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REACH

1,3-diethyl-2-thiourea

EC number: 203-308-5 | CAS number: 105-55-5

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Several studies were available to evaluate the genotoxic potential of DETU.
Negative results were observed during Ames test, in vitro chromosomal aberration test, in vitro SCE test, SLRL test.
Positive results were observed during Mouse lymphoma assay and in vitro Comet assay on human thyroid cells.
The in vitro UDS test on human thyroid cells is equivocal.

The genotoxicity of the registered substance was further investigated using in vivo studies.

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

DETU was assayed for the ability to induce DNA damage in primary cultures of human thyroid cells : DNA fragmentation was evaluated by the Comet assay.

GLP compliance:

Type of assay:

comet assay

Species / strain / cell type:

other: human thyroid cells

Details on mammalian cell type (if applicable):

Samples of human thyroid were obtained from discarded surgical material during the course of prescribed surgery. Informed consent was obtained from each subject, and before signing the patient was told that a fragment of his thyroid wouldbe used for a scientific research. Donors 1, 5, 8, 11 and 12 were males, ranging from 49- to 68-year old; donors 2, 3, 4, 6, 7, 9,10 and 13 were females, ranging from 49- to 74-year old; all under went surgery for simple multinodular goiter. The simple multinodular goiter do not cause hormonal dysfunction.The tissue sample was obtained from areas in which haemorrhage,fatty degeneration, and fibrosis were absent. Thyroid cells wer eisolated as previously described within 2 h from thyroidectomy. The percentage of viable cells, as measured by the trypan blue exclusion test, ranged from 90 to 95%. Morphologically, the cells appeared to be more than 90% follicular cells; theother cells were mainly fibroblasts.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

1.25, 2.5 and 5.0 mM

Vehicle / solvent:

DMSO

Untreated negative controls:

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: 75µM

Details on test system and experimental conditions:

PRIMARY CULTURES:
Aliquots of cell suspensions were plated in 24-well uncoated TC plates (2 x 10^5cells per 14.5 mm well) for the Comet assay, and in 35-mm dishes coated with rat tail collagen (1 x 10^6cells per dish) for determination of cytotoxicity and DNA repair synthesis. After an attachment period of 3 hat 37 °C in an atmosphere of 95% air- 5% C02, cell cultureswere washed and incubated for 20h with serial concentrations of the test compounds and the positive control methyl methenesulfonate (MMS) in serum-free medium. In these short-term assays, exposure of cultures to test compounds for 16-20h is recommended; shorter exposure times might be inappropriate, and a longer exposure results in a reduction of cellmetabolic competence and of cell viability. The media containing methimazole and potassium bromate were freshly preparedfrom stock solutions in distilled water and the media containingethylenethiourea fromstock solutions in dimethyl sulfoxide; the maximum dimethylsulfoxide concentration (0.5%) was present in correspondingcontrol cultures. MMS was directely dissolved in the mediumjust prier to use, [Methyl-3H]thymidine (10 µCi/ml) was addedto thyroid cell cultures to be used for the DNA repair assay andleft in the culture for the entire treatment period. At the end oftreatment, cells were immediately examined for cytotoxicityby trypan blue exclusion, for DNA fragmentation by the Cometassay, and for DNA repair synthesis by quantitative autoradiography. Compound was assessed for DNA damage and repair in three independent experiments using cultures from three different donors.

Statistics:

Statistical analysis of data was performed by the use of ANOVA andthe two samples compared were values of tail length and tail moment in 50 cells from each dose point. The p <0.05 level was considered to be statistically significant.

Key result

Species / strain:

other: human thyroid cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The DNA damaging concentrations ranged from 1.25 and 5 MM for DETU.
Results show that in three independent experiments on cells from three different donors a statistically significant dose-dependent increase of both tail length and tail moment, indicative of DNA single-strand breaks and/or alkali-labile sites was consistently produced by a 20h-exposure ti subtoxic concentrations of DETU.

Table of results: Damage of nuclear DNA induced in primary cultures of human thyroid cells by a 20h-exposure to DETU

Treatment conditions	Donor no.	Relative survival	Comet assay
Treatment conditions	Donor no.	Relative survival	Tail length (µm)	Tail moment
Solvent control (DMSO)	8 (M, 49)	(0.94)	2.4+/-0.6	186+/-51
DETU 1.25 mM		0.98	2.8+/-0.6 a	239+/-49 a
DETU 2.5 mM		0.94	3.6+/-1.6ª	274+/-85a
DETU 5 mM		0.90	3.7+/-1.9a	288+/-129a
MMS 75 mM		0.98	27.2+/-2.5a	2211+/-286a
Solvent control (DMSO)	9 (F, 49)	(0.94)	2.0+/-0.4	204+/-35
DETU 1.25 mM		1.02	3.0+/-2.7a	305+/-216 a
DETU 2.5 mM		1.01	4.1+/-2.5a	342+/-227a
DETU 5 mM		0.86	4.6+/-3.9a	358+/-307a
MMS 75 mM		1.00	22.2+/-8.1a	1998+/-724a
Solvent control (DMSO)	10 (F, 74)	(0.96)	1.9+/-0.4	171+/-31
DETU 1.25 mM		0.99	2.6+/-1.2a	238+/-110 a
DETU 2.5 mM		0.97	2.7+/-0.9a	260+/-87a
DETU 5 mM		0.98	2.8+/-1.0a	267+/-95a
MMS 75 mM		0.98	19.5+/-6.8a	1766+/-530a

a = comet assay, significance level was determined by use of ANOVA (p<0.05)

Conclusions:

DETU induced in primary cultures of human thyroid cells a significant dose-dependent increase in the tail length.

Executive summary:

DETU was assayed for the ability to induce DNA damage a in primary cultures of human thyroid cells : DNA framentation/Comet assay was performed. Cells were exposed at 1.25, 2.5 and 5.0 mM of DETU.

Results show that in three independent experiments on cells from three different donors a statistically significant dose-dependent increase of both tail length and tail moment, indicative of DNA single-strand breaks and/or alkali-labile sites was consistently produced by a 20h-exposure ti subtoxic concentrations of DETU.

DETU induce in primary cultures of human thyroid cells a significant dose-dependent increase in the frequency of the tail length.

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

DETU was assayed for the ability to induce DNA damage and DNA repair synthesis in primary cultures of human thyroid cells.
DNA repair synthesis was demonstrated autoradiographically according ti the criteria indicated by guideline performance of UDS test in vitro.

GLP compliance:

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Species / strain / cell type:

other: human thyroid cells

Details on mammalian cell type (if applicable):

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

1.25, 2.5 and 5.0 mM

Vehicle / solvent:

DMSO

Untreated negative controls:

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: 75µM

Details on test system and experimental conditions:

PRIMARY CULTURES
Aliquots of cell suspensions were plated in 24-well uncoated TC plates (2 x 10^5cells per 14.5 mm well) for the Comet assay, and in 35-mm dishes coated with rat tail collagen (1 x 10^6cells per dish) for determination of cytotoxicity and DNA repair synthesis. After an attachment period of 3 hat 37 °C in an atmosphere of 95% air- 5% C02, cell cultureswere washed and incubated for 20h with serial concentrations of the test compounds and the positive control methyl methenesulfonate (MMS) in serum-free medium. In these short-term assays, exposure of cultures to test compounds for 16-20h is recommended; shorter exposure times might be inappropriate, and a longer exposure results in a reduction of cellmetabolic competence and of cell viability. The media containing methimazole and potassium bromate were freshly preparedfrom stock solutions in distilled water and the media containingethylenethiourea fromstock solutions in dimethyl sulfoxide; the maximum dimethylsulfoxide concentration (0.5%) was present in correspondingcontrol cultures. MMS was directely dissolved in the mediumjust prier to use, [Methyl-3H]thymidine (10 µCi/ml) was addedto thyroid cell cultures to be used for the DNA repair assay andleft in the culture for the entire treatment period. At the end oftreatment, cells were immediately examined for cytotoxicityby trypan blue exclusion, for DNA fragmentation by the Cometassay, and for DNA repair synthesis by quantitative autoradiography. Compound was assessed for DNA damage and repair in three independent experiments using cultures from three different donors.

UDS Test in vitro :
DNA repair synthesis was demonstrated autoradiographically according to the criteria indicated by the guidelines for the performance of UDS test in vitro. One hundred cells per data point were counted manually in two autoradiographs. Net nuclear grains (NNG) of each cell were determined by subtracting from silver grains over the nucleus (NUC) the grains of an equal size area in the cytoplasm (CYT). Cell in S-phase were easily recognized by the very dense labelling of silver grains over the nucleus.

Evaluation criteria:

A response was considered frankly positive in the presence of a dose-dependent increase of NNG over at least two consecutive concentrations.
To exclude marginal affects, the increase was considered positive if both NNG values exceeded our lab-specific threshold. A confirming criterion was the occurrence of a dose-dependent increase in the percentage of repairing cells. The data are expressed as the mean±S.D. of the 100 net nuclear counts.

Statistics:

The data are expressed as the mean +/-SD of the 100 net nuclear counts.

Key result

Species / strain:

other: human thyroid cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The DNA damaging concentrations ranged from 1.25 and 5 MM for DETU: the percentage of repair is higher than control percentage.
See table of results.

