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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A lot of reliable in vitro tests are available and were used to evaluate the genotoxic potential of Catechol: 3 valid Ames test, 3 studies similar to OECD 476 with reliability 2, 2 references of Mouse lymphoma assays with mouse lymphoma L5178y cells with reliability 2, 2 studies similar to OECD 487 with reliability 2, 12 studies regarding DNA damage with reliability 2 and other in vitro studies on topoisomerase inhibition.

Taken together, all the reliable in vitro studies showed a genotoxic effect of Catechol.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
other information
Study period:
19 april 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Ames test according to micromethod, and not GLP. no repetition performed
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
micromethod
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA1537, TA98, TA100 and TA102
Metabolic activation:
with and without
Metabolic activation system:
liver and kidney S9 fraction
Test concentrations with justification for top dose:
0, 0.38, 1.14, 3.43, 10.29, 30.86, 92.59, 277.78, 833.33, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: solvent usually used in Ames test
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see details
Details on test system and experimental conditions:
Positive controls:
Without S9-mix: TA100: MNNG (0.25 µg/pl); TA1537: 9-aminoacridine (1.56 µg/pl); TA98: 2-nitrofluorene (0.5 µg/pl); TA102: mitomycine C (0.0625 µg/pl)
With liver S9-mix: 2-anthramine for TA98, TA100 and TA1537 (0.5 µg/pl); benzo[a]pyrene for TA102 (4 µg/pl)
With Kidney S9-mix: streptozotocin for TA98, TA100 and TA102 (25 µg/pl); aristolochic acids for TA1537 (25 µg/pl)

METHOD OF APPLICATION: in agar (plate incorporation), micromethod

DURATION
- incubation period: 90 minutes at 37°C under stirring
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATES: 3

METABOLIC ACTIVATION SYSTEM:
The S9 fraction was prepared according to method of Ames et al. (1975). Rat male Sprague Dawley OFA of 7 to 8 weeks receieved a single intraperitoneal injection of Aroclor 1254 (origin- Monsanto, Saint-Louis, USA, batch N° KD 06-618) at dose of 500 mg/kg in corn oil solution. 5 days later, animala were sacrified by cervical rupture and exsanguinated. the livers or kidneys are removed and S9 extracted.
The sterility and activity assessment of S9 are checked and S9 mix prepared as follow:
S9: 0.1 mL
0.4 M Mg Cl2: 0.01 mL
1.65 M KCL: 0.01 mL
0.2 M pH 7.4 phosphate buffer: 0.5 mL
0.1 M NADP: 0.04 mL
1M glucose-6-phosphate: 0.005 mL
H2O: 0.335 mL
Sterility of S9 mix was also checked.

DETERMINATION OF CYTOTOXICITY: the increase of the bacteria

OTHER:
Validity criteria for the test:
1- the test product must be sterile: at the highest dose, after spreading out under the conditions of the test and after 48 hour-incubation at 37°C, no colony must be visible.
2- The mean frquency of spontaneous revertants for each strain (both with and without metabolic activation) is between the limit historical control values.
3- The mean frequency of revertants induced by the positive reference products (both with and without metabolic activation), for each strain, must be greater than the lowest limit historical values.
Evaluation criteria:
Strain TA1537:
A product causing a positive response proportional to the dose for at least 3 concentrations with, for the highest increase, a value greater than or equal to 3 times the value for the solvent control, is considered positive in the assay.

Strains TA98, TA100 and TA102:
A product causing a positive response proportional to the dose for at least 3 concentrations with, for the highest increase, a value greater than or equal to 2 times the value for the solvent control, is considered positive in the assay.
Species / strain:
S. typhimurium, other: TA1537, TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
for TA102 without S9-mix and with kidney S9-mix
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the assay without metabolic activation in the strain TA102, statistically significant increases in the mean number of revertants were observed at the doses ranging from 30.86 to 0.38 µg/pl, with induction ratios between 2.2 and 1.7. The biologically threshold for a positive response set at 2 in this strain was reached at the doses of 10.29, 3.43 and 1.14 µg/pl.

In the assay with metabolic activation with liver S9-mix in strain TA102, a statiscally significant increase in the mean number of revertants was noted at the dose of 1.14 µg/pl, with an induction ratio of 1.2. Nevertheless, the biologically threshold for a positive response was not reached. This increase could therefore not be attributed to a mutagenic effect.

In the assay with metabolic activation with kidney S9-mix in strain TA102, statistically significant increases in the mean number of revertants were observed at the doses ranging from 833.33 to 0.38 µg/pl, with induction ratios between 2.5 and 1.9. The biologically threshold for a positive response set at 2 in this strain was reached at all doses between 833.33 to 1.14 µg/pl.
The test compound Catechol induced a clear mutagenic activity in strain TA102 without metabolic activation and in presence with kidney S9-mix.

The test compound Catechol induced no statistically or biologically significant increase in the mean number of revertants in the Salmonella typhimurium strains TA1537, TA98 and TA100 tested in absence or in presence of metabolic activation system by means of liver or kidney S9-mix.

Tables of results:

 

 

TA 1537

TA 98

TA 100

TA 102

 

Doses (µg/plate)

Revertants/ plate

Induction ratio

Revertants/ plate

Induction ratio

Revertants/ plate

Induction ratio

Revertants/ plate

Induction ratio

Positive control

(a)

2022.3

179

518

14.9

1475.3

7.7

1907.3

9.6

Solvent control

0

11.3

-

34.7

-

191.3

-

199.3

-

 

Catechol

 

WITHOUT S9-mix

0.38

9.7

0.9

34.7

1

211

1.1

343.3

1.7

1.14

9.7

0.9

32.7

0.9

209.3

1.1

423.3

2.1

3.43

8.3

0.7

32.7

0.9

198.3

1

427

2.1

10.29

8.3

0.7

28

0.8

209

1.1

447.7

2.2

30.86

11

1

26.7

0.8

203

1.1

384.7

1.9

92.59

13.3

1.1

25

0.7

230.3

1.2

316.7

1.6

277.78

14.3

1.3

28.3

0.8

197

1

265.3

1.3

833.33

9

0.8

27.7

0.8

197

1

116

0.6

2500

3.3

0.3

0

0

0

0

0

0

5000

0

0

0

0

0

0

0

0

 

 

 

 

TA 1537

TA 98

TA 100

TA 102

 

Doses (µg/plate)

Revertants/ plate

Induction ratio

Revertants/ plate

Induction ratio

Revertants/ plate

Induction ratio

Revertants/ plate

Induction ratio

Positive control

(b)

156.3

24.8

1642.7

30.6

1898.7

8.9

1362.3

3.7

Solvent control

0

6.3

-

53.7

-

213.2

-

365.8

-

 

Catechol

 

