3.3, 10, 33.3, 100 and 333.3 µg per plate in an Ames test for genotoxicity in the strains Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and E. coli uvrA-. Thiourea appeared to be of weak to moderate cytotoxicty but no genotoxic effects could be observed. Furhtermore a DNA repair assay conducted with the strains E.coli W3110/polA+ and p3478/polA- did not show a genetoxic potential of thiourea. There was no dose-response relationship.

In this study thiourea is not considered to have genotoxic potential. This study is not adequate because there is no justification for the selection of the doses of thiourea in this study. The concentration range which was chosen might be to low to determine genotoxic effect of the substance.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Deviations:
not specified
GLP compliance:
no
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Target gene:
n.a.
Species / strain / cell type:
hepatocytes: Wistar rats
Details on mammalian cell type (if applicable):
- Type and identity of media: Waymouth Medium MB 752/1 + 2mM GLutamin, Penicillin (100 U/ml)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
No details reported on test concentrations; concentration range: 0.3 to 30 mM
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Methylmethansulfonate (MMS), 2-Acetamidofluoren (2-AAF)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: Incubation of the cells with BrdUrd and FdUrd for 1h
- Exposure duration: Incubation of the cells BrdUrd, FdUrd, ³H-dCyd for 2.5 h
- Expression time (cells in growth medium): 2.5 h
- Selection time (if incubation with a selection agent): n.a.
- Fixation time (start of exposure up to fixation or harvest of cells): no data
Evaluation criteria:
Determination of the cpm-profile to identify DNA repair. DNA repair becomes visible through a peak at the UV absoprtion maximum
Statistics:
not reported
Key result
Species / strain:
hepatocytes: Wistar rats
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
no additional information on results
Conclusions:
Interpretation of results: negative
Thiourea was evaluated for mutagenic potential in the DNA-repair test and did not induce a positive mutagenic response under the conditions of this study.
Executive summary:

Thiourea was tested in a DNA repair assay. Therefore primary hepatocytes isolated from rats (WIstar) were incubated with the test substance in concentrations ranging from 0.3 -30 mM and the thymidine analogon BrdUrd and radioactive labelled Desoxycytidine ( ³H-dCyd). Based on the results of these experiments, it is concluded that Thiourea did not induce DNA repair in hepatocytes.

The method applied in this study is described in detail. But there is no detailed information of the results available.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: other: genome rearrangements
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Qualifier:
no guideline followed
Principles of method if other than guideline:
A system screening for intrachromosomal recombination that results in genome rearrangement has been constructed for potential use as a short-term test in the yeast Saccharomyces cerevisiae.
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
no data
Species / strain / cell type:
Saccharomyces cerevisiae
Details on mammalian cell type (if applicable):
no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
not specified
Metabolic activation system:
no data
Test concentrations with justification for top dose:
Thiourea was tested at concentrations of 0, 5, 10, 20 and 30 mg/ml.
Vehicle / solvent:
no data
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
no data
Evaluation criteria:
no data
Statistics:
no data
Key result
Species / strain:
other: Saccharomyces cerevisiae
Metabolic activation:
not specified
Genotoxicity:
other: possibly positiv,but cytotoxicity might interfere with result
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
no data

no data

Conclusions:
Interpretation of results: ambiguous genotoxic effect might be due to cytotoxicity of the substance
It cannot be excluded that the positive effect on genotoxicity is based on the cytotoxic effect of thiourea because the survival rate was only 14% (20 mg/l) and 0.68% (30mg/l), respectively in comparison to the control (100%).
Executive summary:

A system screening for intrachromosomal recombination that results in genome rearrangement has been constructed for potential use as a short-term test in the yeast Saccharomyces cerevisiae. Thiourea was tested at concentrations of 0, 5, 10, 20 and 30 mg/ml. In case of a dose dependent 2fold induction of the DEL recombination and intrachromosomal recombination in comparison to the control the results were treated as positive in terms of genotoxicity. This was reported for thiourea at concentrations of 20 and 30 mg/ml. The mutation frequency for His+/104 cells was reported to be 3.4fold and 22.4fold, respectively, and for ADE+/105 cells 1.86 fold and 18.5fold, respectively in comparison to the control. It cannot be excluded that these result are based on the cytotoxic effect of thiourea because the survival rate was only 14% (20 mg/l) and 0.68% (30mg/l), respectively in comparison to the control (100%).

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
Deviations:
yes
Remarks:
20 instead of 25 metaphases were analysed
GLP compliance:
no
Type of assay:
sister chromatid exchange assay in mammalian cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not reported
- Periodically checked for karyotype stability: not reported
- Periodically "cleansed" against high spontaneous background: not reported
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (aroclor 1254)
Test concentrations with justification for top dose:
0, 0.755*10^-3 µg/mL, 7.8*10^-3 µg/mL, 83.2*10^-3 µg/mL 0.832 mg/mL, 8.6 mg/mL and 0, 5, 20, 50, 100 and 250 mg/10mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ham`s F10 medium
- Justification for choice of solvent/vehicle: Ham`s F10 medium is used for cultivation of CHO cells
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
no
Positive control substance:
no
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period: yes; 4 h during which the cells attach themselves to the walls
- Exposure duration: 1 h
- Expression time (cells in growth medium): 24 h
- Selection time (if incubation with a selection agent): no
- Fixation time (start of exposure up to fixation or harvest of cells): 25 h

SELECTION AGENT (mutation assays): no
SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Hoechst 33258 fluorochrome, Giemsa