Table of results: DNA repair synthesis induced in primary cultures of human thyroid cells by a 20h-exposure to DETU

Treatment conditions	Donor no.	Relative survival	DNA repair synthesis			% repair
Treatment conditions	Donor no.	Relative survival	NUC	CYT	NNG	% repair
Solvent control (DMSO)	8 (M, 49)	(0.94)	10.4+/-4.3	9.5+/-5.0	0.9+/-2.5	2
DETU 1.25 mM		0.98	6.5+/-2.2	2.8+/-1.8	3.7+/-1.8	22
DETU 2.5 mM		0.94	7.0+/-2.3	1.9+/-1.5	5.1+/-1.9	54
DETU 5 mM		0.90	6.5+/-2.2	2.1+/-1.8	4.4+/-1.5	54
MMS 75 mM		0.98	31.2+/-8.5	10.3+/-5.2	20.9+/-6.0	100
Solvent control (DMSO)	9 (F, 49)	(0.94)	11.3+/-4.2	10.7+/-4.3	0.6+/-2.6	2
DETU 1.25 mM		1.02	9.8+/-3.0	7.0+/-2.5	2.8+/-2.2	16
DETU 2.5 mM		1.01	11.0+/-3.5	6.7+/-3.1	4.3+/-2.0	52
DETU 5 mM		0.86	11.3+/-3.8	6.0+/-3.2	53+/-2.8	76
MMS 75 mM		1.00	55.2+/-14.7	21.7+/-8.2	33.5+/-10.2	100
Solvent control (DMSO)	10 (F, 74)	(0.96)	11.9+/-4.1	10.7+/-5.4	1.2+/-3.1	4
DETU 1.25 mM		0.99	11.4+/-3.6	7.0+/-3.8	4.4+/-2.7	60
DETU 2.5 mM		0.97	9.7+/-2.8	4.6+/-2.4	5.1+/-2.3	60
DETU 5 mM		0.98	9.3+/-3.2	3.5+/-2.1	5.8+/-2.7	72
MMS 75 mM		0.98	38.2+/-7.4	9.9+/-4.2	28.3+/-6.5	100

NUC = nuclear grain count

CYT = cytoplasmic grain count

nNG = net nuclear grains

The % repair is the percentage of cells with net nuclear labelling > 5 grains.

Conclusions:

DETU induced a DNA repair in primary cultures of human thyroid cells, but the criteria of positivity were not showed, the results were equivocal.

Executive summary:

DETU was assayed for the ability to induce DNA repair synthesis in primary cultures of human thyroid cells. Cells were exposed at 1.25, 2.5 and 5.0 mM of DETU.

DNA repair was observed at all tested dose but not in the control animal.

DETU induced a DNA repair in primary cultures of human thyroid cells, but the criteria of positivity were not showed, the results were equivocal.

Reference 3

Endpoint:: in vitro gene mutation study in mammalian cells
Type of information:: experimental study
Adequacy of study:: key study
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:: no guideline followed
Principles of method if other than guideline:: A mouse lymphoma assay was performed with DETU without metabolic activation only.
GLP compliance:: no
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):: The tk+/tk-, 3.7.2C heterozygote of L5178Y mouse lymphoma cells was obtained from Dr. D. Clive, Burroughs Wellcome Co., Research Triangle Park, NC 27709, and stored in liquid nitrogen.
Metabolic activation:: without
Test concentrations with justification for top dose:: First trial: 0, 600, 1100, 1600, 2100 and 2600 µg/ml,
Second trial: 0, 500, 1000, 1500, 2000 and 2500 µg/ml.
Vehicle / solvent:: DMSO
Untreated negative controls:: yes
Negative solvent / vehicle controls:: yes
True negative controls:: no
Positive controls:: yes
Positive control substance:: methylmethanesulfonate
Details on test system and experimental conditions:: - Number of replicates: 2 (except 4 for negative control)
- Metabolic activation: no
- Vehicle: DMSO
- Volume of vehicle added: no data
- Positive controls: Methylmethanesulfonate (15 µg/ml)
- Pre-incubation time: no
- Incubation temperature: no
- Duration of exposure: 4h
- Cell density adjustment after exposure: 300 000 cells/ml
- Duration of expression period: 2 days
- Cell density adjustment after expression period: 200 000 cells/ml
- Mutant selection support: 30-ml tubes
- Selective agent: TFT (3 µg/ml)
- Duration of selection: 11-14 days
- Selection temperature: 37°C
Evaluation criteria:: A test is posifitive if:
. the assay is valid
. the mutant frequency at one or more doses was significantly greater than that of negative control (p<0.05)
. there is a dose relationship
. the effects are reproducible.
Statistics:: Analysis of variance of pair-wise comparisons of each dose versus the vehicle control.
: Key result
Species / strain:: mouse lymphoma L5178Y cells
Metabolic activation:: without
Genotoxicity:: positive
Remarks:: at 1500 µg/plate and more (in the 2 tests)
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: not examined
Positive controls validity:: valid
Additional information on results:: The lowest observed effect dose for mutagenicity was found to be 1500 µg/ml in the absence of metabolic activation. RTG was 15% at 1500 µg/ml in the second trial, whereas it was about 42% at 1600 µg/ml in the first one. At higher dose levels, there was a sharp increase in mutant numbers and mutant fractions.
Conclusions:: In this mouse lymphoma assay, 1,3-diethylthiourea is mutagenic without metabolic activation.
Executive summary:: DETU was tested for his mutagenic potential in the L5178Y tk+/- mouse lymphoma cell forward mutation assay. Cultures were exposed to the chemical for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 µg/ml. The chemical was tested at least twice. Significant responses were obtained with DETU without S9 (not tested with S9). A dose of 1500 µg was required to demonstrate the mutagenicity of DETU in the absence of S9 mix. The RTG was 15% at 1500µg/ml in this experiment, whereas the RTG was about at 1600 µg/ml in the other experiment. At higher dose levels, there was a sharp increase in mutant numbers and mutant fractions.

Reference 4

Endpoint:: in vitro gene mutation study in bacteria
Type of information:: experimental study
Adequacy of study:: key study
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:: no guideline followed
Principles of method if other than guideline:: Method of Mortelman et al. (1986) Environ Mut, 8 (suppl. 7), 1-119.
GLP compliance:: not specified
Type of assay:: bacterial reverse mutation assay
Species / strain / cell type:: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:: not applicable
Metabolic activation:: with and without
Metabolic activation system:: S9 mix (hamster or rat liver fractions, pretreated with aroclor 1254)
Test concentrations with justification for top dose:: Without metabolic activation: 0, 100, 333, 1000, 3333 and 6667 µg/plate
With metabolic activation: 0, 100, 333, 1000, 3333 and 10000 µg/plate
Vehicle / solvent:: DMSO
Untreated negative controls:: yes
Negative solvent / vehicle controls:: yes
True negative controls:: no
Positive controls:: yes
Positive control substance:: no
Remarks:: Migrated to IUCLID6: see below
Details on test system and experimental conditions:: Positive controls:
-sodium azide : For Strain TA100 and TA 1535; without S9
-9-aminoacridine: For Strain TA 1537; without S9
-4-nitro-o-phenylenediamine : For strain TA 98 ; without S9
-2-aminoanthracene : All strain tested ; With S9

DETU was incubated with the Salmonella typhimurium tester strains aither in buffer or S9 mix (metabolic activation enzymes and cofactors from Aroclor 1254- induced male SD rat or Syrian hamster liver) for 20 minutes at 37 °C. Top agar supplemented with l-histidine and d-biotin was added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidine-independant mutant colonies arising on these plares were counted following 2 days incubation at 37°C.
Each trial consisted of triplicate plates of concurrent positive and negative controls and of at least five doses of DETU. High dose was limited by experimental design to 10.00 µg/plate. All assays were repeated.

All chemical were initially tested with strain TA100 in the presence and the absence pf the metabolic activation systems, over a wide dose range with an upper limit of 10 mg/plate, or less when solubility problems were encountered. Toxicity was evidenced by one or more of the following phenomena: appearance of [His -] pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterian lawn.
Evaluation criteria:: The criteria used for data evaluation are summarized as follows :
1) mutagen response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold;
2) nonmutagenic response:when no increase in the number of revertants was elicited by the chemical ;
3)questionable response : when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproductible; or when the response was of insufficient magnitude to support a determination of mutagenicity.
Statistics:: A positive response in the Salmonella typhimurium assay was defined as a reproducible dose-related increase in histidine-independent (revertant) colonies in a y one strain/activation combinaison. An equivocal response was defined as an increase inrevertants that was not dose related, not reproducible, or of insufficient magnitude to support a determination of mutagenicity. A negative response was obtained when no increase in revertant colonies was observed following chemical treatment. There was no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive.
Species / strain:: S. typhimurium TA 1535
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: other: A slight toxicity was noted at 6667 µg/plate
Vehicle controls validity:: valid
Untreated negative controls validity:: not applicable
True negative controls validity:: not applicable
Positive controls validity:: valid
Species / strain:: S. typhimurium TA 1537
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: other: A slight toxicity was noted at 6667 µg/plate
Vehicle controls validity:: valid
Untreated negative controls validity:: not applicable
True negative controls validity:: not applicable
Positive controls validity:: valid
Species / strain:: S. typhimurium TA 98
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:: valid
Untreated negative controls validity:: not applicable
True negative controls validity:: not applicable
Positive controls validity:: valid
Species / strain:: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: other: A slight toxicity was noted at 6667 µg/plate
Vehicle controls validity:: valid
Untreated negative controls validity:: not applicable
True negative controls validity:: not applicable
Positive controls validity:: valid
Additional information on results:: No genotoxicity was observed (see table).
Remarks on result:: other:
Conclusions:: The preincubation modification the Salmonella assay as used to test DETU in up to four Salmonella strains (TA1535, TA1537, TA100, TA98) on the presence and absence of rat and hamster liver S9. DETU (100 to 10 000 µg/plate) was not mutagenic in Salmonella typhimurium strains in the presence or in absence of induced hamster or rat liver S9.
Executive summary:: The preincubation modification the Salmonella assay as used to test DETU in up to four Salmonella strains (TA1535, TA1537, TA100, TA98) on the presence and absence of rat and hamster liver S9. DETU (100 to 10 000 µg/plate) was not mutagenic in Salmonella typhimurium strains in the presence or in absence of induced hamster or rat liver S9.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A positive response was obtained during a non-standard Comet assay (2006). As this study lacked several key parameters, it was not considered as adequate by ECHA and a Decision on Compliance Check was released, requesting an in vivo Comet assay to be conducted on the registered substance.
Negative results were during an in vivo micronucleus test (2011) and an in vivo Comet assay (2022).
Based on the weight of evidence, DETU is considered to be not mutagenic.

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start Date: 31 August 2021
Experimental Completion Date: 08 November 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Justification for Selection
The comet assay is recommended to assess the genotoxic potential of short-lived reactive mutagens at their site of contact or as a supplementary in vivo test for investigation of genotoxicity (OECD, 2016). This study has been designed to provide data at the request of the European chemical agency (ECHA), based on Article 41 of the REACH regulation (Regulation (EC) No 1907/2006).

The rat was selected as there is a large volume of background data in this species and was specifically requested by ECHA for this comet assay.