WITH LIVER S9-mix

0.38

5.7

0.9

62.7

1.2

193

0.9

397

1.1

1.14

4.7

0.7

61.3

1.1

187.3

0.9

456.7

1.2

3.43

8.3

1.3

63

1.2

187

0.9

417

1.1

10.29

5.7

0.9

62.3

1.2

201.7

0.9

402

1.1

30.86

8

1.3

62.3

1.2

204

1

407.3

1.1

92.59

5.7

0.9

63

1.2

225

1.1

359.3

1

277.78

7.3

1.2

63.3

1.2

238.3

1.1

387.7

1.1

833.33

6

1

66.7

1.2

211.7

1

289

0.8

2500

2

0.3

12.7

0.2

93.3

0.4

101

0.3

5000

0

0

0

0

0

0

0

0

 

 

 

 

TA 1537

TA 98

TA 100

TA 102

 

Doses (µg/plate)

Revertants/ plate

Induction ratio

Revertants/ plate

Induction ratio

Revertants/ plate

Induction ratio

Revertants/ plate

Induction ratio

Positive control

(c)

727.3

83.6

202

5.7

2601.3

13.3

3603

22.1

Solvent control

0

8.7

-

35.3

-

196

-

163.3

-

 

Catechol

 

WITH KIDNEY S9-mix

0.38

12.7

1.5

41.7

1.2

196.3

1

309.3

1.9

1.14

10.3

1.2

37.7

1.1

211

1.1

348.3

2.1

3.43

7.7

0.9

47.7

1.4

242.7

1.2

372.3

2.3

10.29

10

1.1

43.3

1.2

221.3

1.1

412

2.5

30.86

10.3

1.2

42

1.2

206.7

1.1

344

2.1

92.59

11

1.3

33

0.9

233

1.2

336.3

2.1

277.78

10

1.1

35

1

221

1.1

375.7

2.3

833.33

12.3

1.4

41.7

1.2

220.3

1.1

343.3

2.1

2500

12

1.4

0

0

34.3

0.2

5

0

5000

0

0

0

0

0

0

0

0

(a), (b), (c) : Without S9-mix: TA100: MNNG (0.25 µg/pl); TA1537: 9-aminoacridine (1.56 µg/pl); TA98: 2-nitrofluorene (0.5 µg/pl); TA102: mitomycine C (0.0625 µg/pl)

With liver S9-mix: 2-anthramine for TA98, TA100 and TA1537 (0.5 µg/pl); benzo[a]pyrene for TA102 (4 µg/pl)

With Kidney S9-mix: streptozotocin for TA98, TA100 and TA102 (25 µg/pl); aristolochic acids for TA1537 (25 µg/pl)

Conclusions:

Positive TA102 without S9-mix and with kidney S9-mix

Under conditions of this test, Catechol induced a clear mutagenic activity in strain TA102 without metabolic activation and in presence of metabolic activation by kidney S9-mix, in a screening micromethod assay of the Ames's test performed without repetition. No mutagenic activity was observed in strains TA1537, TA98 or TA100 with or without S9-mix.
Executive summary:

In this study (Watzinger, 2007), Catechol was tested in a Ames test using a screening micromethod assay, with Salmonella typhimurium strains TA1537,TA98, TA100 and TA102, with and without metabolic activation system from rat livers or kidneys induced by Aroclor 1254. Doses tested ranging from 0 to 5000 µg/plate.

Catechol induced a clear mutagenic activity in strain TA102 without metabolic activation and in presence of metabolic activation by kidney S9-mix, in this screening micromethod assay of the Ames's test performed without repetition. No mutagenic activity was observed in the Salmonella typhimurium strains TA1537, TA98 or TA100 tested in absence or in presence of metabolic activation system.

The positive controls induced the appropriate responses in the corresponding strains.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Studies according to recognized guidelines, but not GLP.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: Salmonella typhimurium TA 98,TA 100,TA 1535,TA 1537,TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction was from Sprague-Dawley rats pretreated with Aroclor 1254. The S9-Mix contained 100 µl of liver homogenate/ml
Test concentrations with justification for top dose:
3.9 - 15.6 - 62.5 - 250 - 1000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see details below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

POSITIVE CONTROLS:
- Without S9: MNNG for TA 1535 and TA 100 (1.6 µg), 9-aminoacridine for TA 1537 (50 µg), Daunomycine for TA 98 (5 µg) and 4-nitroquinoline-N-Oxide for TA 1538 0.25 µg).
- With S9: 2-anthramine (12.5 µg).

* Culture preparation: The histidine requirement for growth was checked for each frozen stock culture preparation. The presence or absence of the R-factor plasmid, the presence of the rfa mutation through sensitivity to crystal violet and the uvrB mutation through sensitivity to ultraviolet light were also verified.
* Base medium was prepared with agar Bacto-Difco at 1.5 % in Vogel-Bonner minimal medium E and 2 % of glucose. Overlay agar was made of 0.6 % agar Bacto-Difco in 0.6% NaCl containing L-histidine and biotine.
Evaluation criteria:
A positive response was indicated by a reproductible, dose-related increase, whether it be two-fold over background or not.
Statistics:
No data
Species / strain:
S. typhimurium, other: TA 98,TA 100,TA 1535,TA 1537,TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Study 7960 05

 

 

TA 1535

TA 1537

TA 98

µg/plate

+S9

-S9

+S9

-S9

+S9

-S9

0

16

18

18

9

49

25

3.9

27

20

16

8

53

24

15.6

20

25

17

8

53

25

62.5

24

23

13

7

54

31

250

25

25

11

9

54

19

1000

21

11

7

7

54

18

Positive control

315

1720

102

173

2943

209

Ethanol

22

21

15

7

40

19

 

 

TA 100

TA 1538

µg/plate

+S9

-S9

+S9

-S9

0

117

119

20

13

3.9

104

99

21

18

15.6

110

112

24

12

62.5

111

102

19

10

250

118

101

22

9

1000

111

85

22

9

Positive control

1693

2176

2220

222

Ethanol

107

114

22

9

 

 

Study 7961 03

 

 

TA 1535

TA 1537

TA 98

µg/plate

+S9

-S9

+S9

-S9

+S9

-S9

0

16

18

18

9

49

25

3.9

27

29

16

5

55

23

15.6

23

18

12

7

54

23

62.5

26

18

13

6

54

27

250

27

20

12

7

48

26

1000

18

11

13

5

43

28

Positive control

315

1720

102

173

2943

209

Ethanol

22

21

15

7

40

19

 

 

TA 100

TA 1538

µg/plate

+S9

-S9

+S9

-S9

0

117

119

20

13

3.9

109

109

29

11

15.6

114

92

27

12

62.5

103

99

26

10

250

95

108

18

12

1000

82

65

20

13

Positive control

1693

2176

2220

222

Ethanol

107

114

22

9

 

Conclusions:

Negative with and without metabolic activation

The test item presented no mutagenic activity with and without S9 mix in Salmonella Typhimurium in all strains.
Executive summary:

In a reverse gene mutation assay in bacteria (Battelle, 1983), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Catechol at concentrations of 0 to 1000 µg/plate in the presence and absence of rat metabolic activation system (S9 mix).