NUMBER OF REPLICATIONS: not reported

NUMBER OF CELLS EVALUATED: 20 metaphases were scored

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no

Other: Survival of the cells: 500 cells were plated on petri dishes with medium; cell were allowed to attach 5 h; 1 h exposure with test substance; one week after esposure cells were stained with methylene blue and counted; exposure conditions similar to SCE test
Evaluation criteria:
Dose-response curves were assumed to be linear (y=ax+b; x= dose; y= number of SCE per metaphase); maximum likelihood method; Likelihood ratio test to test whether the coefficient , a, of the calculated line differed significantly from zero
Statistics:
maximum likelihood method
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
small but significant increase in the number of sister chromatid exchanges per metaphase
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
the highest dose (25mg/mL) appeared to be toxic to the cells
Vehicle controls validity:
not specified
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality:no data
- Evaporation from medium:no data
- Water solubility:no data
- Precipitation:no data
- Other confounding effects:no data

RANGE-FINDING/SCREENING STUDIES: initially concentrations up to 8.6 mg/mL were tested; they did not influence survival of the cells

Remarks on result:
other: strain/cell type: CHO

Table 1: Survival of CHO cells after 1 h exposure to thiourea
























































Dose mg/mL



0



0.755*10-3



7.8*10-3



83.2*10-3



0.832



8.6



Clones per plate



375



368



401



319



395



398



411



363



363



405



405



347



368



343



436



 



 



425



390



392



 



 



 



394



334



381



 



 



 



435



 


 


Table 2: Induction of SCE by thiourea

































































Dose mg/10 mL



+ S9



- S9



 



X



s



n



X



s



n



0



10.70



4.43



20



8.75



3.21



20



5



9.55



3.26



20



9.50



4.39



20



20



11.28



4.75



20



12.70



4.95



21



50



13.05



5.72



20



13.75



3.51



20



100



13.05



3.86



20



12.70



3.33



20



+ S9 = with S9-Aroclor 1254


X= average number of SCE/metaphase when n metaphases are conducted


s= standard deviation of the mean


 

Conclusions:
Interpretation of results: positive
Thioharnstoff induces a very small but significant increase (p>0.005) in SCE per metaphase under the conditions of the test.
Executive summary:

In a mammalian cell cytogenetics assay (sister chromatid exchange; SCE), CHO cell cultures were exposed to Thiourea in Ham`s F10 medium at concentrations of 0, 5, 20, 50, 100 and 250 mg/10mL with and without metabolic activation (S9-aroclor 1254). Thiourea was tested up to the cytotoxic concentration 250 mg/10 mL. The dose of 250 mg/10mL caused gross toxic effects which ruled out scoring for SCE induction. Thiourea induced a very small but significant increase (p<0.005) in SCE per metaphase under the conditions of the test. The increase is independent of metabolic activation. The effect is slight even at high doses. This study is classified as acceptable and satisfies the requirement for in vitro cytogenetic mutagenicity data.

Endpoint:
genetic toxicity in vitro, other
Remarks:
Type of genotoxicity: other: DNA damage/repair, chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
In this study the outcome of several studies on genotoxic effects of the thiourea metabolite FASA are summarized. No detailled information on the results is available. Tests for determination of cytotoxicity, mutagenicity, micronucleus induction and DNA repair were conducted.
GLP compliance:
no
Type of assay:
other: Determination of cytotoxicity, mutagenicity and micronucleus induction
Target gene:
no data
Species / strain / cell type:
other: V79 cells and hepatocytes of the rat
Details on mammalian cell type (if applicable):
- Type and identity of media: V79-cells DMEM (Dulbecco`s Medium modified (Eagle) + 10% fetal calf serum + antibiotics)
- Properly maintained: yes (7% CO2/93% Air, 37°C)
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
5-100 mM
Vehicle / solvent:
no data
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
no data
Evaluation criteria:
no data
Statistics:
no data
Key result
Species / strain:
hepatocytes: V79
Metabolic activation:
not specified
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Thiourea metabolite FASA is cytotoxic
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
Interpretation of results: positive
The thiourea metabolite FASA (formamidine sulfinate) is considered to be responsible for the genotoxic effects of thiourea in V79 hepatocytes. FASA induces DNA repair, micronuclei formation and gene mutation.
Executive summary:

In this study the thiourea metabolite FASA (formamidine sulfinate) was tested for its genotoxic effects on V79 hepatocytes of rats. FASA is formed enzymatically by FMO (FAD-dependent monooxygenase) and is inducing several genotoxic effects like induction of gene mutation, micronuclei formation and DNA repair. The author concluded that FASA is responsible for the genotoxic effects elicited by thiourea.

Endpoint:
genetic toxicity in vitro, other
Remarks:
Type of genotoxicity: other: gene mutation and DNA damage
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Qualifier:
no guideline followed
Principles of method if other than guideline:
not applicable
GLP compliance:
not specified
Type of assay:
other: genotoxicity in human thyroid follicle cells
Target gene:
no data
Species / strain / cell type:
other: human thyroid follicle cells
Details on mammalian cell type (if applicable):
no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
not specified
Metabolic activation system:
no data
Test concentrations with justification for top dose:
no data
Vehicle / solvent:
no data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
no data
Details on test system and experimental conditions:
no data
Evaluation criteria:
no data
Statistics:
no data
Key result
Species / strain:
other: human thyroid follicle cells
Metabolic activation:
not specified
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
no data

no data

Conclusions:
Interpretation of results other: marginally genotoxic

In this study Thiourea is considered to be marginally genotoxic due to the slight increase in the frequency of DNA strand bracks. Thiourea is not associated with the formation of thytoid tumours due to genotoxic effects.
Executive summary:

The results obtained in the PhD thesis of Timme Madlon (1998) on "Studies on the mechanism and significance of thiourea genotoxicity in mammalian cells in vitro and in vivo – The genotoxicity and metabolism of thiourea in human thyroid follicle cells" showed that thiourea was marginally genotoxic to human thyroid follicle cells. The genotoxicity was indicated by a slight increase in the frequency of DNA strand breaks in the cells and possibly due to slight auto oxidation of thiourea to electrophilic S oxygenated products. The investigations yielded no indication of organ-specific metabolic activation of thiourea in thyrocytes. It can therefore be assumed that human exposure to thiourea is not associated with a relevant risk of thyroid tumours due to genotoxic effects.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
L5178Y mouse lymphoma cell mutagenesis assay
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
no data
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
The concentrations tested were in the range of 0 and 5000 µg/mL without and between 0 and 6000 µg/mL with S9 mix.
Vehicle / solvent:
no data
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
no data
Evaluation criteria:
no data
Statistics:
no data
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
no data

no data

Conclusions:
Interpretation of results: ambiguous with metabolic activation
The results of this study are ambiguous and it cannot be excluded that thiourea has a genotoxic potential.
Executive summary:

The L5178Y mouse lymphoma cell mutagenesis assay is used to detect the mutagenic activity of chemicals in a mammalian cell system. To evaluate this assay the results of assays performed independently on 63 chemicals by laboratories at SRI International and Litton Bionetics, Inc. were evaluated. The two laboratories (A and B) used similar protocols. The assay was performed with and without metabolic activation (S9 mix). The concentrations tested were in the range of 0 and 5000 µg/ml without and between 0 and 6000 µg/ml with S9 mix. Without metabolic activation thiourea showed no mutagenic and toxic potential whereas with metabolic activation in laboratory B the test result was positive. Because there was no information on possible toxic effects associated with the positive result in the mouse lymphoma assay it cannot be excluded that cytotoxicity of thiourea is the reason for the genotoxic effect observed.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
the mouse lymphoma TK+/(-)-TK-/- forward mutation assay.
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
0, 0.068, 0.68, 1.37, 2.05, 2,74 mg/mL without metabolic activation and 0, 0.63, 0.95, 1.26, 1.89 and 2.52 mg/mL with metabolic activation
Vehicle / solvent:
no data
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
no data
Evaluation criteria:
no data
Statistics:
no data
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
no data

no data

Conclusions:
Interpretation of results: ambiguous depending on evaluation of results
In this study thiourea was considered to be slightly mutagenic.
Executive summary:

Thiourea was tested in concentrations of 0, 0.068, 0.68, 1.37, 2.05, 2,74 mg/mL without metabolic activation and 0, 0.63, 0.95, 1.26, 1.89 and 2.52 mg/mL with metabolic activation. Without metabolic activation a mutation frequency of 1.3 (p≤0.01) in comparison to the control was reported for concentrations of 1.37 and 2.05 mg/mL. With metabolic activation a mutation frequency of 1.6 (p≤0.01) was reported at the highest concentration (2.52 mg/mL). This positive effect was due to the statistical evaluation of the results. Underlying the criterion of a twofold induction of mutation frequency as positive result the results reported in this study would not be considered as positive. In this study thiourea was considered to be slightly mutagenic.

Endpoint:
genetic toxicity in vitro, other
Remarks:
Type of genotoxicity: other: gene and genome mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Qualifier:
no guideline followed
Principles of method if other than guideline:
no data
GLP compliance:
not specified
Type of assay:
other: Forward mutations (methionine suppressors), mitotic crossing-over and chromosome malsegregation
Target gene:
no data
Species / strain / cell type:
other: Aspergillus nidulans
Details on mammalian cell type (if applicable):
no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Metabolic activation system:
n.a.
Test concentrations with justification for top dose:
65.7 and 197.1 mM (equal to 5 and 15 mg/mL)
Vehicle / solvent:
no data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
no data
Evaluation criteria:
no data
Statistics:
no data
Key result
Species / strain:
other: Aspergillus nidulans
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
no data

no data

Conclusions:
Interpretation of results: negative
Thiourea did not induce forward mutations and chromosome malsegregation in Aspergillus nidulans. In this study thiourea is not considered to have mutagenic potential.
Executive summary:

The genotoxicitiy of thiourea was tested in Aspergillus nidulans at concentration of 5 and 15 mg/ml. Forward mutations (methionine suppressors), mitotic crossing-over and chromosome malsegregation were the end-points scored. Thiourea did not induce genotoxic effects despite being tested at cytotoxic concentration.

Endpoint:
genetic toxicity in vitro, other
Remarks:
Type of genotoxicity: other: gene conversion
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Qualifier:
no guideline followed
Principles of method if other than guideline:
gene conversion within the trp-locus
GLP compliance:
not specified
Type of assay:
other: gene conversion in Saccharomyces cerevisiae
Target gene:
trp-locus
Species / strain / cell type:
Saccharomyces cerevisiae
Details on mammalian cell type (if applicable):
no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
not specified
Metabolic activation system:
no data
Test concentrations with justification for top dose:
0,12 to 0.4 M (about 9.13 to 30.44 mg/ml)
Vehicle / solvent:
no data
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
no data
Evaluation criteria:
no data
Statistics:
no data
Species / strain:
Saccharomyces cerevisiae
Metabolic activation:
not specified
Genotoxicity:
positive
Remarks:
gene conversion
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
no data
Remarks on result:
other: all strains/cell types tested

no data

Conclusions:
Interpretation of results: positive
Thiourea led to an increase of gene conversion within the trp-locus in comparison to the control. In this study thiourea is considered as mutagenic.
Executive summary:

Gene conversion frequency was tested in Saccharomyces cerevisiae in the presence of 0.12 to 0.4 M thiourea. An increase of gene conversion up to a degree of 1.5 to 7.5 fold in comparison to the control within the trp-locus was reported. Therefore, in this study thiourea is considered to have mutagenic potential.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
trp+-revertant
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
trp-locus
Species / strain / cell type:
Saccharomyces cerevisiae
Additional strain / cell type characteristics:
other: permeable mutant C658-K42
Metabolic activation:
with and without
Metabolic activation system:
no data
Test concentrations with justification for top dose:
Thiourea was test in concentration of 0, 0.5, 1 and 2 mg/mL.
Vehicle / solvent:
no data
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
no data
Evaluation criteria:
no data
Statistics:
no data
Key result
Species / strain:
other: Saccharomyces cerevisiae mutant C658-K42
Metabolic activation:
with and without
Genotoxicity:
other: no dose response relationship
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
no data

no data

Conclusions:
Interpretation of results: other: no dose-response relationship
In this study thiourea induces the incidence of trp+-revertant. No conclusion can be drawn for genotoxicity as there is no dose-response relationship.
Executive summary:

The permeable Saccharomyces cerevisiae mutant C658-K42 was employed for mutagenicity tests on 12 carcinogens which had been reported to be non-mutagenic in Salmonella/microsome tests.Thiourea was tested at concentration of 0, 0.5, 1 and 2 mg/mL with and without metabolic activation. Without metabolic activation no effect was observed whereas with metabolic activation at a concentration of 0.5 mg/mL thiourea a 6.7 fold and at a concentration of 1 mg/mL a 4.5 fold increase of trp+-revertants could be observed. In contrast to that 2.0 mg/ml thiourea did not lead to any effect. Cytotoxicity could be ruled out to influence the results of this study.Therefore a dose-response relationship can not be derived.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Qualifier:
no guideline followed
Principles of method if other than guideline:
A differential DNA repair test was evaluated in vitro, using derivatives of E. coli K-12 343/113 with the genotype uvrB-/recA- and uvrB+/recA+. The aim of this study was to characterize the sensitivity of the assay to different compounds in vitro and thereby provide information on the usefulness of this end-point as an indicator of genotoxicity in a host-mediated assay
GLP compliance:
not specified
Type of assay:
other: DNA repiar in E.coli K-12
Target gene:
no data
Species / strain / cell type:
E. coli, other: K-12 343/113
Details on mammalian cell type (if applicable):
n.a.
Additional strain / cell type characteristics:
other: uvrB-/recA- and uvrB+/recA+
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
up to 329 mM (equals to 25 mg/mL); concentration range not specified
Vehicle / solvent:
no data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
no data
Evaluation criteria:
no data
Statistics:
no data
Key result
Species / strain:
E. coli, other: K-12 343/113
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
E. coli, other: K-12 343/113
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
no data

no data

Conclusions:
Interpretation of results: negative with metabolic activation
In a differential DNA-repair test in E. coli K-12 343/113 Thiourea showed positive results without and negative results with metabolic activation.
Executive summary:

A differential DNA repair test was evaluated in vitro, using derivatives of E. coli K-12 343/113 with the genotype uvrB-/recA- and uvrB+/recA+. The aim of this study was to characterize the sensitivity of the assay to different compounds in vitro and thereby provide information on the usefulness of this end-point as an indicator of genotoxicity in a host-mediated assay. Thiourea was tested in concentrations up to 329 mM (equal 25 mg/ml) with and without metabolic activation. Thiourea showed positive results without and negative results with metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
It would not be possible to detect cross-linking ad oxidizing agents because strain S. typhimurium TA 102 or E.coli WP2 uvrA is missing.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
strain S. typhimurium TA 102 or E.coli WP2 uvrA is missing;
Qualifier:
according to guideline
Guideline:
other: Entwurf der Prüfungsvorschrift UBA "Screening-Test auf Mutagenität" vom März 1979
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
genes in the histidine operon
Species / strain / cell type:
S. typhimurium, other: TA1535, TA100, TA1537, TA1538, TA98
Details on mammalian cell type (if applicable):
n.a.
Additional strain / cell type characteristics:
other: histidine autotrophy; uvrA-, rfa-
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
No details reported on test concentrations; five dosed of the test substance according the guideline.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9-mix: Sodium azide, 9-Aminoacridine, 4-Nitro-o-phenylendiamine; with S9-mix: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period:n.a.
- Exposure duration: 72 h
- Selection time (if incubation with a selection agent): n.a.
Evaluation criteria:
The positive controls showed with and without the expected mutation frequency.
Statistics:
not reported
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
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:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
no additional information on results
Conclusions:
Interpretation of results: negative
Thiourea was evaluated for mutagenic potential in the Ames test, both in the presence and the absence of metabolic activation, and did not induce a positive mutagenic response under the conditions of this study.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537, and 1538 of S. typhimurium were exposed to Thiourea. The test was performed with and without the addition of S-9 mix as an extrinsic metabolic activation system. The compound was dissolved in DMSO and tested at a five concentrations in the presence and absence of a metabolic activation system. The test was conducted in fourfold replicate. The experiments with and without metabolic activation were performed as plate incorporation assay.