The liver is recommended as the primary site of xenobiotic metabolism and is often highly exposed to both the test article and any metabolites. The stomach and duodenum are recommended tissues to examine for site of contact effects after oral exposure. Male gonadal cells were collected for potential analysis if induction of DNA strand breaks was determined in any somatic tissue. The analysis of gonadal cells may have been relevant for the overall assessment of possible germ cell mutagenicity including classification and labelling according to CLP Regulation. As such, this study was performed in male rats only.

Sex:

male

Details on test animals or test system and environmental conditions:

Species, Strain and Supplier
36 male young adult out-bred Sprague Dawley rats (Crl:CD(SD)) were obtained from Charles River (UK) Ltd., Margate, UK

Specification
9 male animals were dosed during the Range-Finder Experiment. They were approximately 7 to 9 weeks old and 240-302 g on the first day of dosing.

27 male animals were dosed during the Main Experiment. They were approximately 7 to 8 weeks old and 280-322 g on the first day of dosing.

Environment
Animals were housed in wire topped, solid bottomed cages, with three animals per cage.

The animals were housed in rooms air-conditioned to provide a minimum of 15 air changes/hour and set to maintain temperature and relative humidity in the range 19-25¿C and 40 70%, respectively. Fluorescent lighting was controlled automatically to give a cycle of 12 hours light (0600 to 1800) and 12 hours dark. The animals were routinely kept under these conditions except for short periods of time where experimental procedures dictated otherwise.

Diet, Water, Bedding and Environmental Enrichment
Throughout the study the animals had ad libitum access to 5LF2 EU Rodent Diet. Each batch of diet was analysed for specific constituents and contaminants.

Mains water was provided ad libitum via water bottles. The water supply was periodically analysed for specific contaminants.

Bedding was provided on a weekly basis to each cage by use of clean European softwood bedding (Datesand Ltd., Manchester). The bedding was analysed for specific contaminants.
In order to enrich both the environment and the welfare of the animals, they were provided with wooden Aspen chew blocks and rodent retreats.

No contaminants were present in any of the above at levels that might interfere with achieving the objective of the study. Results of any analyses performed are held centrally at Labcorp Early Development Laboratories Ltd.

Allocation to Treatment Group
On arrival, animals were randomly allocated to cages. Range-Finder animals were allocated to groups of three and Main Experiment animals were randomised to groups of six (three for the positive control).

Checks were made to ensure the weight variation of Main Experiment animals prior to dosing was minimal and did not exceed ±20% of the mean weight.

Identification of the Test System
The animals were individually identified by uniquely numbered tail mark (Range Finder) or subcutaneous electronic transponder (Main Experiment). Cages were appropriately identified (using a colour-coded procedure) with study information including study number, study type, start date, number and sex of animals, together with a description of the dose level and proposed time of necropsy.

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

Preparation
Range-Finder Experiment formulations were freshly prepared prior to each dosing occasion and Main Experiment formulations were prepared once by formulating 1,3-diethyl-2-thiourea in corn oil as follows:

The test article was weighed into a formulation bottle and vehicle was added to achieve the final volume. Formulations were stirred to mix, silversoned to homogenise and aliquoted (as required).

Stability data generated under Labcorp Study Number 8468464 (Smith, 2022) indicated that the test article formulations were stable for at least 15 days when protected from light and stored at 15 25°C.

All Main Experiment formulations used for animal dosing were protected from light and stored at 15- 25°C when not required for dosing. All formulations were used within 2 days of preparation.

Homogeneity
To ensure homogeneity, dose formulations were stirred continuously (on a magnetic stirrer) before and throughout dosing.

Formulations Analysis
Samples were taken from each test article formulation used in the Main Experiment together with concurrent vehicle controls.

Two sets of duplicate (2 x 1 mL) samples were taken from the top, middle and bottom of each test article formulation together with duplicate 1 mL samples (taken from the middle) of the vehicle control. Each set of duplicate samples (2 x 1 mL) and a single 1 mL vehicle control sample was sent to the Test Site and one set was analysed for achieved concentration and a determination of homogeneity. Reserve samples were not required for analysis so were held at the Test Site until they were discarded with Study Director approval prior to study finalisation.
Samples were stored at 15-25°C prior to dispatch to the analytical test site (under ambient conditions).

Duration of treatment / exposure:

24 hours

Frequency of treatment:

The test article and vehicle control were given as two administrations, at 0 and 21 hours; the positive control was administered once only at 21 hours. All surviving animals were sampled at approximately 24 hours.

Post exposure period:

Observation times were as follows:

Animals Day Approximate Observation Time
Range-Finder Experiment 1 Prior to dose, immediate, 0.5, 1, 2 and 4-6 hours post dose
2 Prior to dose, immediate, 0.5, 1, 2 and 4-6 hours post dose
Main Experiment 1* Prior to dose, immediate, 1, 2 and 4 hours post dose
2 Prior to dose, immediate and prior to necropsy
* Excluding positive control group

Dose / conc.:

320 mg/kg bw/day (actual dose received)

Remarks:

Range-finder: Group 1

Dose / conc.:

450 mg/kg bw/day (actual dose received)

Remarks:

Range-finder: Group 2

Dose / conc.:

630 mg/kg bw/day (actual dose received)

Remarks:

Range finder: Group 3

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Group 1; Vehicle control

Dose / conc.:

110 mg/kg bw/day (actual dose received)

Remarks:

Group 2

Dose / conc.:

225 mg/kg bw/day (actual dose received)

Remarks:

Group 3

Dose / conc.:

450 mg/kg bw/day (actual dose received)

Remarks:

Group 4

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

Group 5: Positive control. Administered as a singe dose approximately 3 hours prior to sample time

No. of animals per sex per dose:

Male rats only due to ethical consideration of animal use as there was potential analysis of gonadal cells

Animals per group: Six (three for the positive control group)

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulfonate 200 mg/kg, single oral administration at 21 hours (Day 2)

Tissues and cell types examined:

The liver, stomach, duodenum and gonad were removed from each control (vehicle and positive) and test article treated animal.

For histopathology, a sample of liver, stomach, duodenum and gonad from vehicle control and test article treated animals only was removed. Liver, stomach and duodenum samples were immediately preserved in neutral buffered formalin and stored at room temperature. Gonad samples were immediately preserved in modified Davidson’s fluid and stored at room temperature. No histopathology samples were preserved for the positive control animals.

Details of tissue and slide preparation:

Justification for Dose Selection
The following information on the in vivo toxicity of 1,3-diethyl-2-thiourea was provided.

An in vivo rat bone marrow micronucleus test (Simar, 2011) consisting of a toxicity dose ranging finding experiment and a micronucleus genotoxicity experiment orally dosed Sprague Dawley rats once on two consecutive days at 10 mL/kg with 1,3-diethyl-2-thiourea in 0.5% carboxymethylcellulose in distilled water.

The toxicity assay dosed 2 males and 2 females at 320, 500 and 800 mg/kg/day with mortality observed in both sexes 6 hours after the first or second dose at 500 and 800 mg/kg/day. The lower dose at 320 mg/kg/day was tolerated and accepted as the maximum tolerated dose (MTD) for that study with clinical signs of toxicity limited to decreased activity/slight prostration from 2 hours after each dose.

The genotoxicity assay dosed 5 males and 5 females at 80, 160 and 320 mg/kg/day. There was no morbidity or mortality observed. Similarly, to the toxicity assay, clinical signs of toxicity were limited to decreased activity between 2 and 4 hours post dosing at 320 mg/kg/day.

There were no sex differences in toxicity or micronucleus response.

Based on this information an initial dose of 320 mg/kg/day was administered in a Range-Finder Experiment. Additional higher doses of 450 mg/kg/day and 630 mg/kg/day were tested until an estimate of the MTD (450 mg/kg/day) was determined (OECD, 2016). Animal morbidity was observed following dosing at 630 mg/kg/day.

From the results of the Range-Finder Experiment dose levels of 110, 225 and 450 mg/kg 1,3-diethyl-2-thiourea (equivalent to approximately 25% MTD, 50% MTD and MTD respectively) were tested in the Main Experiment.

The MTD was defined as the highest dose that was tolerated without evidence of study-limiting toxicity, relative to the duration of the study period (for example, clear clinical signs such as abnormal behaviour or reactions, body weight depression or target tissue cytotoxicity, but not death or evidence of pain, suffering or distress necessitating humane euthanasia).

As gonadal cells were collected for potential analysis, to optimise the use of animals, male rats only were used in this study to ethically reduce animal testing.

Histopathology
Preserved liver, stomach, duodenum and gonad samples were embedded in wax blocks and sectioned at 5 µm nominal. Liver, stomach and duodenum slides were stained with haematoxylin and eosin and examined by the Study Pathologist. Gonad samples were not examined as the somatic tissues did not show genotoxic potential.

Preparation of Cell Suspensions
The comet liver samples were washed thoroughly in Merchants solution and placed in fresh buffer. The samples were cut into small pieces in Merchants solution and the pieces of liver were then pushed through bolting cloth (pore size of 150 µm) with approximately 4 mL of Merchants solution to produce single cell suspensions.

The comet stomach samples were washed in Merchants solution and then incubated on ice for 15 minutes, covered in fresh Merchants solution. After incubation the stomach samples were removed and placed in 200 µL of fresh ice cold Merchants solution. Cells were gently scraped from the inside surface of the stomach using the back of a scalpel blade to produce single cell suspensions.

The comet duodenum samples were washed thoroughly in ice cold Merchants solution and placed into fresh buffer. Each sample was vortexed in ice cold Merchants solution for approximately 15 seconds. The tissue was removed from the Merchants solution and the inner surface gently scraped (released material discarded) using the back of a scalpel blade. The tissue was vortexed in ice cold Merchants solution for a further 15 seconds prior to gently scraping the inside of the duodenum with the back of a scalpel blade in 150 µL of fresh ice cold Merchants solution to produce single cell suspensions.

The comet gonad samples were washed thoroughly in Merchants solution and placed in fresh buffer. The samples were cut into small pieces in Merchants solution and the pieces of gonad were then pushed through bolting cloth (pore size of 150 µm) with approximately 10 mL of Merchants solution to produce single cell suspensions.

All cell suspensions were held on ice prior to slide preparation.