Catechol was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains.

There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not indicated
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study according to OECD guideline, but not GLP.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared from liver Rat induced with Aroclor
Test concentrations with justification for top dose:
62.5 - 125 - 250 - 500 - 1000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see test conditions
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
A preliminar test was studied with one strain : TA 100 and 10  concentrations: 0 - 1 - 5 - 10 - 50 - 100 - 500 - 1000 - 2500 - 5000  µg/plate with three replicates and without metabolic activation system. The  cytotoxic reaction was observed with three concentrations: 5000 - 2500  µg/plate (total cytotoxic action) and 1000 µg/plate (marked cytotoxic  action).

Positive controls tests used for the three  lots of catechol were:
- Sodium azide with strain TA 1535, TA 100, without S9
- Hycanthone with strain TA 1537, TA 1538, TA 98, without S9
- Ethidium bromide with strain TA 98, with and without S9.

For each concentration, the number R was calculated. R corresponds to total number of revertants for three plate divided by total number of spontaneous revertants for three plates (negative  control). 

Three validity criteria was compared with historical data: 
- the frequency of spontaneous revertant for each strain -
- S9 activity (mean of 3 plates)
-frequency of revertants induced by references products.
Evaluation criteria:
When R is less than 2, the substance is considered as non mutagenic. When R is superior to 2 and follow a relationship dose-effect, the substance is considered as mutagenic.
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 1000 µg/plate
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
For all the lots of catechol tested, with and without metabolic activation, the frequency of revertants is less than 2.
Conclusions:


Negative with and without metabolic activation in Ames test.
Executive summary:

In a reverse gene mutation assay in bacteria (Cordier et al., 1983), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Catechol at concentrations of 0 to 1000 µg/plate in the presence and absence of mammalian metabolic activation.

Catechol was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains.

There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Positive control used for TA 98 strain without S9 is not the one recommended by the OECD guidelines. Furthermore 2-AA should not be used as the sole indicator of the efficacy of the S9 Mix
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
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
Metabolic activation:
with and without
Metabolic activation system:
S9 RLI = induced male Sprague Dawley rat liver; S9 HLI = induced male Syrian hamster liver S9
Test concentrations with justification for top dose:
Lab. 1: 0 - 11 - 35 - 104 - 333 - 1000 µg/plate
(0 - 0.1 - 0.32 - 0.94 - 3.02 - 9.08 µmol/plate)
Lab. 2: 0 - 33.3 - 100 - 333.3 - 1000 - 3333.3 µg/plate
(0 - 0.30 - 0.91 - 3.03 - 9.08 - 30.27 µmol/plate)
Vehicle / solvent:
Water (Lab. 1) and DMSO (Lab. 2).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see details below
Details on test system and experimental conditions:
DETAILS ON TEST SYSTEM AND CONDITIONS:
METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours
- Nb of replicates: 3

Positive controls:
2-Aminoanthracene (2-AA) (concentrations ranging from 0.75 to 1.5 µg/plate) was tested on all strains in the presence of rat and hamster S-9. 
4-nitro-o-phenylediamine (NOPD) was tested on TA 98 without S9 (5 - 12 µg/plate). 
Also without S9, sodium azide (SA) was tested on TA 100 and TA 1535 (1.0 - 2.5 µg/plate), and 9-aminoacridine (9-AAD) was tested on TA 1537 (50 - 80 µg/plate). 
The actual concentration for each positive control chemical used for each strain and activation condition was selected by the individual laboratory based on dose response curves generated at the beginning of the testing program.
Evaluation criteria:
A positive response was indicated by a reproductible, dose-related increase, whether it be two-fold over background or not.
Statistics:
No data
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:
cytotoxicity
Remarks:
(-S9): 3333.3 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Study 181204, vehicle : water

 

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

10% RLI
(Negative)

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

19

2

7

1.5

11

3.2

7

1.8

11

1

6

0.6

11     

13

2.7

8

1.9

9

1.2

8

1.8

7c

2.5

5

1.9

35     

13

1.2

13

0.6

10

0

8

1.3

11

0.3

5

1.2

104     

14

2.1

11

2.4

11

1.5

7

0.7

8

1.5

11

0.9

333     

9

1.5

5s

0.9

10

2.7

8

0.6

12

1.7

9

2

1000     

8s

0.9

4s

0.9

15

1.2

13

3

16

2.1

12

2.1

Positive Control

1133

5.6

783

22.9

154

10.4

163

18.8

84

8.7

129

8.8

Strain: TA100

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

10% HLI
(Equivocal)

10% RLI
(Negative)

10% RLI
(Negative)

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

67

6.5

92

3.4

91

6.8

53

2.2

89

4.4

55

3.3

11     

72

2.5

96

4.2

86

2.2

68

2.8

78

2.6

55

1.2

35     

78

4.9

89

4.3

78

5.3

72

7.1

84c

8

59

4

104     

80

7.4

110

4.4

76

2

78

3.4

92

2.5

60

4.5

333     

87

13

89s

6.4

86

8.1

85

8.6

84

3.5

59

2.1

1000     

53s

10.5

81s

0.9

84

10.1

73

5.2

92

5.8

66

6.2

Positive Control

1557

21.4

1383

57

2698

105

2080

121

2068

82

1232

22.9

Strain: TA98

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

10% RLI
(Negative)

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

17

3

15

0.6

25

2.6

17

2.8

22

3

10

0.7

11     

14

2.3

17

1.5

27

3.3

14

2.9

25

4.7

10

2

35     

13

3.1

15

3.5

21

2

21

3.5

20

3

13

2.3

104     

12

2.2

14

0.7

22

1.3

17

2.3

27

2.8

7

0.6

333     

10s

2

6s

0.3

22

0.7

19

3

23

2.7

8

2.6

1000     

6s

0.9

0s

0

27

1.8

13

0.7

19

2

13

1.9

Positive Control

1628

35.5

1789

26.9

1926

74.1

2237

91.1

1429

29.7

1316

37

 

Strain: TA1537

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

10% RLI
(Negative)

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

6

0.7

3

0.3

11

2.7

7

0.9

4

1.3

3

1

11     

7

1.2

4

1

7

0.7

4

0.9

5

2.3

4

1

35     

5

0.6

5

1.2

6

0.7

5

2.6

5

1.5

3

0.9

104     

6

1.2

5

1.2

8

2.6

5

2

5

0.3

3

0.9

333     

6

1.7

3s

1.2

7

0.3

4

1.5

6

1.2

4

0.7

1000     

4s

1

4s

0.7

7

1.5

5

2.2

8

1.2

2

0.9

Positive Control

406

45.3

369

24

136

5.8

140

5.6

81

5.9

71

6.7

 

Study 113194, Vehicle : DMSO.

