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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
DNA fragmentation and DNA repair sysnthesis 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 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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

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

Administration / exposure

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
Doses / concentrations
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.

Examinations

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.

Results and discussion

Test resultsopen allclose all
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.

Any other information on results incl. tables

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).

Applicant's summary and conclusion

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.