Slide Preparation
Three slides, labelled ‘A’, ‘B’ and ‘C’ were prepared per single cell suspension per tissue. Slides were labelled with the study number, appropriate animal tag number and tissue. Slides were dipped in molten normal melting point agarose (NMA) such that all of the clear area of the slide and at least part of the frosted area was coated. The underside of the slides was wiped clean and the slides allowed to dry. 40 µL of each single cell suspension was added to 400 µL of 0.7% low melting point agarose (LMA) at approximately 37°C. 100 µL of cell suspension/agarose mix was placed on to each slide. The slides were then coverslipped and allowed to gel on a chiller plate with ice packs.

Cell Lysis
Once gelled the coverslips were removed and all slides placed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH adjusted to pH 10 with NaOH, 1% Triton X 100, 10% DMSO) overnight at 2-8°C, protected from light.

Unwinding and Electrophoresis
Following lysis, slides were washed in purified water for 5 minutes, transferred to electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH>13) at 2-8°C and the DNA unwound for 20 minutes (stomach and duodenum) or 30 minutes (liver and gonad). At the end of the unwinding period the slides were electrophoresed in the same buffer at 0.7 V/cm for 20 minutes (stomach and duodenum) or 40 minutes (liver and gonad). As not all slides could be processed at the same time a block design was employed for the unwinding and electrophoretic steps in order to avoid excessive variation across the groups for each electrophoretic run; i.e. for all animals the same number of triplicate slides was processed at a time.

Neutralisation
At the end of the electrophoresis period, slides were neutralised in 0.4 M Tris, pH 7.0 (3 x 5 minute washes). After neutralisation the slides were dried and stored at room temperature prior to scoring.

Staining
Prior to scoring, the slides were stained with 100 µL of 2 µg/mL ethidium bromide and coverslipped.

Slide Analysis
Scoring was carried out using fluorescence microscopy at an appropriate magnification and with suitable filters.

A slide from a vehicle and positive control animal were checked for quality and/or response prior to analysis. All slides were allocated a random code by an individual not connected to scoring of the study.

A minimum of five animals per group were analysed.

Measurements of tail intensity (%DNA in tail) were obtained from 150 cells/animal/tissue. In general, this was evenly split over three slides.

The number of ‘hedgehogs’ (a morphology indicative of highly damaged cells often associated with severe cytotoxicity, necrosis or apoptosis) observed during comet scoring was recorded for each slide. To avoid the risk of false positive results ‘hedgehogs’ were not used for comet analysis. Each slide was scanned starting to the left of the centre of the slide.

The following criteria were used for analysis of slides:
1. Only clearly defined non overlapping cells were scored
2. Hedgehogs were not scored
3. Cells with unusual staining artefacts were not scored.
Comet slides were retained until report finalisation; at this time the slides were discarded with SD approval. Due to the nature of the slides, long term storage is not recommended as comet integrity cannot be assured.

Evaluation criteria:

For valid data, the test article was considered to induce DNA damage if:
1. A least one of the test doses exhibited a statistically significant increase in tail intensity, in any tissue, compared with the concurrent vehicle control
2. The increase was dose related in any tissue
3. The increase exceeded the laboratory’s historical control data for that tissue.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met and target tissue exposure was confirmed.
Results which only partially satisfied the criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example comparison of the response against the historical control data, consistency of response within and between dose levels.

Statistics:

Please refer to "Any other information on materials and methods"

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MAIN EXPERIMENT RESULTS
Formulations Analysis
Results of the analyses demonstrated that the formulations were homogeneous with a relative standard deviation (RSD) =10% (values of 1.73-3.37%) and that the achieved concentrations were within 100±15% of the nominal test article concentrations (mean values of 96.4-100%). The formulations were therefore considered acceptable. No test article was detected in the vehicle sample.

Post Dose Observations
There were no clinical observations of toxicity for any animal dosed with the vehicle control, 110 mg/kg/day 1,3-diethyl-2-thiourea or the positive control in the Main Experiment.

Following dose administration of 225 mg/kg/day 1,3-diethyl-2-thiourea, there were no clinical observations of toxicity on Day 1. On Day 2, mild decreased activity and piloerection were noted post dosing at the final observation occasion prior to necropsy.

Following dose administration of 450 mg/kg/day 1,3-diethyl-2-thiourea, there were no clinical observations of toxicity on Day 1. Prior to dosing on Day 2, there were observations for only animal R0305 including moderate decreased activity, ataxia and a mild hunched posture.
Following Day 2 dosing, observations also included being observed in a lateral recumbent posture with laboured breathing for R0305. The animal was euthanised in extremis approximately one hour post dosing on Day 2. The remaining animals administered 450 mg/kg/day were noted with mild decreased activity and piloerection at the final observation occasion prior to necropsy only.

Necropsy of decedents identified no obvious cause of death.

Range-Finder Experiment data showed that dosing higher than 450 mg/kg/day would induce mortality with the dosing regimen for this study within the sampling time. In the Main Experiment, one animal (out of six) was euthanised with severe clinical observations at 450 mg/kg/day indicating that this dose level was at the limit of dose tolerability and therefore maximized assay sensitivity.

Body Weights
There was a test article-related effect on animal body weight between Day 1-Day 2 with group mean body weight change values of -2.3%, -3.0% and -3.8% at 110, 225 and 450 mg/kg/day, respectively, compared to +1.4% in the concurrent vehicle control group.

Bioanalysis
Plasma was processed from whole blood samples as a contingency. Analysis of these samples was not performed as clinical chemistry and histopathology data demonstrated liver exposure. Stomach and duodenum exposure was considered assured as site-of-contact tissues following oral gavage dosing.

Clinical Pathology
Clinical Chemistry
Reduced alanine aminotransferase (ALT) activity and increased cholesterol and albumin, with a concomitant increase in the albumin:globulin ratio, was recorded for animals in all groups administered 1,3-diethyl-2-thiourea.

Decreased aspartate aminotransferase (AST) activity and increased calcium and phosphate was recorded for animals administered 225 or 450 mg/kg/day.

Decreased chloride and increased glucose, indicative of stress, was recorded for animals administered 450 mg/kg/day.

The clinical chemistry changes were generally very small, based on the acute nature of this study (24 hours) at the maximum-tolerated dose and because the values in 1,3-diethyl-2-thiourea-treated animals were outside the control range and often noted with a dose relationship, the findings described were considered related to 1,3-diethyl-2-thiourea. Increased albumin may have been due to mild dehydration, which resulted in an associated increase in calcium.

Other differences in individual clinical pathology parameters were observed in animals administered the test article; however, they were considered not test article related due to the negligible magnitude of the change, individual animal variability, and overlap of values for test article treated animals with concurrent control values.

Histopathology
Macroscopic and Microscopic findings are presented in the Pathology Contributory Report.
On macroscopic examination, the duodenum was distended for some animals administered 225 or 450 mg/kg/day, with no microscopic correlation. Other tissues were considered macroscopically unremarkable or the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age.

On microscopic examination, changes related to 1,3-diethyl-2-thiourea were recorded for the liver and duodenum.
In the liver, decreased hepatocyte glycogen was recorded for animals administered 225 or 450 mg/kg/day, with a dose relationship, when compared with control group. Minimal centrilobular hepatocyte vacuolation was recorded in two animals administered 450 mg/kg/day. Fewer inflammatory cell infiltrates were recorded for animals administered 450 mg/kg/day when compared with control group.

In the duodenum, minimal or slight mucosal vacuolation was noted for two animals administered 450 mg/kg/day.

The decreased inflammatory cell infiltrates recorded for animals administered 450 mg/kg/day may have correlated with decreased ALT and AST activities as these infiltrates are usually associated with necrotic hepatocytes, which may have released ALT and AST, although reduced
ALT and AST activities are generally of no toxicological significance.

Glycogen is normally stored in hepatocytes in large, often perinuclear vacuoles with a granular or feathery appearance. Decreased glycogen vacuolation was noted in animals administered 1,3-diethyl-2-thiourea. Animals were not fasted prior to necropsy and were also sacrificed in replicate order (within 1 hour of each other); as such, this decrease in glycogen was attributed directly or indirectly to the effects of 1,3-diethyl-2-thiourea. This decrease may have indicated an increased utilization of glycogen, possibly due to the increased metabolism in the liver or decreased food consumption.

Other tissues were considered microscopically unremarkable or the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age.

RANGE-FINDER RESULTS
Groups of three male rats were dosed at 0 hours and approximately 21 hours with 1,3-diethyl-2-thiourea at 320, 450 and 630 mg/kg/day.

After both doses at 320 mg/kg/day, decreased activity and piloerection was observed from 0.5 hours after dosing which persisted to the approximately 4 hour observation occasions. The animals recovered overnight between Day 1 and Day 2 such that they were in a normal state prior to dosing on Day 2. Salivation was observed sporadically on each day (all 3 males: 2 hours after dosing on Day 1 only, 1 hour after dosing on Day 2 only). Body weight loss between Day 1 and Day 2 was observed for all 3 males (group mean body weight change of -4.4%).

At 450 mg/kg/day, decreased activity was observed from 0.5 hours after the first dose which persisted until scheduled termination on Day 2. The animals did not recover overnight between Day 1 and Day 2. Salivation was observed on Day 1 from 1-hour post dosing and piloerection observed on Day 2, for one out of the three males prior to dose on Day 2 and two out of three males at the final observation occasion on Day 2. Body weight loss between Day 1 and Day 2 was observed for all 3 males (group mean body weight change of -2.1%).

At 630 mg/kg/day, clinical observations were observed from 0.5 hours after the first dose, which included decreased activity (3/3 animals) and semi-closed eyes (1/3 animals). From approximately 1-hour post dose, all 3 animals were observed with piloerection, semi-closed eyes and decreased activity. These observations persisted throughout Day 1. At the AM health check of Day 2, one animal was prone with semi-closed eyes and piloerection and was therefore euthanised in extremis as it was unsuitable for dosing. Piloerection, semi-closed eyes and decreased activity persisted overnight for the other 2 males; due to these persistent observations the two remaining males were also euthanised. Body weight loss between Day 1 and Day 2 was observed for all 3 males (group mean body weight change of -5.1%).

From these results 450 mg/kg/day was considered to be an appropriate estimate of the MTD and was therefore selected as the maximum dose for the Main Experiment. Two lower doses of 110 and 225 mg/kg/day were also selected.