Strain: TA1535

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

10% RLI
(Equivocal)

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

24

1.8

25

6

16

0.7

14

0.9

17

2.2

12

2.9

2.5   

24

4.2

 

 

 

 

 

 

 

 

 

 

33.3   

 

 

22

1.3

 

 

13

2.9

 

 

15

0.9

100     

26

2.8

20

3.5

15

1.5

14

2.5

17

5

10

1.7

333.3   

21

2.2

18

0.3

14

3.9

13

2.1

12

2.3

16

0.3

1000     

12s

2.6

5s

1.3

13

2.3

16

1.9

20

4.8

23

4

3333.3   

t

 

t

 

29

4.1

20

3.2

19

3.5

20

2

6666.7   

t

 

 

 

11s

3.3

 

 

17

2.1

 

 

Positive Control

407

15.2

438

5.3

286

12

367

6.2

264

6.8

334

51.3

s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; T = Toxic; c = Contamination
Conclusions:

Negative with and without metabolic activation

Regarding results and applying actual criteria evaluation, the test item presented no mutagenic activity with and without S9 mix in Salmonella Typhimurium in all strains.
Reproductibility between the experiment and positive control results in accordance with positive criteria, validated the study.
Executive summary:

In a reverse gene mutation assay in bacteria (Haworth et al., 1983), strains TA 98, TA 100, TA 1535, TA 1537 of S. typhimurium were exposed to Catechol at concentrations of 0 to 3333.3 µg/plate in the presence and absence of mammalian metabolic activation.

Catechol was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains.

There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well described. Unusual E. coli strains used. No details about controls.
Principles of method if other than guideline:
WP2 Mutoxitest
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
E. coli, other: WP2uvrA/pKM101; IC203
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate was prepared from induced rats.
Test concentrations with justification for top dose:
At least 5 doses, but only highest doses are reported (with no observable toxicity): 1000 - 2000 - 3000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Remarks:
t-butyl hydroperoxide
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: menadione
Details on test system and experimental conditions:
- Tester strain: WP2uvrA/pKM101 (IC188) carries the trpE65 ochre mutation and is widely used. A new tester strain IC203, a derivative of WP2uvrA/pKM101 deficient in the OxyR function was also used. The OxyR deficiency prevents the oxidative stress-induced synthesis of antioxydant enzymes: this defect is assumed to determine an enhancement in the mutagenesis resulting from DNA lesions caused by reactive oxygen species (ROS).

- Plate Reverse Mutation assays: plate incorporation assays were performed by mixing 100 µL of overnight grown culture of each tester strain, 100 µL of a suitable dilution of catechol, 50µL of S9 when indicated and 2.5 mL of molten top agar. The plate were incubated 2 days at 37°C.  * Concentrations and replicates: Catechol was tested at least twice with at least 5 dose levels, including a toxic dose, and the means of Trp+ revertants per plate are presented. 

- Cytotoxicity tests were performed.
Species / strain:
E. coli, other: WP2uvrA/pKM101; IC203
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: At the dose of 2000 µg/disc, the inhibition (millimeters) was 6 mm for IC188, 12 mm for IC203 and 7 mm for IC 203+S9.
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
- Catechol was found to be an oxidative mutagen, the mutagenicity response being positive in the IC203 strain and negative in the IC188 strain.
- Mutagenesis by catechol is completely inhibited by S9.
- Cytotoxic effects: IC203 is more sensitive to the toxic effect than IC188 and the presence of S9 had a protective effect.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: Escherichia coli; WP2uvrA/pKM101; IC203

Table of results:

 

Dose (µg/plate)

Number of revertant per plate

 

IC188

IC203

IC203 + S9

0

145

144

151

1000

124

241

158

2000

124

414

150

3000

94

793

171

Menadione (30µg)

130

957

138

 

Conclusions:

Positive without metabolic activation
Executive summary:

A plate mutation reverse assay was performed with Escherichia coli WP2uvrA/pKM101; IC203 and ICI 188. The bacteria were exposed to at least 5 doses of catechol, but only highest doses are reported (with no observable toxicity):1000 - 2000 - 3000 µg/plate with and without metabolic activation system from rat liver induced. Catechol was found to be an oxidative mutagen, the mutagenicity response being positive in the IC203 strain and negative in the IC188 strain. Mutagenesis by catechol is completely inhibited by S9. Cytotoxic effects: IC203 is more sensitive to the toxic effect than IC188 and the presence of S9 had a protective effect.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Even if the GLP are not stated, the test conditions are similar to the EC and OECD guidelines. No metabolic activation and no positive control were used.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Metabolic activation system:
none
Test concentrations with justification for top dose:
2.5 - 4 - 5.5 - 7 - 8.5 µg/mL
Details on test system and experimental conditions:
CELLS:
tk+tk- 3.7.2.C heterozygote of L5178Y mouse lymphoma cells, obtained from Dr. D. Clive, burroughs Wellcome Company, Research Triangle Park, NC.
Samples were cultured and used for up to 3 months, then discarded.
Laboratory cultures were confirmed as free of mycoplasma and maintained in Fisher's medium at 37°C. Fisher's medium (Fo) was supplemented with 2 mM L-glutamine, sodium pyruvate, 0.05% pluronic F68, antibiotics, and 10% heat-inactivated donor horse serum (designated as F10P).

EXPOSURE:
Each exposed culture consisted of 6 x 10E6 cells in a final volume of 10 mL F5P (F10P, but contaning only 5% serum). Superoxide dismutase (SOD) or catalase was added, to give a concentration of 100 units/mL.
The tubes were incubated for 4 h, sedimented by centrifugation (500g for 10 min), washed, and finally resuspended in 20 mL F10P.
These cell suspensions (3 x 10E5 cells/mL) were incubated for 2 days. After 48 h, cell density was adjusted to 2 x 10E5 cells/mL.

CLONING EFFICIENCY:
0.1 mL sample of the cell suspension was withdrawn and diluted 1:100.
Three 0.1-mL samples of the diluted cultures were mixed with 25 mL cloning medium (F20P) containing 0.35% Noble agar, and poured into Petri dishes. Each plate contained 200 cells.

MUTANT SELECTION:
3 aliquots (10E6 cells) of the remaining culture were mixed with 20 mL F20P to give a final concentration of 0.35% Noble agar and 3 µg trifluorothymidine/mL, then poured into Petri dishes.

INCUBATION:
The agar was gelled at 4°C for 5-10 min, then the plates were incubated for 11-14 days on 5% CO2:95% air at 37°C.

Colonies were counted with an automated colony counter.