In both experiments, performed with and without metabolic activation, none of the tested concentrations of Thiourea induced a positive mutagenic response under the conditions of this study.

Based on the results of these experiments, it is concluded that Thiourea did not induce gene mutations in the strains of S. typhimurium used.

This study is classified as acceptable. Hoewever, it would not be possible to detect cross-linking and oxidizing agents because strain S. typhimurium TA 102 or E.coli WP2 uvrA is missing.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Qualifier:
no guideline followed
Principles of method if other than guideline:
A comparison was made for 40 compounds belonging to different chemical classes of mutagenicity as measured by the Salmonella assay and of the SOS-inducing potency as measured by the SOS chromotest kit procedure.
GLP compliance:
not specified
Type of assay:
other: Salmonella/microsome test and SOS chromotest
Target gene:
no data
Species / strain / cell type:
S. typhimurium, other: TA 97, TA 98, TA 100
Details on mammalian cell type (if applicable):
n.a.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
100-1000 µg per plate (Salmonella/microsome assay)
7.6 ng/mL to 7.6 mg/mL (SOS chromotest kit procedure)
Vehicle / solvent:
no data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
no data
Details on test system and experimental conditions:
incubation time: 48 h (Salmonella/microsome assay), 2 h (SOS chromotest)
Evaluation criteria:
no data
Statistics:
no data
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
not applicable
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
not examined
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
not applicable
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
not examined
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:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
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:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
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:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
no data

no data

Conclusions:
Interpretation of results: negative
Thiourea is considered to be not mutagenic in this study.
Executive summary:

A comparison was made for 40 compounds belonging to different chemical classes of mutagenicity as measured by the Salmonella assay and of the SOS-inducing potency as measured by the SOS chromotest kit procedure. It was found that most (78%) of the chemicals described as mutagens/carcinogens (14 compounds) were detected with a simplified Ames test procedure, using 3 strains (TA 97, TA 98, TA 100) and 3 concentrations of the tested material. Thiourea was tested in the concentrations 100-1000 µg per plate with and without metabolic activation (S9 mix) and 48 h incubation. In this Salmonella/microsome assay thiourea did not show mutagenic potential neither with nor without metabolic activation.


In the SOS chromotest concentrations in the range of 7.6 ng/mL to 7.6 mg/mL were tested with 2 h incubation. Neither with nor without metabolic activation a mutagenic potential of thiourea could be shown.


Thiourea is considered to be not mutagenic in this study.

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
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
Target gene:
S. typhimurium: his;
E. coli: trp
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
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
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
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.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Salmonella/Microsome test with the strains TA 97, TA 98, TA 100 and TA 1535 with and without metabolic activation
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
no data
Species / strain / cell type:
S. typhimurium, other: TA 97, TA 98, TA 100, TA 1535
Details on mammalian cell type (if applicable):
n.a.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0, 100, 333, 1000, 3333 and 10000 µg per plate.
Vehicle / solvent:
no data
Details on test system and experimental conditions:
no data
Evaluation criteria:
no data
Statistics:
no data
Species / strain:
S. typhimurium TA 1537
Remarks:
not tested
Metabolic activation:
not applicable
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
not examined
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:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
not specified
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:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
not specified
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:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
not specified
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:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
not specified
Additional information on results:
no data

no data

Conclusions:
Interpretation of results: negative
Thiourea is not considered to have genotoxic potential.
Executive summary:

Thiourea was tested in a Salmonella/Microsome test with the strains TA 97, TA 98, TA 100 and TA 1535 with and without metabolic activation in concentrations of 0, 100, 333, 1000, 3333 and 10000 µg per plate. Neither with nor without metabolic activation thiourea showed mutagenic potential. In this study thiourea is not considered to be a mutagen.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Thiourea did not induce genotoxicity in an in vivo micronucleus test (equivalent to OECD No. 474).


In insects weak positive results were obtained.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
February 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
400 instead of 200 erythrocytes were examined
GLP compliance:
no
Type of assay:
micronucleus assay
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Central institute for the breeding of laboratory animals TNO, Zeist
- Age at study initiation: 4-5 weeks
- Assigned to test groups randomly: the animals were assigned to three test groups of 5 males and 5 females according body weight
- Fasting period before study: deprivation of food for 14-15 h
- Housing: screen bottom cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24+/-1°C
- Humidity (%): 40-60 %
- Air changes (per hr): no data. air conditioned rooms
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle: 350 mg/kg bw in 5 mL water per kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: freshly prepared immediately before treatment of the animals

Duration of treatment / exposure:
The test samples were administered twice with an interval of 24 h. 6 hours after the last treatment the animals were killed.
Frequency of treatment:
twice
Post exposure period:
6 hours
Dose / conc.:
350 mg/kg bw/day (nominal)
Remarks:
Basis: nominal in water
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
2,3,5-tris-ethyleneiminobenzoquinone (Trenimon; Bayer) was used as a solution in physiological saline
- Justification for choice of positive control(s): no data
- Route of administration: oral
- Doses / concentrations: 0.0625 mg/kg body weight
Tissues and cell types examined:
bone marrow of the femora; erythrocytes
Details of tissue and slide preparation:
The bone marrow of the femora was flushed into centrifuge tubescontaining foetal calf serum and centrifuged. The excess serum was removed. The cells were than resuspended by mixing gently with a pasteur pipette. A drop was placed on a slide cleaned with methanol overnight and spread with a haemocytometer cover glass. Five slides were prepared of each animal. The smears were air dried, fixed in methanol and stained according to May-Grünwald.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
The dose-levels used in the present study were based on acute oral LD50 of the test material for rats (CIVO letters 29/12/75)

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Oral administration of thiourea did not affect the incidence of micronucleated erythrocytes
- Ratio of PCE/NCE (for Micronucleus assay):Oral administration of thiourea did not affect the ratio of poly and normochromatic erythrocytes
- Appropriateness of dose levels and route:
- Statistical evaluation: standard deviations were determined

No mortality or abnormalities of condition or behaviour, attributable to treatment were observed in any of the animals during the exposure period.