Validity of Data

The data generated in this study confirm that:

The vehicle control data were comparable to laboratory historical control data for each tissue

The positive control induced responses that were compatible with the laboratory historical control data and are statistically significant compared to the concurrent vehicle control

Adequate numbers of cells and doses were analysed

The high dose was considered to be the MTD.

As dosing was via oral gavage, exposure to the stomach and duodenum was assured. There were test article-related changes in liver enzymes (decreases) which correlated with microscopic changes (fewer inflammatory cell infiltrates). Although of no toxicological significance, there were clear adaptive changes and perturbations in the liver.

The assay data were therefore considered valid.

Data Analysis

There was no dose-related increases in %hedgehogs in liver, stomach or duodenum, thus demonstrating that treatment with 1,3-diethyl-2-thiourea did not cause excessive DNA damage that could have interfered with comet analysis.

Animals treated with 1,3-diethyl-2-thiourea at 110, 225 and 450 mg/kg/day exhibited group mean and individual animal tail intensities in the liver that fell within the 95% reference range of the laboratory's historical vehicle control data. There was statistical significance (p<0.05) associated with the low dose group (110 mg/kg/day) compared to the concurrent vehicle control group; however, the group mean tail intensity value (0.25%) fell within the 95% reference range (0.04-1.80%) of the laboratory’s historical vehicle control data (and fell below the mean tail intensity value of 0.41% in the historical vehicle control data) and the individual animal values in the low dose group (0.20-0.30%) were similar to the vehicle control group (0.08-0.23%) and the other dose groups (225 mg/kg/day: 0.06-0.26% and 450 mg/kg/day: 0.14-0.42%). Furthermore, there was no evidence of a dose response in either group mean tail intensity, 0.25%, 0.14% and 0.23% at 110, 225 and 450 mg/kg/day, respectively, compared to 0.12% in the vehicle control group, and no statistically significant (p>0.05) linear trend test. As such, the statistical significance at the low dose only, for values within the 95% reference range of the laboratory’s historical vehicle control data was considered of no biological relevance and a clear lack of genotoxic potential in the liver.

Animals treated with 1,3-diethyl-2-thiourea at 110, 225 and 450 mg/kg/day exhibited group mean and individual animal tail intensities in the stomach and duodenum that were similar to the concurrent vehicle control group and that fell within, or marginally below, the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control, with no evidence of a dose response.

These data were considered clearly negative in the duodenum and stomach.

Text Table 1: 1,3-diethyl-2-thiourea: Summary of Group Mean Data – Liver

Group/Dose Level (mg/kg/day)	Tail Intensity						Mean % Hedgehogs
Group/Dose Level (mg/kg/day)	Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance	Mean % Hedgehogs
1/ Vehicle (0)	0.12	0.02	-	-	-	-	1.33
2/ 1,3-diethyl-2-thiourea (110)	0.25	0.02	2.04	U	0.0125	*	0.69
3/ 1,3-diethyl-2-thiourea (225)	0.14	0.03	1.05	U	0.6844	NS	0.94
4/ 1,3-diethyl-2-thiourea (450)	0.23	0.05	1.75	U	0.0545	NS	0.89
5/ EMS (200)	17.34	0.87	163.01	U	<0.0001	***	2.64
Dose response: (groups 1,2,3,4 )				U	0.1154	NS	NA

Text Table 2: 1,3-diethyl-2-thiourea: Summary of Group Mean Data – Stomach

Group/Dose Level (mg/kg/day)	Tail Intensity						Mean % Hedgehogs
Group/Dose Level (mg/kg/day)	Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance	Mean % Hedgehogs
1/ Vehicle (0)	0.59	0.13	-	-	-	-	6.77
2/ 1,3-diethyl-2-thiourea (110)	0.36	0.23	0.38	U	0.9951	NS	9.00
3/ 1,3-diethyl-2-thiourea (225)	0.39	0.15	0.57	U	0.9650	NS	6.69
4/ 1,3-diethyl-2-thiourea (450)	0.54	0.29	0.70	U	0.9176	NS	7.72
5/ EMS (200)	8.01	0.92	17.34	U	<0.0001	***	10.60
Dose response: (groups 1,2,3,4 )				U	0.6404	NS	NA

Text Table 3: 1,3-diethyl-2-thiourea: Summary of Group Mean Data – Duodenum

Group/Dose Level (mg/kg/day)	Tail Intensity						Mean % Hedgehogs
Group/Dose Level (mg/kg/day)	Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance	Mean % Hedgehogs
1/ Vehicle (0)	0.23	0.05	-	-	-	-	9.12
2/ 1,3-diethyl-2-thiourea (110)	0.15	0.05	0.65	U	0.9358	NS	10.06
3/ 1,3-diethyl-2-thiourea (225)	0.34	0.11	2.14	U	0.2251	NS	7.75
4/ 1,3-diethyl-2-thiourea (450)	0.29	0.08	1.39	U	0.5323	NS	9.42
5/ EMS (200)	6.50	0.26	50.84	U	0.0015	**	6.56
Dose response: (groups 1,2,3,4 )				U	0.1362	NS	NA

EMS	Ethyl methanesulfonate
NA	Not applicable
NS	Not significant (P>0.05)
SEM	Standard Error of Mean
U	Unranked
*	P≤0.05
**	P≤0.01
***	P≤0.001

Table 9.3: Summary of Body Weight Percentage Change

Summary of Body Weight Percent Change

Test Article Vehicle DETU EMS

Group 1 2 3 4 5

Dose level (mg/kg/day) 0 110 225 450 200

---------------------------------------------------------------------

Data Presented in "%" Interval X through X

Phase DSNG

Group/ ------------

Sex Day 1 - 2

---------------------------------------------------------------------

1/M Mean 1.4

SD 1.97

N 6

2/M Mean -2.3

SD 1.72

N 6

3/M Mean -3.0

SD 1.98

N 6

4/M Mean -3.8

SD 2.76

N 6

5/M Mean 4.9

SD 1.28

N 3

---------------------------------------------------------------------

Liver: Animal Comet Data

Group/ Dose Level	Animal	Total	Tail Intensity (%)		Hedgehogs
(mg/kg/day)	Number	Comets	Mean	SD	(%)
1/ Vehicle (0)	R0001	150	0.23	0.02	0.61
	R0002	150	0.13	0.12	0.59
	R0003	150	0.11	0.08	2.25
	R0004	150	0.08	0.02	1.69
	R0005	150	0.09	0.03	1.52
	R0006	150	0.10	0.01	1.19
2/ 1,3-diethyl-2-thiourea (110)	R0101	150	0.30	0.20	0.00
	R0102	150	0.27	0.24	0.00
	R0103	150	0.30	0.15	0.60
	R0104	150	0.20	0.04	1.12
	R0105	150	0.20	0.09	0.62
	R0106	150	0.22	0.07	1.74
3/ 1,3-diethyl-2-thiourea (225)	R0201	150	0.14	0.03	0.59
	R0202	150	0.19	0.02	1.62
	R0203	150	0.10	0.09	0.64
	R0204	150	0.26	0.20	1.05
	R0205	150	0.11	0.04	0.00
	R0206	150	0.06	0.01	1.53
4/ 1,3-diethyl-2-thiourea (450)	R0301	150	0.15	0.02	1.08
	R0302	150	0.29	0.14	0.56
	R0303	150	0.14	0.08	0.59
	R0304	150	0.42	0.31	0.50
	R0305	D	D	D	D
	R0306	150	0.17	0.17	1.85
5/ EMS (200)	R0401	150	15.61	2.54	2.21
	R0402	150	18.25	2.14	2.63
	R0403	150	18.17	2.14	3.05

D	Animal died
SD	Standard Deviation
EMS	Ethyl Methanesulfonate

Stomach: Animal Comet Data

Group/ Dose Level	Animal	Total	Tail Intensity (%)		Hedgehogs
(mg/kg/day)	Number	Comets	Mean	SD	(%)
1/ Vehicle (0)	R0001	150	0.97	0.32	6.76
	R0002	150	0.41	0.15	4.76
	R0003	150	0.40	0.23	8.02
	R0004	150	0.27	0.21	6.33
	R0005	150	1.00	0.95	10.71
	R0006	150	0.46	0.15	3.93
2/ 1,3-diethyl-2-thiourea (110)	R0101	150	0.09	0.03	4.09
	R0102	150	1.48	0.65	17.98
	R0103	150	0.06	0.01	4.35
	R0104	150	0.27	0.22	6.35
	R0105	150	0.16	0.09	10.65
	R0106	150	0.12	0.07	8.50
3/ 1,3-diethyl-2-thiourea (225)	R0201	150	0.31	0.04	10.84
	R0202	150	0.18	0.10	3.83
	R0203	150	0.43	0.19	6.11
	R0204	150	0.26	0.05	5.00
	R0205	150	0.07	0.05	4.84
	R0206	150	1.07	0.15	9.05
4/ 1,3-diethyl-2-thiourea (450)	R0301	150	0.11	0.05	2.31
	R0302	150	0.51	0.22	6.70
	R0303	150	0.17	0.02	5.13
	R0304	150	0.23	0.08	11.76
	R0305	D	D	D	D
	R0306	150	1.67	0.28	11.29
5/ EMS (200)	R0401	150	8.87	0.65	9.51
	R0402	150	9.00	2.61	11.11
	R0403	150	6.17	1.14	11.38

D	Animal died
SD	Standard Deviation
EMS	Ethyl Methanesulfonate

Duodenum: Animal Comet Data

Group/ Dose Level	Animal	Total	Tail Intensity (%)		Hedgehogs
(mg/kg/day)	Number	Comets	Mean	SD	(%)
1/ Vehicle (0)	R0001	150	0.33	0.25	11.80
	R0002	150	0.36	0.13	10.16
	R0003	150	0.24	0.08	9.55
	R0004	150	0.04	0.05	7.53
	R0005	150	0.28	0.15	5.42
	R0006	150	0.14	0.15	10.70
2/ 1,3-diethyl-2-thiourea (110)	R0101	150	0.07	0.06	8.38
	R0102	150	0.14	0.11	7.81
	R0103	150	0.10	0.07	9.42
	R0104	150	0.05	0.05	15.38
	R0105	150	0.35	0.27	11.06
	R0106	150	0.20	0.14	8.33
3/ 1,3-diethyl-2-thiourea (225)	R0201	150	0.19	0.14	7.11
	R0202	150	0.87	0.29	5.65
	R0203	150	0.15	0.06	7.37
	R0204	150	0.30	0.12	5.92
	R0205	150	0.27	0.02	11.33
	R0206	150	0.27	0.10	8.60
4/ 1,3-diethyl-2-thiourea (450)	R0301	150	0.19	0.10	14.59
	R0302	150	0.22	0.14	8.85
	R0303	150	0.59	0.46	2.60
	R0304	150	0.35	0.15	6.81
	R0305	D	D	D	D
	R0306	150	0.10	0.13	14.36
5/ EMS (200)	R0401	150	6.04	2.61	5.42
	R0402	150	6.54	0.56	7.69
	R0403	150	6.92	1.23	6.55

D	Animal died
SD	Standard Deviation
EMS	Ethyl Methanesulfonate

Historical Control Ranges: Comet Assay Data

Data generated from studies performed within the GLP laboratory, by GLP trained staff, whether a claim of GLP compliance was made or not, were included in the compilation of the historical control ranges without bias.