CALCULATIONS:
* Relative Total Growth (RTG) = (total suspension growth x cloning efficiency) in dosed culture / (total suspension growth x cloning efficiency) in control culture

* Mutation Fraction (MF) = 200 x (mutant number per plate / viable number per plate)
Evaluation criteria:
A significant response was defined as a 2-fold increase in MF at 1 of the 3 highest dose levels, where the RTG was > 1% and mutant number was increase over the vehicle control value.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2.5 µg/ml
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
- The lowest effective dose producing cytotoxic effect in the absence of metabolic activation was the lowest dose tested, i.e. 2.5 µg/ml. The mutagenic effects of catechol were not dose dependent.
- The mutagenic potential of catechol was completely negated by coincubation with SOD. It was noticeable that SOD had little effect upon toxicity.

Table of results:

 

Doses (µg/ml)

CE

RTG

MC

MF

Fisher’s medium(F0P)

72

106

92

43

F0P

103

94

84

27

2.5

40

30

650

543

4

55

6

888

543

5.5

55

9

707

433

7

43

7

675

529

8.5

64

9

866

455

 

CE: Cloning Efficiency

RTG: Relative total growth

MF: Mutation Fraction

 

Conclusions:

Positive without metabolic activation
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well described.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Metabolic activation system:
none
Test concentrations with justification for top dose:
1.145 - 2.874 - 5.516 - 11.450 - 28.736 µg/mL
(10.4 - 26.1 - 50.1 - 104 - 261 µM)
Details on test system and experimental conditions:
DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time: at the end of the expression period 3.4 x 10E6 cells from each treatment tube were centrifugated and resuspended in 1 ml of remaining medium by vigorous pipetting.

SELECTION AGENT (mutation assays): trifluorothymidine. It was added to the three undiluted cell suspensions to a final concentration of 1 µg/ml.

OTHER:
Following the 4 hours treatment with catechol, the cells were resuspended in Fisher's medium containing 10 % horse serum to which Hepes was added and the concentration of NaHCO was reduced to 0.8 g/L.
Catechol was tested up to a concentration that reduced total growth to 10-20 % of the solvent control.
The cells were cloned by adding 3.3 ml 2% molten agar (maintained at 45°C) to give a final concentration of 0.2% in each tube, which was vortexed directly and poured onto 90-mm Petri dishes and allowed to solidify.
Colonies formed on the plates, containin the selective agent, were counted manually and those without using an Artek 880 automatic colony counter.

The spontaneous mutation frequency was 76 +/- 25 x 10E6 cells.
Evaluation criteria:
A 2-fold or greater increase in mutation frequency, at 10% or higher total growth, as the criterion for a positive result.
Statistics:
A pairwise two-tailed Student's t test was performed on each set of treated replicates versus the corresponding solvent control replicates.
The statistical analyses were performed using the SAS computer programming package (1985).
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Catechol was found to increase the mutation frequency without metabolic activation.

Table of results:

 

Doses (µg/ml)

S9 mix

Total growth (a)

Mutation frequency (b)

Mutation index (c)

0

-

 

73/79

 

1.145

-

12

636***

8.4

2.874

-

10

558***

7.4

5.516

-

9

560***

7.4

11.45

-

8

614***

8.1

28.736

-

5

725***

9.6

(a) : Suspension growth x cloning efficiency

(b): Mutants/10E6 surviving cells

(c): Mutation frequency of treated culture/average mutation frequency of control cultures

***: .0001< P <= 0.001

Conclusions:

Positive without metabolic activation
Executive summary:

In this Mouse lymphoma assays, mouse lymphoma L5178Y cells were tested without metabolic activation system at doses of 1.145 - 2.874 - 5.516 - 11.450 - 28.736 µg/mL for 4 hours with 48 hours expression time.

Catechol was found to increase the mutation frequency in a non dose dependent manner.

Endpoint:
in vitro transformation study in mammalian cells
Remarks:
Type of genotoxicity: other: cell transformation
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The test was not performed with metabolic activation. The purity of the substance is unknown. Negative control group (untreated culture) was used but positive control groups are not mentioned.
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.21 (In Vitro Mammalian Cell Transformation Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
other: cell transformation
Species / strain / cell type:
mammalian cell line, other: Syrian Hamster Embryo (SHE) cells
Metabolic activation:
without
Metabolic activation system:
none
Test concentrations with justification for top dose:
0.11- 0.33 - 1.1 - 3.3 µg/mL (1 - 3 - 10 - 30 µM)
Vehicle / solvent:
none
Details on test system and experimental conditions:
SHE cell cultures were established from 13-day-gestation foetuses collected from inbred Syrian hamster, strain LSH/ss LAK. The cell culture medium (complete medium) used was IBR Dulbecco's modified Eagle's reinforced medium supplemented with 10 % foetal bovine serum.
Catechol was first dissolved in serum free culture medium to give the concentration of 10 mM. Aliquots of this freshly made solution were added to the culture to give the appropriate final concentration.

Cell growth: Cells in logarithmic phase were plated in triplicate, and after overnight incubation, treated at 1 -100 µM catechol for 48 h.

Cell transformation: 
Cells were plated, and after overnight incubation, treated at 1 -100 µM catechol for 48 h. 3000 cells were plated on each of 20 dishes per group and incubated for 7 days for colony formation. The cells were fixed with absolute methanol and stained with 10 % Giemsa solution. The number of surviving colony and morphologically transformed colonies were scored by criteria established by Berwal and Sachs, 1965.
Species / strain:
mammalian cell line, other: Syrian Hamster Embryo (SHE) cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 10 µM (cell growth inhibition)
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
- Catechol induced morphological transformations of SHE cells at concentrations of 1-30 µM. The frequency of transformation increased with increasing dose of catechol.
- Catechol at the concentration of 10 µM decreased cell survival to 28.8 % of untreated cells and the percentage of surviving  cell was 1.4% of untreated cells at 30µM catechol.