 


 


Table 1: Average body weights of groups of 5 rats



























Group No



Treatment/kg body weight



Average body weight +/- SD



males



females



390



2x5mL distilled water



99+/-3



79+/-7



392



2x5 mL 7% Thioharnstoff



96+/-4



81+/-5



 


 


 


Table 2: Mean numbers of micronucleated erythrocytes and percentage polychromatic erythrocytes in bone marrow of rats












































Treatment



Sex



Incidence of micronucleated cells per 2000 erythrocytes per rat



Percentage polychromatic erythrocytes +/-SD



mean



range



Vehicle control



Male



4.4



2-7



74.2+/-3.5



Thioharnstoff



Male



5.4



4-8



72.0+/-3.7



Vehicle control



Female



5.0



2-8



63.8+/-5.1



Thioharnstoff



female



4.0



4-6



62.9+/-3.5


Conclusions:
Interpretation of results: negative
Thiourea did no increase the frequency of micronucleated polychromatic erythrocytes in bone marrow. In this study thiourea did not reveal any evidence for mutagenic activity.
Executive summary:

In a Wistar rats bone marrow micronucleus assay, 5 male and 5 female animals per dose were treated by oral gavage with Thiourea (7 % a.i.) at doses of 0 and 350 mg/kg bw. Bone marrow cells were harvested at 6 hours post-treatment. The vehicle was water. There were no signs of toxicity during the study. Thiourea was tested at an adequate dose based on the results of an oral acute toxicity test. The positive control induced the appropriate response. There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.

Endpoint:
in vivo mammalian germ cell study: gene mutation
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Qualifier:
no guideline followed
Principles of method if other than guideline:
Zeste-white (UZ) somatic mutation assay
GLP compliance:
not specified
Type of assay:
somatic mutation and recombination test in Drosophila
Species:
Drosophila melanogaster
Strain:
not specified
Sex:
not specified
Details on test animals or test system and environmental conditions:
no data
Route of administration:
other: no data
Vehicle:

- Vehicle(s)/solvent(s) used: water
Details on exposure:
Concentrations of 0.1 and 0.5 mM in water (equals to 7.6 and 38.1 µg/mL) were used.
Duration of treatment / exposure:
no data
Frequency of treatment:
no data
Post exposure period:
no data
Dose / conc.:
0.1 other: mM
Remarks:
Basis: nominal conc.
equals 7.6 µg/mL
Dose / conc.:
0.5 other: mM
Remarks:
Basis: nominal conc.
equals 38.1 µg/mL
No. of animals per sex per dose:
no data
Control animals:
not specified
Positive control(s):
no data
Tissues and cell types examined:
no data
Details of tissue and slide preparation:
no data
Evaluation criteria:
no data
Statistics:
no data
Key result
Sex:
not specified
Genotoxicity:
positive
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
no data

no data

Conclusions:
Interpretation of results: positive
In this study thiourea induced somatic mutations in Drosophila melanogaster. However, from the availabel data it is not possible to exclude a cytotoxic effect.
Executive summary:

Thiourea was tested in Drosophila melanogaster in a zeste-white assay. Thiourea induced somatic mutations at concentrations of 0.1 and 0.5 mM (equals to 7.6 and 38.1 µg/mL). The results of this study showed that Thiourea produces significant increases in the eye spot frequency. In this study Thiourea is considered to have genotoxic potential. However, from the availabel data it is not possible to exclude a cytotoxic effect.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
Eye mosaic white/white + (w/w+) assay of Drosophila (in vivo short-term test measuring genetic damage in somatic cells of Drosophila after treatment of larvae).
GLP compliance:
yes
Type of assay:
other: white/white+ somatic assay
Species:
Drosophila melanogaster
Strain:
other: Hikone-R, Haag 79-R
Sex:
male/female
Route of administration:
other: chronic exposure
Duration of treatment / exposure:
three days of mating then during all three instar stages of larval development
Frequency of treatment:
chronic
Post exposure period:
1-5 days
Dose / conc.:
0 other: mM
Remarks:
Basis: nominal in diet
Dose / conc.:
0.25 other: mM
Remarks:
Basis: nominal in diet
Dose / conc.:
0.5 other: mM
Remarks:
Basis: nominal in diet
Dose / conc.:
1 other: mM
Remarks:
Basis: nominal in diet
No. of animals per sex per dose:
15 pairs mated, hatched females were taken and scored
Control animals:
yes, concurrent vehicle
Tissues and cell types examined:
inspected for the occurrence of white in their compound eyes
Key result
Sex:
female
Genotoxicity:
negative
Remarks:
chemical biotransformed and detoxified
Toxicity:
yes
Remarks:
induced a drop in spot frequencies at highest concentration due to pronounced toxicity
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Conclusions:
Interpretation of results: negative
Thiourea is negative under conditions of this w/w + somatic assay of Drosophila
Executive summary:

Thiourea proved to be non-genotoxic in an eye mosaic white/white + (w/w+) assay of Drosophila. The authors of this study concluded that the chemical was probably biotransformed and detoxified. This assumption was supported by a drop in spot frequencies at the highest concentration tested, an effect probably due to the pronounced toxicity of the test compound.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Qualifier:
no guideline followed
Principles of method if other than guideline:
Drosophila assay; monitoring interchromosomal mitotic recombination; in vivo short-term test measuring genetic damage in somatic cells of Drosophila after treatment of larvae
GLP compliance:
not specified
Type of assay:
other: Drosophila eye mosaic assay
Species:
Drosophila melanogaster
Strain:
not specified
Sex:
not specified
Details on test animals or test system and environmental conditions:
no data
Route of administration:
other: no data
Vehicle:
water
Details on exposure:
no data
Duration of treatment / exposure:
no data
Frequency of treatment:
no data
Post exposure period:
no data
Dose / conc.:
0.5 other: mM
Remarks:
Basis: nominal conc.
38.1 µg/mL
Dose / conc.:
1 other: mM
Remarks:
Basis: nominal conc.
76.2 µg/mL
No. of animals per sex per dose:
no data
Control animals:
not specified
Positive control(s):
no data
Tissues and cell types examined:
no data
Details of tissue and slide preparation:
no data
Evaluation criteria:
no data
Statistics:
no data
Key result
Sex:
not specified
Genotoxicity:
positive
Toxicity:
yes
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
no data

no data

Conclusions:
Interpretation of results: other: weak positive results were associated with toxicity
Thiourea induced somatic mutations in Drosophila melanogaster. Those were associated with signs of cytotoxicity and therefore could not be evaluated.
Executive summary:

Thiourea was tested at concentration of 0.5 mM and 1 mM (38.1 and 76.2 µg/mL) in an in vivo short-term test measuring genetic damage in somatic cells of Drosophila after treatment of larvae. Thiourea induced somatic mutations which were associated with signs of toxicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:


 


Key Study in vitro bacterial reverse mutation assay (OECD 471)


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.


 


Supporting Studies


Additional supplementary studies were evaluated. Thiourea did not induce gene mutation in bacteria, but mixed results were obtained in mammalian cells. It consistently induced chromosomal recombination in yeast and insects and induced mammalian cell transformation. Thiourea is reported to elicit cytotoxicity in mammalian cell cultures. In several in vitro tests cytotoxicic effects have not been reported making it difficult to verify any positive result. Thiourea did not induce genotoxicity in an in vivo micronucleus test in rats.


Based on the available data the MAK commission concluded in 1990 that Thiourea is weak genotoxic. The WHO/IPCS concluded in 2003 that thiourea is not considered to be a genotoxic carcinogen and that the development of thiourea-induced thyroid tumours may involve inhibition of peroxidase in the thyroid gland, leading to decreased thyroid hormone production and increased proliferation as a result of an increase in the secretion of thyroid-stimulating hormone.


Based on the overall assessment of the available data Thiourea is not considered being genotoxic. This is in agreement with the WHO/IPCS conclusion (2003).


Andrae (1989) tested the Thiourea metabolite FASA (formamidine sulfinate) for its genotoxic effects on V79 hepatocytes of rats. FASA is formed enzymatically by FMO (FAD-dependent monooxygenase) and is inducing several genotoxic effects like induction of gene mutation, micronuclei formation and DNA repair. The author concluded that FASA would be responsible for the genotoxic effects elicited by thiourea if it would be formed in vivo, which was not verified in this study.


The results obtained in the PhD thesis of Timme Madlon (1998) on "Studies on the mechanism and significance of thiourea genotoxicity in mammalian cells in vitro and in vivo – The genotoxicity and metabolism of thiourea in human thyroid follicle cells" showed that thiourea was marginally genotoxic to human thyroid follicle cells. The genotoxicity was indicated by a slight increase in the frequency of DNA strand breaks in the cells and possibly due to slight auto oxidation of thiourea to electrophilic S oxygenated products. The investigations yielded no indication of organ-specific metabolic activation of thiourea in thyrocytes. It can therefore be assumed that human exposure to thiourea is not associated with a relevant risk of thyroid tumours due to genotoxic effects.


 


The following tables summarize several of the available test reports, some are not included as individual IUCLID study records, but to emphasise the amount of available, ambiguous data.


 


 


Genotoxicity and related endpoints: in vitro


Table1: summary of available studies on genotoxicity in vitro























































































































































































































































































































Endpoint



Results and references



Species, strain



Result



Metabolic activation



Reference



Genotoxicity and related endpoints:in vitro



Gene mutation



Salmonella typhimurium TA 97



Negative



+/-



Brams et al. 1987
Zeiger et al. 1988



Salmonella typhimurium TA1535, TA100, TA1537, TA1538, TA98



Negative



+/-



Eurofins, 2021


Korte und Greim, 1981


Brusick, 1977



Salmonella typhimurium TA 98



Negative



+/-



Simmon 1979a*
Dunkel et al. 1984*
Brams et al. 1987
Zeiger et al. 1988



Salmonella typhimurium TA 100



Negative



+/-



Eurofins, 2021


Simmon 1979a*
Yamaguchi 1980*
Dunkel et al. 1984*
Brams et al. 1987
Zeiger et al. 1988



Salmonella typhimurium TA1535



Negative



+/-



Eurofins, 2021


Simmon 1979a*
Rozenkranz and Poirier 1979*
Dunkel et al. 1984*
Zeiger et al. 1988



