Registration Dossier

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 on Ames test, in vitro chromosomal aberration test, in vitro SCE test, SLRL test.
Positive results were observed on Mouse lymphoma assay, on in vitro Comet and assay on human thyroid cells.
The in vitro UDS test on human thyroid cells is equivocal.

Link to relevant study records

Referenceopen allclose all

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:
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:
no
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:
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.
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

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.

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:
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:
no
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):
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:
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.

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

NUC

CYT

NNG

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.

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.

Tables of results: Mouse lymphoma assay with DETU

 

Non activation trial: 1

 

 

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

82

104

210

86

1100

96#

77

363

126

130

85#

65

340.5

134

1600

68#

43

1344

657

628*

71#

40

1276.5

599

2100

31

16

2487

2689

2408*

42

18

2660

2128

2600

31

9

1842

2002

0*

MMS (positive control)

15

34#

34

262.5

259

260*

36#

28

283.5

261

 

Non activation trial: 2

 

 

Conc. (µg/ml)

Cloning efficiency

Relative total growth

Mutant colonies

Mutant frequency

AVG Mutant frequency

DMSO (vehicle control)

0

71

93

70

33

40

97

101

106

36

85

108

96

38

69

99

110

53

DETU

500

64

95

77

40

37

72

83

75

35

1000

76

54

105

46

46

95

64

129

45

1500

73

15

326

148

182*

72

15

468

216

2000

63

7

868

460

600*

50

6

1105

739

2500

LETHAL

MMS (positive control)

15

42

37

222

177

177*

51

32

269

176

 

*: significant response

#: reduced sample size because of the loss of one culture dish due to contamination

 

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.

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, 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 in TA1535, TA1537 and TA100.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No genotoxicity was observed (see table).
Remarks on result:
other:

Table of results : Mutagenicity of DETU in Salmonella typhimurium

 

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

* cytotoxicité

 

 

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

* cytotoxicité

 

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

Negative results were observed on in vivo micronucleus test, but positive results were obtained on in vivo Comet assay (rat).

Based on the weight of evidence, DETU is considered to be not mutagenic

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
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:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
DETU was administered p.o. in rats in a single dose corresponding to 1/2 LD50. Rats were sacrified for the evaluation of DNA fragmentation 16h after treatment. Cells of thyroid, kidney and liver were studied.
GLP compliance:
no
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Harlan (Correzzana, Italy)
- Age at study initiation:no data
- Weight at study initiation:120-150g
- Fasting period before study: yes, 12 hours before treatment
- Housing: no data
- Diet (e.g. ad libitum): rat chow (TRM, Harlan), ad libitum
- Water (e.g. ad libitum): not precised, ad libitum
- Acclimation period:1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22+/-2°C
- Humidity (%):50+/-10%
- Air changes (per hr): oui but not precised number of charges per hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
DMSO
Details on exposure:
DETU was administered in 0.1 ml/g body weight.
Duration of treatment / exposure:
1 single exposure
Frequency of treatment:
1 single exposure
Post exposure period:
16 hours after treatment
Dose / conc.:
158 mg/kg bw/day
Remarks:
= 1/2 LD50 (ETU)
No. of animals per sex per dose:
one group of 3 rats
Control animals:
no
Positive control(s):
Rats were sacrified for the evaluation of DNA fragmentation 16h after treatment.
Tissues and cell types examined:
Thyroid, liver and kidney cells.
Details of tissue and slide preparation:
Thyroid, Iiver and kidneys were quickly removed and then separately processed as follows. Suspensions of thyroid cells were obtained as previously described for human cells. Kidney cells were isolated by the method described by Bruggeman et al. Liver was briefly minced in Merchant's solution (0.14 M NaCl, 2.7 mM KCI, 8.1 mM Na2HPO4, 1.47 mM H2PO4, 0.53 mM disodium EDTA; pH 7.4). Fragments of liver were then homogenized using a loosely fitting Potter-Elvehjem homogenizer; after sedimentation of the large tissue fragments, single cells remaining in the supernatant were pelleted at 50 x g for 4 min. The cells of the three organs were finally resuspended in a suitable volume of Merchant's solution and counted in a hemocytometer. The fraction of viable cells, determined with the trypan blue exclusion method, was, in each rat and for all organs, higher than 80%. The degree of DNA fragmentation was evaluated with the Comet assay, using the saine procedure described for the in vitro assay.
Statistics:
The pooled mean ± S.D. was based on the mean data generated from individual animals.
Key result
Sex:
male
Genotoxicity:
positive
Remarks:
Thyroid and kidneys
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not examined
Sex:
male
Genotoxicity:
negative
Remarks:
Liver
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
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 (DETU).
DNA fragmentation was absent or of minimum degree in both liver and kidney of rats treated with DETU.

Table of results: Comet assay (in vivo)

 

treatment

Rat no.