Relative cell survival and frequency of morphologically transformed colonies by treatment of SHE cells with Catechol for 48h:

Test compound

Dose (µM)

Percent of survivala

No. of colonies scoredb

No. of transformed colonies

Percent of transformation (c)

Catechol

1

85.2 ± 6.5

4160

10

0.24e

3

70.2 ± 5.2

3426

11

0.32e

10

28.8 ± 3.8

1404

12

0.85e

30

1.4± 0.5

68

2

1.26e

aCalculated by using untreated cultures as a control. The actual colony forming efficiency of the cells was11.05± 0.61 (SD)%.

bTotal number of colonies per twenty 100-mm dishes

c(Total No. of morphologically transformed colonies / Total No. colonies scored)x100

ep<0.01 as compared to untreated cultures (x² test)

Conclusions:

Positive without metabolic activation in cell transformation test with syrian hamster embryo cells (SHE cells).
Executive summary:

Syrian Hamster Embryo (SHE) cells were treated with catechol concentration of : 0.11-0.33- 1.1 - 3.3 µg/mL (1 - 3 - 10 - 30 µM) without metabolic activation system. The cells were fixed with absolute methanol and stained with 10 % Giemsa solution. The number of surviving colony and morphologically transformed colonies were scored. Catechol induced morphological transformations of SHE cells at concentrations of 1-30 µM. The frequency of transformation increased with increasing dose of catechol. Catechol at the concentration of 10 µM decreased cell survival to 28.8 % of untreated cells and the percentage of surviving cell was 1.4% of untreated cells at 30µM catechol.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well described, but no details about controls.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
livers (300 µl/ml S9 mix) from Aroclor 1254-pre-treated Fisher male rats
Test concentrations with justification for top dose:
50 µg/mL (454 µM)
Vehicle / solvent:
MEM supplemented with 2.5 % fœtal calf serum at 37°C, pH 7.4.
Details on test system and experimental conditions:
The aim of this study was to examine the capacity of catechol to induce chromatid breaks and exchanges in CHO cells. In addition, the modulating effect of Cu2+ and Mn2+ was examined.

Cell culture:
CHO cells were grown in Eagle's Minimal Essential Medium (MEM) supplemented with 10% foetal calf serum, antibiotics and sodium bicarbonate. The stock cultures were maintained at 37°C in a water-saturated CO2 incubator.
Approximately 140 000 CHO cells were seeded on 22 mm2 cover slips in 3.5 cm plastic dishes and kept in MEM with 10% foetal calf serum at 37°C for 2-3 days. Experiments were begun when cells were 40-60% confluent. The tissue culture medium was removed and replaced by 1 ml of the catechol solution.
- Exposure time was 3 hours.

- For estimating the frequency of chromosome aberrations, 0.1 mL of colchicine was added at 16 h post exposure to the chemicals and left for 4 hours. Cells were then treated with 1% sodium citrate solution for 20 min, followed immediately with fixation in ethanol/acetic acid (3:1) for 20 min.
For each sample, 200 metaphase plates were analysed for chromosome aberrations. The frequency of two types of chromosome aberrations was estimated: (a) chromatid breaks, (b) chromatid exchanges. Chromatid exchanges occurred between homologous and non-homologous chromosomes and between two or more chromosomes.
Solutions of catechol were prepared by dissolving the compound in 2.5% MEM at 37°C and adjusting the pH to 7.4.
The presented concentration is half the dose which induces mitotic inhibition, which is defined as one metaphase or less in 6000 cells.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 100 µg/mL (908 µM)
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
- Percentage of CHO metaphase plates with at least one chromatid break or exchange:
Controls: 0.7 %Catechol
(-S9): 23.5 %
(+S9): 2.0 %
Aflatoxine B1 +S9 induced aberration in 84.2 % of the metaphase plate.
- The average numbers of chromatid break/cell were 0.17 (-S9) and 0.00 (+S9) and the average numbers of chromatid exchange/cell were 0.67 (-S9) and 0.03 (+S9). Control values were 0.01 break/cell and 0.00 exchange/cell.
- Catechol exhibits a chromosome-damaging potential. The addition of an S9 mixture reduced the clastogenic activity of catechol.
Conclusions:
Positive without metabolic activation
Negative with metabolic activation

Executive summary:

The capacity of catechol to induce chromatid breaks and exchanges in CHO cells were evaluated after treatment of the cells at doses of 50 µg/mL (454 µM) with and without metabolic activation system induced by Aroclor 1254-pre-treated Fisher male rats. In addition, the modulating effect of Cu2+ and Mn2+ was examined.

Catechol exhibits a chromosome-damaging potential (chromatid break or exchange). The addition of an S9 mixture reduced the clastogenic activity of catechol.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well described, no details about controls.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
mammalian cell line, other: Syrian Hamster Embryo (SHE) cells
Metabolic activation:
without
Metabolic activation system:
none
Test concentrations with justification for top dose:
1 - 3 - 10 - 30 µM for chomosomal aberration test.
3 - 10 - 30 µM for aneuploidy test
Vehicle / solvent:
None
Details on test system and experimental conditions:
- SHE cell cultures were established from 13-day-gestation foetuses collected from inbred Syrian hamster, strain LSH/ss LAK. The cell culture medium (complete medium) used was IBR Dulbecco's modified Eagle's reinforced medium supplemented with 10 % foetal bovine serum.
Catechol was first dissolved in serum free culture medium to give the concentration of 10 mM. Aliquots of this freshly made solution were added to the culture to give the appropriate final concentration.
-Cell growth: Cells in logarithmic phase were plated in triplicate, and after overnight incubation, treated at 1 -100 µM catechol for 48 h.

- Chromosome aberration and chromosome number:
Cells were plated, and after overnight incubation, treated with catechol for 6, 24 or 48 h. Three hours before the end of treatment time, colcemid was administered at 0.2 µg/ml, and metaphase chromosomes were prepared. For determination of both chromosome aberration (gaps, breaks, exchanges, dicentrics O-ring, and fragmentation) and chromosome number, 100 metaphases per experimental group were scored.
Statistics:
Statistical analysis was performed by x² test.
Species / strain:
mammalian cell line, other: Syrian Hamster Embryo (SHE) cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 10 µM
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
mammalian cell line, other: SHE cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 10 µM
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
CHROMOSOMAL ABERRATION TEST:
- 6 h: Few chromosome aberrations were exhibited when the cells were treated for 6 h with catechol (data not shown, not significative at all concentrations).

- After 24 h, statistical analysis by the Pearson chisquare test or a trend test showed that a dose-dependant increase in the frequencies of chromosomal aberrations (mainly gaps and breaks) in SHE cells was induced by catechol at doses of 3, 10, and 30 µM.

- After 6, 24 or 48 h, no effects were observed at the dose of 1 µM.

ANEUPLOIDY TEST:
- Slight aneuploidy in the near diploid range of SHE cells was significantly induced by catechol (30 µM, 48 h). No significant increase was observed at 3 and 10 µM.
- No significant increase in the number of metaphases with tetraploid and a near tetraploid number of chromosomes was induced by treatment for 24 and 48 h catechol.(94 % of the 300 metaphases from untreated cultures had a diploid number of chromosomes, but 4.2 were in the tetraploid and near tetraploid range. 1.8 % were in the near diploid range or other).

- Catechol at the concentration of 10 µM decreased cell survival to 28.8 % of untreated cells.