RAT LIVER COMET HISTORICAL CONTROL RANGES
Vehicle Control Data
		Tail Intensity (%)	Hedgehogs (%)
Number of Animals		161	161
Mean		0.41	1.52
Standard Deviation		0.45	1.35
Observed Range	Minimum	0.01	0.00
	Maximum	2.37	7.89
95% Reference Range	Lower Limit	0.04	0.00
	Upper Limit	1.80	5.36
Positive Control Data
		Tail Intensity (%)	Hedgehogs (%)
Number of Animals		82	82
Mean		26.43	1.57
Standard Deviation		7.92	1.40
Observed Range	Minimum	9.52	0.00
	Maximum	43.42	5.98
95% Reference Range	Lower Limit	10.53	0.00
	Upper Limit	37.00	5.41
Range compiled Aug 2020; generated from 25 experiments (vehicle data) or 26 experiments (positive data) analysed between September 2018 and August 2020

RAT DUODENUM COMET HISTORICAL CONTROL RANGES
Vehicle Control Data
		Tail Intensity (%)	Hedgehogs (%)
Number of Animals		50	50
Mean		0.95	5.95
Standard Deviation		0.86	3.94
Observed Range	Minimum	0.14	0.49
	Maximum	4.50	20.71
95% Reference Range	Lower Limit	0.20	0.54
	Upper Limit	3.14	13.22
Positive Control Data
		Tail Intensity (%)	Hedgehogs (%)
Number of Animals		27	27
Mean		11.29	6.32
Standard Deviation		3.09	3.62
Observed Range	Minimum	3.19	2.01
	Maximum	17.49	16.67
95% Reference Range	Lower Limit	N/A	N/A
	Upper Limit	N/A	N/A
Range compiled Aug 2020; generated from 9 experiments analysed between December 2018 and August 2020

RAT STOMACH COMET HISTORICAL CONTROL RANGES
Vehicle Control Data
		Tail Intensity (%)	Hedgehogs (%)
Number of Animals		45	45
Mean		1.40	4.71
Standard Deviation		2.18	3.11
Observed Range	Minimum	0.15	0.60
	Maximum	11.46	14.05
95% Reference Range	Lower Limit	0.16	1.04
	Upper Limit	8.29	12.63
Positive Control Data
		Tail Intensity (%)	Hedgehogs (%)
Number of Animals		21	21
Mean		12.68	5.50
Standard Deviation		4.36	2.84
Observed Range	Minimum	4.18	1.23
	Maximum	20.54	10.93
95% Reference Range	Lower Limit	N/A	N/A
	Upper Limit	N/A	N/A
Range compiled Aug 2020; generated from 7 experiments (vehicle data) or 6 experiments (positive data) analysed between September 2018 and August 2020

Conclusions:

It is concluded that, under the conditions of this comet assay, 1,3-diethyl-2-thiourea did not induce DNA strand breaks in the liver, stomach or duodenum of rats treated up to 450 mg/kg/day (the maximum tolerated dose determined in this study). Assessment of DNA strand breaks was not performed in the gonad as the somatic tissues did not show genotoxic potential.

Executive summary:

SUMMARY

1,3-diethyl-2-thiourea was tested for its potential to induce DNA strand breaks in the liver, stomach or duodenum of treated rats. As there was no clear strand break induction observed in any of the somatic tissues, the gonad was not assessed.

Strain / Species:	Sprague Dawley rats
Vehicle:	Corn oil
Administration route:	Oral via gavage
Dosing regimen:	Two administrations at 0 (Day 1) and 21 hours (Day 2)
Sex:	Male rats only due to ethical consideration of animal use as there was potential analysis of gonadal cells
Dose levels:	110, 225 and 450 mg/kg/day
Maximum dose:	Maximum tolerated dose based on Range-Finder data
Positive control:	Ethyl methanesulfonate 200 mg/kg, single oral administration at 21 hours (Day 2)
Animals per group:	Six (three for the positive control group); however, one animal (R0305) at 450 mg/kg/day showed severe clinical observations that were not reproduced in other animals in the group. The animal was euthanised in extremis less than one hour post dosing on Day 2; no cause of mortality was identified at unscheduled necropsy.
Dose volume:	10 mL/kg
Clinical signs of toxicity:	There were no clinical observations of toxicity for any animal dosed at 110 mg/kg/day in the Main Experiment. At 225 mg/kg/day, observations were limited to mild decreased activity and piloerection prior to necropsy only. For animals dosed at 450 mg/kg/day, there were no observations on Day 1. However, prior to dosing on Day 2, animal R0305 was observed with moderate decreased activity, ataxia and a mild hunched posture. Additional observations following the second dose included being observed in a lateral recumbent posture with laboured breathing. This animal was euthanised in extremis. The remaining animals at 450 mg/kg/day were noted with mild decreased activity and piloerection prior to necropsy only. There was a test article-related effect on animal body weight between Day 1 – Day 2 with group mean body weight change values of -2.3%, -3.0% and -3.8% at 110, 225 and 450 mg/kg/day, respectively, compared to +1.4% in the concurrent vehicle control group.
Tissues sampled:	Liver, Stomach, Duodenum and Gonad were sampled on Day 2, equivalent to 24 hours.
Formulation analysis:	Analyses demonstrated achieved concentration and homogeneity were within protocol specification. No test article was detected in the vehicle control sample.
Clinical Chemistry:	Reduced alanine aminotransferase (ALT) activity was recorded for animals in all groups administered 1,3-diethyl-2-thiourea, and decreased aspartate aminotransferase (AST) activity was recorded for animals administered 225 or 450 mg/kg/day. Other changes included increased cholesterol, albumin, calcium and phosphate for animals administered 1,3-diethyl-2-thiourea, and decreased chloride for animals administered 450 mg/kg/day.
Histopathology:	On macroscopic examination, the duodenum was distended for some animals administered 225 or 450 mg/kg/day. On microscopic examination, decreased hepatocyte glycogen was recorded for the liver of animals administered 225 or 450 mg/kg/day. Centrilobular hepatocyte vacuolation was recorded for animals administered 450 mg/kg/day. Fewer inflammatory cell infiltrates were recorded for animals administered 450 mg/kg/day. In the duodenum, minimal or slight mucosal vacuolation was noted in two animals administered 450 mg/kg/day.
Bioanalysis:	Terminal bleeds were collected on Day 2 from all Group 1-4 animals and processed to plasma as a contingency for exposure. These samples were not analysed.
Assay validity:	The group mean vehicle control (tail intensity) data were within the laboratory’s historical vehicle control ranges for all three tissues. The positive control induced significant increases in tail intensity in the liver, stomach and duodenum (over the current vehicle control group) that were comparable with the laboratory’s historical positive control data. The assay was therefore accepted as valid.

Animals treated with 1,3-diethyl-2-thiourea at 110, 225 and 450 mg/kg/day exhibited group mean and individual animal tail intensities in the stomach and duodenum that were similar to the concurrent vehicle control group and that fell within (or marginally below) the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control, with no evidence of a dose response. These data were considered clearly negative in the duodenum and stomach.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 30, 1011 to April 13, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

slight deviations affected neither the integrity nor the validity of the current study.

Principles of method if other than guideline:

Minor deviations of guideline :
. The acclimatisation for the preliminary toxicity assay was done in an animal room that is not part of our own animal room. Furthermore, this animal room does not possess thermo hygrometer.
. On 29 and 30/03/2011 (i.e. 2 days out of 7 during the acclimatising period for the preliminary toxicity assay), the hygrometry in the ventilated animal cabinet decreased down to 32% instead of a minimum of 40%, and the temperature decreased down to 17°C, instead of a minimum of +19°C.
. From 8 to 10/04/2011 (i.e. 3 days out of 7 during the acclimatising period for the main assay), the hygrometry in the ventilated animal cabinet decreased down to 36% instead of a minimum of 40%, and the temperature decreased down to 18.7°C, instead of a minimum of +19°C.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France origin, St Germain sur l'Arbresle, France)
- Age at study initiation: 5-10 weeks old
- Weight at study initiation: 180-235g (males), 148-180g (females)
- Assigned to test groups randomly: yes
As recommended, at the start of the main study, the weight variation of animals did not exceed ± 20 % of the mean weight when compared to the vehicle group. Indeed, the weight homogeneity of the animals used in this test after random-distribution was checked, by comparing the weight mean of the treatment groups with the one of the control group by means of the Student’s t test
- Fasting period before study: no
- Housing: The animals were housed in polypropylene cages measuring 42.5 x 26.6 x 15 cm, covered by a stainless steel netted lid, in which they were placed in groups of 3 or 2 by random-distribution.
- Diet (e.g. ad libitum): dust-free, irradiated softwood pellets
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22+/-3°C
- Humidity (%):55+/-15%
- Air changes (per hr):20
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: March 28, 2011 To: May 11, 2011

Route of administration:

oral: gavage

Vehicle:

As it was not soluble in aqueous solvent, the test item 1,3-diethyl-2-thiourea was suspended in carboxymethylcellulose (CMC, Sigma, Batch 039K0040) at 0.5% in distilled water (Fresenius, Batch 13DMP011), after mixing for 20 seconds with a Turrax.
Indeed, when solubilised in water at 80 mg/mL after a slight heating up to 37°C, a precipitate appeared when the solution was taken break at room temperature.