Salmonella typhimuriumTA1535/pSK1002 umu test



Negative



+/-



Nakamura et al. 1987



Salmonella typhimuriumTA1537



Negative



+/-



Eurofins, 2021


Simmon 1979°*
Dunkel et al. 1984*



Salmonella typhimuriumTA1538



Negative



+/-



Rozenkranz and Poirier 1979*
Simmon 1979a*
Dunkel et al. 1984*



Escherichia coli SOS repair



Negative



+/-



Brams et al. 1987
Kevekordes et al. 1999*



E. coli WP2 uvrA (pKM101)



Negative



+/-



Eurofins, 2021



Escherichia coliWP2uvrA



Negative



+/-



Dunkel et al. 1984*



Escherichia colipol A



Negative



+



Rosenkranz and Poirier 1979*
McCarroll et al. 1981



-



McCarroll et al. 1981



Escherichia coliuvr/rec strains



Positive



-



Hellmér and Bolcsfoldi 1992



Negative



+



Hellmér and Bolcsfoldi 1992



Escherichia coliK12



Negative



+



Mamber et al. 1984*



Not tested



-



Mamber et al. 1984*



Escherichia coliRK



Negative



+/-



Hayes et al. 1984*



Aspergillus nidulans



Negative



-



Crebelli et al. 1986



Saccharamyces cerevisiae



Positive



-



Schiestl et al. 1989
Galli and Schiestl 1995, 1996, 1998*



+



Galli and Schiestl 1998*
Egilsson et al. 1979*
Wilkie and Gooneskera 1980*



ambigous



+/-



Zoushu, 1989



Saccharamyces cerevisiaetrp locus



Positive



+



Morita et al. 1989



Negative



-



Morita et al. 1989



Saccharamyces cerevisiaeD7 trp locus



Positive



Not indicated



Jiang et al. 1989*



Drosophila melanogaster



Positive



-



Batiste-Alentorn et al. 1991*



Weakly positive



-



Vogel & Nivard 1993*



Inconclusive



-



Batiste-Alentorn et al. 1994, 1995*



Negative



-



Rodriguez-Arnaiz 1997*



Mouse L5178Y cells



Weakly positive



+



Caspary 1988
Wangenheim and Bolcsfoldi 1988



-



Wangenheim and Bolcsfoldi 1988



Negative



+



Mitchell et al. 1988*



-



Mitchell et al. 1988*
Myhr and Caspary 1988*



Chinese hamster ovary cells (V79), hprtlocus



Negative



-



Bradley et al. 1982*



Positive



+/-



Ziegler-Skylakakis et al. 1985*



Chinese hamster ovary cells (V79), cell sub-line Sp5



Negative



-



Helleday et al. 1998*



Sister chromatid exchange



Chinese hamster V79 cells



Negative



-



Bradley et al. 1982*



Chinese hamster Ovary (CHO)



Positive



+/-



TNO 1978; de Raat



Unscheduled DNA synthesis



Rat liver cells



Weakly positive



-



Ziegler-Skylakakis et al. 1985*



Rat liver cells



Negative



-



Lonati-Galligani et al. 1983*
Fautz et al. 1991



Mitotic recombination



Saccharamyces cerevisiaeD3



Negative



+/-



Simmon 1979b*



Recombination



Transformed human lymphoblastoid GM6804 cells



Positive



-



Aubrecht et al. 1995*



Micronucleus formation



Syrian hamster embryo cells



Positive



-



Fritzenschaf et al. 1993



Chinese hamster V79



Weakly positive



 



Ziegler-Skylakakis et al. 1998*



DNA strand breaks



Rat hepatocytesin vitro



Positive



-



Sina et al. 1983*



Rat hepatocytesin vitro



 Negative



-



Fautz et al. 1991



Cell transformation



Syrian hamster embryo cells



Positive



-



Pienta et al. 1977*



Rauscher virus-infected rat embryo cells



Positive



-



Dunkel et al. 1981*



Bovine papilloma virus DNA-enhanced C3H10T1/2 cells



Weakly positive



-



Kowalski et al. 2000*



DNA synthesis inhibition



Human fibroblast cells



Positive



-



Painter 1977*



* studies referred to in IPCS 2003, Health Canada 2008, IARC 2001, or MAK 1990


 


Genotoxicity and related endpoints: in vivo


Table2: summary of available studies on genotoxicity in vivo
















































Endpoint



Results and references



Species, strain



Result



Metabolic activation



Reference



Gene mutation



Drosophila melanogaster



Positive



n.a.



Batiste-Alentorn et al. 1991



Drosophila melanogaster



Weakly Positive



n.a.



Vogel and Nivard 1993



Drosophila melanogaster



Negative



n.a.



Rodriguez-Arnaiz 1997



Drosophila melanogaster



Inconclusive



n.a.



Batiste-Alentorn et al. 1994, 1995*



Micronucleus test



Rats treated with two successive oral doses within 24 hours



Negative



n.a.



TNO 1979b




 


Further investigations


According ECHA Decision from 19th January 2021 (No. CCH D-2114539794-36-01/F) for the fulfilment of information requirement under Annex VIII (Section 8.4.2) for the substance, either an in vitro cytogenicity study in mammalian cells (OECD TG 473) (or the thymidine kinase gene (OECD TG 490)) or/and an in vitro micronucleus study (OECD TG 487) are required.


An in vitro MNT according to OECD No. 487 is on-going. After finalization of this test, this section will be updated accordingly.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the overall assessment of available data the test item is not considered a mutagen, but further investigations are on-going (see above). The substance does not need to be classified for this endpoint according to to Regulation (EC) No 1272/2008 (CLP), as amended for the 17th time in Regulation (EU) 2021/849.