Table of results:

Dose (µM)

Type of aberration

Aberrant metaphases (%)

G*

B

Ex

D

O

F

1

2.5

1.5

0

0

0

0

3.5

3

11

2

0

0

0

0

13b

10

11

4

0

0

0

0

15c

30

29

19

0

0

0

0

42c

*G : gaps ; B : breaks ; Ex : exchanges ; D : dicentric ; O : O-ring ; F : fragmentation

b: p<0.05 as compared to untreated cultures (x² test)

c: p<0.01 as compared to untreated cultures (x² test)

Conclusions:

Positive without metabolic activation in chromosomal aberration test with SHE cells.
Negative without metabolic activation in aneuploidy test with SHE cells.
Executive summary:

Syrian Hamster Embryo (SHE) cells were treated with catechol at doses : 1 - 3 - 10 - 30 µM for 6, 24 or 48 h, for chromosome aberration or aneuploidy and without metabolic activation system. After 24 h, a dose-dependant increase in the frequencies of chromosomal aberrations (mainly gaps and breaks) in SHE cells was induced by catechol at doses of 3, 10, and 30 µM. Slight aneuploidy in the near diploid range of SHE cells was significantly induced by catechol (30 µM, 48 h). No significant increase was observedat 3and 10 µM.

 

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Purity of the test substance is unknown. It's unclear wether all doses have been tested with and without metabolic activation for each pH value.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
Principles of method if other than guideline:
This study deals with the induction of chromosomal aberrations by phenol, catechol and pyrogallol in V79 cells at different pH values (6.0, 7.4 and 8.0). At the same pH values, the production of hydroxyl radicals was assessed by measuring the degradation of deoxyribose.
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
From Wistar rat liver induced with Aroclor 1254
Test concentrations with justification for top dose:
0 - 20 - 40 - 60 - 80 µM
pH : 6.0 - 7.4 - 8.0
Metabolic activation at pH 7.4
Vehicle / solvent:
no data
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Cyclophosphamide (+S9mix); Mitomycine C (-S9mix)
Details on test system and experimental conditions:
In order to clarify the involvement of radical species in the genotoxicity of catechol, the following studies were carried out:
1. induction of chromosomal aberrations in V79 cells
2. production of hydroxyl radicals at different pH values (6.0, 7.4 and 8.0)

1. Chromosomal aberration assay:
* V79 cells were cultured in 5 mL Ham's F-10 medium supplemented with 10% new born calf serum and 1% Fungibact solution, and incubated at 37°C under one atmosphere of 5% CO2. 22 hour cultures (approx. 106 cells) were washed with Ham's F-10 medium reconstituted in phosphate buffer 0.01 M, pH 6.0, 7.4 or 8.0 and grown in 5 mL of this medium for 2h in the presence of the compounds studied. The pH was checked after the 2h in control cultures with no added mutagen and shown to be 6.0, 7.1 and 7.3, respectively.
* For positive controls, 0.5 µg/mL mitomycine C (Sigma) was used.
* When metabolic activation was required, 500 µL of S9mix (10% v/v) and 4.5 mL of medium were used. 42.9 µM of cyclophosphamide was used as positive control in the experiments in presence of S9mix.
* After the treatment cells were washed with culture medium and grown for another 16h, Colchicine was added at a final concentration of 0.56 µg/mL, and cells were grown for a further 3h. cells were then harvested by trypsinisation. After 2min hypotonic treatment with KCl (0.56%, w/v) at 37°C, cells were fixed with methanol/acetic acid (3:1) and slides were prepared and stained with Giemsa (4% in phosphate buffer 0.01M pH 6.8) for 10min. Two independant experiments were carried out with each test compound and 100 metaphases were scored for each dose-level treatment group in each experiment. The cytotoxicity of each compound was assessed by calculating the mitotic indices (MIs) (% of metaphases in 2000 cells). When the dose tested induced a decrease in the MI greater than 75% when compared with the control, it was considered to be cytotoxic.

Induction of chromosomal aberrations in the presence of SOD and catalase:
These experiments were performed as described previously for the dose of 80 µM and at pH 7.4. SOD and catalase were added to the cultures during the 2h treatment period with the coumpound studies. Quantities of SOD and catalase (74.6 U and 265 U respectively) corresponded to the activities of SOD and catalase present in 500µL of S9mix. Experiments with catalase and SOD alone were also performed.

2. production of hydroxyl radicals at different pH values (6.0, 7.4 and 8.0)
Hydroxyl radicals were measured by the deoxyribose assay. Briefly, 1.2 ml of a reaction mixture composed of 10 mM potassium phosphate buffer, pH 6, 7.4 and 8, Catechol at 27 µM, deoxyribose (2.8 mM), FeCl3 (20 µM) and EDTA (100 µM), was incubated for 2 h at 37°C.
Hydrogen peroxide was used as positive control for hydroxyl radicals generation.
The deoxyribose degradation by hydroxyl radicals was measured by the TBA method using 1 ml of trichloroacetic acid (2.8%) and 1 ml of TBA (1%) in 0.05 M NaOH. The mixture was incubated at 100°C for 15 min, cooled and the absorbance measured at 532 nm.
Negative controls were performed: iron plus EDTA and no polyphenol, without iron and EDTA, and the controls without phenolic compound as well the values for the controls were subtracted in each experiment (6 in total).
Statistics:
The statistical analysis of differences in chromosomal aberration frequency in the different experiments was carried out using the t-test. All analyses were performed with an SPSS statistical package (version 10.5) (SPSS Inc., Chicago, IL.)
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid

The clastogenic effect of catechol is dependent on the pH: non-significant increase in chromosomal aberrations at pH 6.0, at any dose-level; at pH values 7.4 and 8.0, catechol induced significant levels of chromosomal aberrations at lower doses (< 80 µM); at higher doses and in a pH-dependent manner, it showed a significant induction of multi-aberrant cells (with more than 10 chromosomal aberrations), which represent in some cases more than 50% of the aberrant cells.

Experiments carried out at pH 7.4 in the presence of S9mix with 80 µM dose show that the genotoxicity of catechol is not inhibited by the addition of S9mix, SOD, catalase and SOD+catalase, suggesting that superoxyde anion and hydrogen peroxide are not associated with the genotoxicity of catechol. However, SOD+catalase seem to decrease the levels of multi-aberrant cells, when compared with the results obtained in the absence of this enzymatic system.

Values and comparison regarding

The ability of catechol to generate hydroxyl radicals was measured by the deoxyribose assay, at different ph values in the presence of Fe3+/EDTA or in the presence of Fe3+: catechol can generate OH° radicals in a pH-dependent way and this effect is more pronounced in the presence of FE3 +/EDTA.