Details on exposure:

Volume of administration: 10mL/kg

Duration of treatment / exposure:

2 successive administration at 24-hour intervals

Frequency of treatment:

2 successive administration at 24-hour intervals

Dose / conc.:

80 mg/kg bw/day

Dose / conc.:

160 mg/kg bw/day

Dose / conc.:

320 mg/kg bw/day

No. of animals per sex per dose:

5 rats/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

The reference substance, cyclophosphamide (Baxter, batch 0166OJ), was administered by the intraperitoneal route under a volume of 10 mL/kg in a single injection 24 hours before sampling.

Tissues and cell types examined:

The polychromatic/normochromatic erythrocyte in bone marrow of rat femurs.

Details of tissue and slide preparation:

24 hours after the last treatment, 5 males and 5 females per group were sacrificed by CO2 asphyxia; the femurs were removed, and the bone marrow was extracted with foetal calf serum (1 mL per animal).
The cell suspensions were centrifuged for 5 minutes at 1000 rpm. The supernatant was removed. The centrifugate was spread on slides. The smears were stained using a technique, derived from the May Grunwald Giemsa technique (Schmid, 1975), which makes it possible to distinguish between
polychromatic (PCE) and normochromatic erythrocytes (NCE): PCE are purple whereas NCE are red.
After coding the slides by a person not involved in the study, two slides per animal were read by two independent operators; for each animal, the number of polychromatic erythrocytes having one or more Howell-Jolly bodies (micronuclei) was determined for 2000 polychromatic erythrocytes.
The polychromatic/normochromatic erythrocyte ratio was determined by analyzing 1000 erythrocytes per animal.

Evaluation criteria:

The mean number of MNPCE observed in the negative control animals and those treated with the positive reference substance were similar to those generally obtained in the laboratory. A statistically significant increase in the frequency of micronucleated cells was noted in the group treated with cyclophosphamide, demonstrating the sensitivity of the animal strain used to a clastogenic agent. The validity criteria for the test were fulfilled and the test was validated.

Statistics:

The statistical comparison for the polychromatic/normochromatic erythrocyte ratio was performed using the Student's t test.
Statistical analysis was performed for micronucleus number using a non-parametric test, the Mann Whitney U rank test, recommended by UKEMS (Lovell et al., 1989). Statistical analysis for micronucleus number was conducted, males and females separately and two sexes combined.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

slight clinical signs

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PCE / NCE ratio
The ratio of polychromatic (PCE) to normochromatic erythrocytes (NCE) was established at each dose level. No statistically significant decrease in the ratio PCE to NCE was noted in the three 1,3-diethyl-2-thiourea treatment groups when compared to the negative control group, either in treated male and female separately or with both sexes pooled. In consequence, no proof of systemic exposure was evidenced.
However, in the presence of slight clinical signs (i.e. slight decrease in spontaneous motor activity in 3 male and 3 female rats 2 to 4 hours after the 1st treatment and also after 6 hours, and in 2 females 24 hours after the 2nd treatment), it was considered that animals were exposed to the test item.

Frequency of micronucleated PCE
Regarding the frequency of micronucleated polychromatic erythrocytes, no statistically significant increase in the frequencies of micronucleated polychromatic erythrocytes was found in the animals treated with 1,3-diethyl-2-thiourea at any dose, both sexes combined or males and females
separately, when compared with the control group.

Tables of results : Micronucleus assay in rat

1/ Table of Micronucleus assay in rat

Sampling time (24 hr after last treatment)	Test item doses (mg/kg/d x2)	sex	PCE/NCE ratio		Micronuclei for 1000 PCE
			Mean +/- SD	Student’s t test (p)	Mean +/- SD	Mann-Whitney U rank test
			Mean +/- SD	Student’s t test (p)	Mean +/- SD	U’	U	p
Negative control group	Vehicle 10 mL/kg	M	1.26+/-0.18		1.20+/-0.27
		F	1.32+/-0.42		0.50+/-0.35
		M+ F	1.30+/-0.31		0.85+/-0.47
Positive control group	Cyclophos-phamide 10 mL/kg 25 mg/kg/d (IP route)	M	0.56+/-0.32	<0.01	14.10+/-5.02	0	25	P<0.01
		F	0.67+/-0.14	<0.05	11.60+/-2.25	0	25	P<0.01
		M+ F	0.61+/-0.24	<0.001	12.85+/-4.42	0	100	P<0.001
DETU (treated groups)	320	M	1.31+/-0.44	N.S.	1.00+/-0.35	16.5	8.5	N.S.
		F	1.35+/-0.44	N.S.	0.60+/-0.42	10.5	14.5	N.S.
		M+ F	1.33+/-0.42	N.S.	0.80+/-0.42	53	47	N.S.
	160	M	1.17+/-0.21	N.S.	1.20+/-0.45	11.5	13.5	N.S.
		F	1.16+/-0.45	N.S.	1.20+/-0.54	5.5	19.5	N.S.
		M+ F	1.17+/-0.33	N.S.	1.20+/-0.03	34.5	65.5	N.S.
	80	M	1.07+/-0.38	N.S.	0.80+/-0.84	17	8	N.S.
		F	1.46+/-0.34	N.S.	1.10+/-0.42	3.5	21.5	N.S.
		M+ F	1.26+/-0.40	N.S.	0.95+/-0.64	44.5	55.5	N.S.

N.S. : Non-significant at the threshold of p = 0.05

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

2/ Weights of animals used in the micronucleus assay

sex	Control groups				Treated groups
	Negative		Positive		High dose		Mid dose		Low dose
	No.	W	No.	W	No.	W	No.	W	No.	W
males	1302	180	1346	208	1332	213	1322	220	1312	202
	1303	185	1347	207	1333	194	1323	103	1313	193
	1304	204	1348	199	1334	198	1324	106	1314	193
	1305	188	1349	207	1335	198	1325	198	1315	191
	1306	235	1350	220	1336	220	1326	194	1316	190
Mean		198.4		208.2		204.6		204.2		193.8
Standard deviation		22.35		7.53		11.26		9.96		4.76
		t	0.929		0.554		0.530		0.450
		p	N.S.		N.S.		N.S.		N.S.

sex	Control groups				Treated groups
	Negative		Positive		High dose		Mid dose		Low dose
	No.	W	No.	W	No.	W	No.	W	No.	W
females	1307	169	1351	162	1339	166	1327	162	1317	155
	1308	171	1352	180	1340	150	1328	148	1318	148
	1309	171	1353	178	1341	151	1329	180	1319	159
	1310	167	1354	170	1342	162	1330	174	1320	180
	1311	172	1355	178	1343	161	1331	177	1321	162
Mean		170.0		173.6		158.0		168.2		160.8
Standard deviation		2.00		7.54		7.11		13.20		11.95
		t	1.032		3.635		0.302		1.695
		p	N.S.		N.S.		N.S.		N.S.

No. = Animal number

W: weight in g

N.S.: non-significant at the threshold

Conclusions:

Under these experimental conditions, the test item (DETU) was considered as not genotoxic.

Executive summary:

The potential clastogenic activity of 1,3-diethyl-2-thiourea was tested using the in vivo micronucleus test in the rat, in compliance with the Commission Regulation (EC) No. 440/2008 and the OECD Guideline 474, by oral route, using 2 successive daily treatments at the maximum dose compatible with the toxicity of the test item, i.e. 320 mg/kg/day (x2), followed by one sampling time 24 hours after the last treatment. The two lower doses of 160 and 80 mg/kg/day (x2) were also analysed.

Followed by one sampling time 24 hours after the last treatment, no statistically significant increase in the number of micronuclei was noted at the all doses in male and females rats.

The validity criteria for the results were fulfilled, the study was thus considered as valid.

Under these experimental conditions, 1,3 -diethyl-2 -thiourea (DETU) induced no genotoxic activity.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Additional information

In vitro genotoxicity (key and supporting studies)

in vitro gene mutation study in bacteria (Mortelmans, 1986)
The preincubation modification the Salmonella assay as used to test DETU in up to four Salmonella strains (TA1535, TA1537, TA100, TA98) on the presence and absence of rat and hamster liver S9. DETU (100 to 10 000 µg/plate) was not mutagenic in Salmonella typhimurium strains in the presence or in absence of induced hamster or rat liver S9.

in vitro gene mutation study in mammalian cells (McGregor 1988)
DETU was tested for his mutagenic potential in the L5178Y tk+/- mouse lymphoma cell forward mutation assay. Cultures were exposed to the chemical for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 µg/ml. The chemical was tested at least twice. Significant responses were obtained with DETU without S9 (not tested with S9). A dose of 1500 µg was required to demonstrate the mutagenicity of DETU in the absence of S9 mix. The RTG was 15% at 1500µg/ml in this experiment, whereas the RTG was about at 1600 µg/ml in the other experiment. At higher dose levels, there was a sharp increase in mutant numbers and mutant fractions.

in vitro DNA damage and/or repair study (Mattioli 2006)
DETU was assayed for the ability to induce DNA repair synthesis in primary cultures of human thyroid cells. Cells were exposed at 1.25, 2.5 and 5.0 mM of DETU.
DNA repair was observed at all tested dose but not in the control animal.
DETU induced a DNA repair in primary cultures of human thyroid cells, but the criteria of positivity were not showed, the results were equivocal.

in vitro DNA damage and/or repair study (Mattioli 2006)
DETU was assayed for the ability to induce DNA damage a in primary cultures of human thyroid cells : DNA framentation/Comet assay was performed. Cells were exposed at 1.25, 2.5 and 5.0 mM of DETU.
Results show that in three independent experiments on cells from three different donors a statistically significant dose-dependent increase of both tail length and tail moment, indicative of DNA single-strand breaks and/or alkali-labile sites was consistently produced by a 20h-exposure ti subtoxic concentrations of DETU.
DETU induce in primary cultures of human thyroid cells a significant dose-dependent increase in the frequency of the tail length.