Thyroid

Liver

Kidney

Tail length (µm)

Tail moment

Tail length (µm)

Tail moment

Tail length (µm)

Tail moment

Control (distilled water)

1

2.5+/-0.7

146+/-41

3.0+/-0.8

181+/-53

2.8+/-1.2

179+/-80

2

1.6+/-0.6

115+/-47

2.5+/-1.0

178+/-74

2.2+/-0.7

160+/-51

3

1.2+/-0.7

108+/-43

2.1+/-0.6

172+/-39

1.7+/-0.7

145+/-64

pooled

1.8+/-0.7

123+/-20

2.5+/-0.4

177+/-5

2.2+/-0.6

161+/-17

Control (DMSO)

1

2.0+/-0.6

183+/-57

2.3+/-0.8

188+/-65

2.2+/-0.7

182+/-56

2

2.2+/-0.4

181+/-33

1.7+/-0.4

142+/-31

2.0+/-0.5

172+/-39

3

1.5+/-0.5

132+/-41

1.5+/-0.6

136+/-54

1.5+/-0.6

129+/-46

pooled

1.9+/-0.4

167+/-25

1.8+/-0.4

155+/-28

1.9+/-0.4

161+/-28

DETU (158 mg/kg)

1

15.7+/-2.6

1361+/-253

1.3+/-0.3

120+/-30

3.6+/-2.3

294+/-176

2

16.9+/-3.4

1383+/-315

1.1+/-0.4

116+/-38

5.3+/-2.0

400+/-145

3

16.3+/-5.0

1449+/-419

2.1+/-0.6

178+/-47

4.0+/-1.1

319+/-81

pooled

16.3+/-0.6*

1398+/-46*

1.5+/-0.5

138+/-35

4.3+/-0.9

338+/-55*

Values are mean +/- SD.

*: the comparison between treated and control rats given the same vehicle used to dissolve the test compound was performed by means of ANOVA; significant difference (p<0.05).

Conclusions:
The results indicated that DNA fragmentation was present in the thyroid of intact rats treated by the oral route with 1/2 LD50 of DETU, thus supportingthe conclusion that chemicals carcinogenic to the rat thyroid may be preliminary identified by the DNA fragmentation/Comet assay performed on cells from the thyroid of rats treated with a single high dose of DETU.
Executive summary:

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.

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:
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 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

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

A reliable Ames test is available (Mortelmans 1986). The preincubation modification the Salmonella assay as used to test DETU in up to 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 the Mouse lymphoma assay (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. Result of this test is positive.

The potential clastogenic activity of 1,3-diethyl-2-thiourea was tested using the in vivo micronucleus test in the rat (Simar 2011), in compliance with 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.

DETU was assayed for the ability to induce DNA repair synthesis and DNA damage in primary cultures of human thyroid cells (Matiolli 2006). Cells were exposed at 1.25, 2.5 and 5.0 mM of DETU.

In the UDS in vitro test, DETU induced in primary cultures of human thyroid cells a dose-dependent increase of DNA repair. DNA repair was observed at all tested dose but not in the control animal. Result of this test is equivocal because the criteria of positivity are not ensured.

In the DNA fragmentation/Comet (in vitro), the results showed 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 to 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. Result of this test is positive.

 

Two in vitro assays for chromosomal damage were conducted in cloned Chinese hamster ovary cells (CHO-W-B1) to identify chemicals capable of inducing chromosomal aberrations (CA) or sister chromatid exchanges (SCE) in mammalian cells (NTP 1983).

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.Results of both tests are negative: no increase of structural chromosomal damage or SCE was observed in cells treated with DETU (with and without metabolic activation).

 

The experimental plan was to each chemical for sex-linked recessive lethal (SLRL) induction by feeding: drosophila were exposed to 3000 ppm of DETU and no significant mutagenicity was observed. Also, the second test was performed: drosophila were exposed to 10000 ppm of DETU by injection, the result are negative also (1985).

 

SHE cell transformation assay was conducted at pH 6.7 culture conditions with a stardardized, Good Laboratory Practives-quality protocol. Cells were exposed to DETU at 400 -700 µg/ml for 24 -hr or 150 -350 µg/ml for 7 -day.

DETU transformed Syrian hamster embryo cells in culture, in these experimental conditions (pH = 6.7) after 24 -hr or 7 -day of exposure. This test is positive.

 

Justification for classification or non-classification

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

DETU don’t induce chromosomal aberration. Indeed, the tests of chromosomal aberrations or sister chromatid exchanges in mammalian cells are negative, the in vivo micronucleus test on rat is negative, and the in vivo test on drosophila (SLRL) is too negative. Moreover, the Ames test is negative.

However, the mouse lymphoma assay is positive and an equivocal response on UDS on human thyroid cells was observed in vitro. The Comet assay is positive on human thyroid cells and the in vivo Comet assay is positive on thyroid.

Since DNA damage is only the first event of the multistep carcinogenic process, the results of Mattioli 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 is not genotoxic.

Proposed self-classification (Regulation (EC) No 1272/2008:

Not classified