1,3-diethyl-2-thiourea

Genetic toxicity: in vitro

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

DNA fragmentation and DNA repair synthesis induced in rat and human thyroid cells by chemicals carcinogenic to the rat thyroid.

Author:

Mattioli F, Martelli A, Gosmar M, Garbero C, Manfredi V, Varaldo E, Torre GC, Brambilla G.

Year:

2006

Bibliographic source:

Mutation Research 609, 146-153

Materials and methods

Test guideline

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:

no

Type of assay:

comet assay

Test material

Test material information

Constituent 1

Reference substance name:

1,3-diethyl-2-thiourea

EC Number:

203-308-5

EC Name:

1,3-diethyl-2-thiourea

Cas Number:

105-55-5

Molecular formula:

C5H12N2S

IUPAC Name:

1,3-diethyl-2-thiourea

Method

Species / strain

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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
			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)

Applicant's summary and conclusion

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.