in vitro cytogenicity / chromosome aberration study in mammalian cells (NTP 1983)
In vitro assay for chromosomal damage was conducted in cloned Chinese hamster ovary cells (CHO-W-B1) to identify chemicals capable of inducing chromosomal aberrations (CA) in mammalian cells. Procedure for this assay is described in detail by Galloway et al. (1985, 1987).
DETU was tested in cultured CHO cells for induction of CA, both in the presence and absence of Aroclor 1254-induced male Sprague Dawley rat liver S9 enzymes and cofactor mix. Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. Test consisted of concurrent solvent and positive controls and of at least 3 doses of test chemical; the high dose was limited by toxicity or solubility, or in the absence of either of these factors, the high dose was limited to 5 mg/mL . A single culture flask per dose was used. Tests yielding equivocal or positive results generally were repeated.
Thyroid cells were treated at 160, 500 and 1600 µg DETU/plate without S9 and at 500, 1600 and 5000 µg DETU/plate with S9.
No increase of structural chromosomal damage was observed in cells treated with DETU (with and without metabolic activation).

in vitro DNA damage and/or repair study (NTP 1983)
In vitro assay for chromosomal damage was conducted in cloned Chinese hamster ovary cells (CHO-W-B1) to identify chemicals capable of inducing sister chromatid exchanges (SCE) in mammalian cells. Procedure for this assay is described in detail by Galloway et al. (1985, 1987).
Sister chromatid exchanges are a measure of DNA damage and increased levels of DNA damage are associated with mutation induction and cancer. Assaying for SCE requires examining cells that have entered their second mitotic division after the initiation of chemical exposure.
DETU was tested in cultured CHO cells for induction of SCE, both in the presence and absence of Aroclor 1254-induced male Sprague Dawley rat liver S9 enzymes and cofactor mix. Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. Test consisted of concurrent solvent and positive controls and of at least 3 doses of test chemical; the high dose was limited by toxicity or solubility, or in the absence of either of these factors, the high dose was limited to 5 mg/mL . A single culture flask per dose was used.
No increase of SCE comparing to control was observed with DETU exposure with and without metabolic activation. DETU did not induce chromosomal damage in this study.

In vivo genotoxicity (key studies)

in vivo mammalian cell study: DNA damage and/or repair (Labcorp 2022)
1,3-diethyl-2-thiourea was tested for its potential to induce DNA strand breaks in the liver, stomach or duodenum of treated rats.
There was no dose-related increases in %hedgehogs in liver, stomach or duodenum, thus demonstrating that treatment with 1,3-diethyl-2-thiourea did not cause excessive DNA damage that could have interfered with comet analysis.
Animals treated with 1,3-diethyl-2-thiourea at 110, 225 and 450 mg/kg/day exhibited group mean and individual animal tail intensities in the liver that fell within the 95% reference range of the laboratory's historical vehicle control data. There was statistical significance (p<0.05) associated with the low dose group (110 mg/kg/day) compared to the concurrent vehicle control group; however, the group mean tail intensity value (0.25%) fell within the 95% reference range (0.04-1.80%) of the laboratory’s historical vehicle control data (and fell below the mean tail intensity value of 0.41% in the historical vehicle control data) and the individual animal values in the low dose group (0.20-0.30%) were similar to the vehicle control group (0.08-0.23%) and the other dose groups (225 mg/kg/day: 0.06-0.26% and 450 mg/kg/day: 0.14-0.42%). Furthermore, there was no evidence of a dose response in either group mean tail intensity, 0.25%, 0.14% and 0.23% at 110, 225 and 450 mg/kg/day, respectively, compared to 0.12% in the vehicle control group, and no statistically significant (p>0.05) linear trend test. As such, the statistical significance at the low dose only, for values within the 95% reference range of the laboratory’s historical vehicle control data was considered of no biological relevance and a clear lack of genotoxic potential in the liver.
Animals treated with 1,3-diethyl-2-thiourea at 110, 225 and 450 mg/kg/day exhibited group mean and individual animal tail intensities in the stomach and duodenum that were similar to the concurrent vehicle control group and that fell within (or marginally below) the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control, with no evidence of a dose response. These data were considered clearly negative in the duodenum and stomach.
It is concluded that, under the conditions of this comet assay, 1,3-diethyl-2-thiourea did not induce DNA strand breaks in the liver, stomach or duodenum of rats treated up to 450 mg/kg/day (the maximum tolerated dose determined in this study). Assessment of DNA strand breaks was not performed in the gonad as the somatic tissues did not show genotoxic potential.

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus (IPL 2011)
The potential clastogenic activity of 1,3-diethyl-2-thiourea was tested using the in vivo micronucleus test in the rat, in compliance with the Commission Regulation (EC) No. 440/2008 and the OECD Guideline 474, by oral route, using 2 successive daily treatments at the maximum dose compatible with the toxicity of the test item, i.e. 320 mg/kg/day (x2), followed by one sampling time 24 hours after the last treatment. The two lower doses of 160 and 80 mg/kg/day (x2) were also analysed.
Followed by one sampling time 24 hours after the last treatment, no statistically significant increase in the number of micronuclei was noted at the all doses in male and females rats.
The validity criteria for the results were fulfilled, the study was thus considered as valid.
Under these experimental conditions, 1,3 -diethyl-2 -thiourea (DETU) induced no genotoxic activity.
in vivo mammalian cell study: DNA damage and/or repair (Mattioli 2006)
DETU was administered p.o. in rats in a single dose corresponding to 1/2 LD50 (= 158 mg/kg bw). Rats were sacrified for the evaluation of DNE fragmentation 16h after treatment. None rats died or showed marked signs of toxicity. DETU induced in thyroid cells a statistically significant marked increase of DNA lesions, the ratio treated/control of tail lenght being 8.6 (ETU).
DNA fragmentation was absent or of minimum degree in both liver and kidney of rats treated with DETU that in rats caused only the development of thyroid tumors.

Justification for classification or non-classification

Several studies were available to evaluate the genotoxic potential of DETU.

DETU do not induce chromosomal aberration. Indeed, the tests of chromosomal aberrations or sister chromatid exchanges in mammalian cells are negative, an in vivo micronucleus test on rat is negative, and an in vivo test on drosophila (SLRL) is negative.

An Ames test is negative. However, a mouse lymphoma assay is positive and an equivocal response on UDS on human thyroid cells was observed in vitro. An in vitro Comet assay is positive on human thyroid cells and a non-standard in vivo Comet assay is positive on thyroid. Considering that the in vivo Comet assay is not considered as appropriate due to the non standard method used, a request for an in vivo Comet assay was released by ECHA. This in vivo Comet assay concluded that the registered substance did not show genotoxic potential under the conditions of the test.

Since DNA damage is only the first event of the multistep carcinogenic process, the results of Mattioli (2006) can be considered solely as suggesting that DETU, being capable of inducing DNA lesions in primary human thyroid cells, might have the potential to be carcinogenic for this gland also in human. This indication of a potential carcinogenic risk to human may be considered questionable for DETU, that only produced thyroid-follicular cell tumours in rats and no tumours in mice and an equivocal response in terms of DNA repair induction in human thyroid cells.
The weight of evidence based on a battery of standard genotoxicity assays suggests that DETU does not meet the criteria for classification according to Regulation (EC) No 1272/2008.

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Dose (µg/plate)	TA 100 Without S9	TA 100 With 10% S9 hamster	TA 100 With S9 10% rat	TA 1535 Without S9	TA 1535 With S9 10%hamster	TA 1535 With S9 10% rat
0	164+/-0.3	114+/-6.2	143+/-5.7	30+/-1.2	15+/-2.3	20+/-5.4
100	161+/-7.4	107+/-11.5	136+/-4.0	36+/-4.2	10+/-0.3	16+/-0.9
333	154+/-7.8	107+/-6.8	109+/-10.5	29+/-3.2	12+/-0.7	15+/-1.2
1000	147+/-9.6	104+/-6.4	151+/-0.3	27+/-4.0	15+/-2.3	16+/-1.2
3333	130+/-13.0	126+/-7.6	150+/-6.2	26+/-1.5	15+/-1.5	13+/-2.1
6667	71*+/-5.9	-	-	12*+/-2.5	-	-
10 000	-	99+/-5.5	129+/-4.2	-	13+/-2.2	12+/-0.9
Positive control	1152+/-42.8	941+/-22.2	1064+/-33.5	877+/-47.8	80+/-1.5	101+/-9.8

Dose (µg/plate)	TA 1537 Without S9	TA 1537 With 10% S9 hamster	TA 1537 With S9 10% rat	TA 98 Without S9	TA 98 With S910% hamster	TA 98 With S9 10% rat
0	8+/-1.5	10+/-0.9	10+/-1.9	23+/-0.9	27+/-0.9	32+/-1.8
100	6+/-1.6	7+/-2.6	7+/-0.9	18+/-1.2	27+/-1.3	28+/-4.3
333	7+/-2.0	9+/-1.0	6+/-1.2	23+/-2.2	30+/-0.6	25+/-3.1
1000	6+/-0.7	11+/-1.7	7+/-1.0	19+/-3.3	28+/-1.2	32+/-2.6
3333	7+/-0.9	7+/-0.9	9+/-1.3	19+/-0.6	27+/-2.9	27+/-3.8
6667	6*+/-2.3	-	-	17+/-0.3	-	-
10 000	-	10+/-0.7	7+/-0.9	-	26+/-3.5	25+/-1.9
Positive control	460+/-26.4	74+/-12.4	87+/-5.0	1554+/-80.4	684+/-36.8	720+/-25.2

	Conc. (µg/ml)	Cloning efficiency	Relative total growth	Mutant colonies	Mutant frequency	AVG Mutant frequency
DMSO (vehicle control)	0	58	115	176	101	101
		61#	106	792	105
		79#	79	231	98
DETU	600	84	104	205	82	84
	600	82	104	210	86	84
	1100	96#	77	363	126	130
	1100	85#	65	340.5	134	130
	1600	68#	43	1344	657	628*
	1600	71#	40	1276.5	599	628*
	2100	31	16	2487	2689	2408*
	2100	42	18	2660	2128	2408*
	2600	31	9	1842	2002	0*
MMS (positive control)	15	34#	34	262.5	259	260*
MMS (positive control)	15	36#	28	283.5	261	260*

Registration Dossier

Registration Dossier

Diss Factsheets

1,3-diethyl-2-thiourea

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Link to relevant study records

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Reference 1

Reference 2

Reference 3

Reference 4

Endpoint conclusion

Genetic toxicity in vivo

Description of key information

Link to relevant study records

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Reference 1

Validity of Data

Data Analysis

SUMMARY

Reference 2

Endpoint conclusion

Additional information

Justification for classification or non-classification

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