Conclusions:

Positive with and without metabolic activation

Catechol is able to induce chromosomal aberrations in V79 cells. In this cell line, and with this experimental parameters, formation of reactive oxygen seems to be only one part of the mechanism involved in the clastogenic activity. Results of exposure with Catechol + S9mix, catalase + SOD, and catalase or SOD alone do not lead to a significant reduction on the level of chromosomal aberration induced by the catechol alone.
Since the addition of S9mix and SOD+catalase leads to a reduction in the number of multi-aberrant cells and since it is observed that Catechol can produce OH° radicals, a participation of a radical-type mechanism can not be excluded in the genotoxicity of catechol.
Executive summary:

Syrian Hamster Embryo (SHE) cells were treated with catechol at doses: 1 - 3 - 10 - 30 µM for 6, 24 or 48 h, for chromosome aberration or aneuploidy without metabolic activation system. After 24 h, a dose-dependant increase in the frequencies of chromosomal aberrations (mainly gaps and breaks) in SHE cells was induced by catechol at doses of 3, 10, and 30 µM. Slight aneuploidy in the near diploid range of SHE cells was significantly induced by catechol (30 µM, 48 h). No significant increase was observedat 3and 10 µM.

In the study of Do Ceu Silva (2003), the induction of chromosomal aberrations by catchol in V79 cells was studied at different concentration 0 - 20 - 40 - 60 - 80 µM, pH values (6.0, 7.4 and 8.0 with and without metabolic activation system from Wistar rat liver induced with Aroclor 1254 at pH 7.4. At the same pH values, the production of hydroxyl radicals was assessed by measuring the degradation of deoxyribose.The clastogenic effect of catechol is dependent on the pH: non-significant increase in chromosomal aberrations at pH 6.0, at any dose-level; at pH values 7.4 and 8.0, catechol induced significant levels of chromosomal aberrations at lower doses (< 80 µM); at higher doses and in a pH-dependent manner, it showed a significant induction of multi-aberrant cells (with more than 10 chromosomal aberrations), which represent in some cases more than 50% of the aberrant cells.

S9mix, catalase + SOD, and catalase or SOD alone do not lead to a significant reduction on the level of chromosomal aberration induced by the catechol.

The addition of S9mix and SOD+catalase lead to a reduction in the number of multi-aberrant cells and it was observed that it can produce OH° radicals, so a participation of a radical-type mechanism can not be excluded in the genotoxicity of catechol.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The test was performed without metabolic activation. The purity of substance is unknown. Negative control group (untreated culture) was used but positive control groups are not mentioned. Positive responses were obtained only at cytotoxic concentrations, thus making these results not relevant.
Qualifier:
according to guideline
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay in mammalian cells
Species / strain / cell type:
mammalian cell line, other: Syrian Hamster Embryo (SHE) cells
Metabolic activation:
without
Metabolic activation system:
none
Test concentrations with justification for top dose:
0.11- 0.33- 1.1 - 3.3 - 11 µg/L (1 - 3 - 10 - 30 - 100 µM)
Vehicle / solvent:
none
Details on test system and experimental conditions:
* SHE cell cultures were established from 13-day-gestation fetuses collected from inbred syrian hamster, strain LSH/ss LAK. The cell culture medium (complete medium) used was IBR Dulbecco's modified Eagle's reinforced medoium supplemented with 10 % fetal bovine serum.
Catechol was first dissolved in serum free culture medium to give the concentration of 10 mM. Aliquots of this freshly made solution were added to the culture to give the appropriate final concentration.

Cell growth: Cells in logarithmic phase were plated in triplicate, and after overnight incubation, treated at 1 -100 µM catechol for 48 h. 

SCE experiment: 
Cells were plated overnight and treated with catechol in presence of BrdU (10 µg/ml) under dark conditions. Three hours before the end of treatment, colcemid was added at 0.2 µg/ml and metaphase chromosomes were prepared by the air drying method. The differential staining of sister chromatids was performed according to a modification of the fluorescence-plus-Giemsa technique. 30 second-division metaphases with the diploid number of chromosomes were analysed for SCE frequency.
Similar experiments were performed 2 or 3 times, and the resulst obtained were reproducible. 
Statistics:
Statistical analysis was performed by Chi-2 test.
Species / strain:
mammalian cell line, other: Syrian Hamster Embryo (SHE) cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 10 µM
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
- Negative results were obtained at 1 and 3 µM. The quantity of SCEs/cell  was not statistically different from that of control (8.37).
- SCE in SHE cells occured with catechol at 10 µM (11.06 SCEs/cell) and  30 µM (15.40 SCEs/cell).  At 100 µM, catechol was overly toxic to obtain SCEs data.
- Catechol at the concentration of 10 µM decreased cell survival to 28.8 % of untreated cells. Cytotoxicity was marked at 30 µM and 100 µM dose dependant.
Conclusions:


positive without metabolic activation in SCE test with syrian hamster embryo cells (SHE cells).
Executive summary:

Syrian Hamster Embryo (SHE) cells were treated without metabolic activation system with catechol at doses of 0.11- 0.33- 1.1 - 3.3 - 11 µg/L (1 - 3 - 10 - 30 - 100 µM) in presence of BrdU. Three hours before the end of treatment, colcemid was added and metaphase chromosomes were prepared. The diploid number of chromosomes were analysed for SCE frequency.

Similar experiments were performed 2 or 3 times, and the results obtained were reproducible. 

Negative results were obtainedat 1and 3 µM. SCE in SHE cells occured with catecholat 10µM (11.06 SCEs/cell) and 30 µM (15.40 SCEs/cell). At 100 µM, catechol was overly toxic to obtain SCEs data. Catchol was also cytotoxic in dose dependent mannerat 10and 30 µM

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study not GLP. Only tested with metabolic activation system, only one donor of lymphocytes.
Principles of method if other than guideline:
Cytokinesis-block micronucleus asay/ anti-kinetochore antibody technique.
This method uses addition of cytochalasin B to cultures in order to block cells in cytokinesis. Such treatment results in multinucleated cell formation. Only binucleated interphase identified and scored for the presence of micronuclei; In adapted from [Eastmond DA and Tucker JD (1989) Environ. Mol. Mutagen., 13, 34-43] to distinguish micronuclei that have a high probability of containing only chromosomes fragments in order to identify the relative potential for induction of aneuploidy and clastogenicity of the chemical. The use of an anti-kinetochore antibody indicated the occurence of numerical as well as structural chromosome aberrations.
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
without
Metabolic activation system:
none
Test concentrations with justification for top dose:
0.5, 5, 50, 100, 200 and 250 µM.
Vehicle / solvent:
none
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
no
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: colchicine (0.025 to 0.1 µM)
Remarks:
Other controls: phospahate buffered saline (PBS)
Details on test system and experimental conditions:
* Lymphocytes were isolated from the blood of the same male adult. Lymphocytes were isolated on Ficoll-Plaque density gradients and were cultured at 37°C for 72h in 5% CO2 atmosphere at initial density of 0.5x10E6 cells/mL. Culture medium consisted of RPMI 1640 supplemented with 2 mM L-glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin, 10% fetal bovine serum, and 1.5% phytohemagglutinin.
Cyclochalasin B was added at 44h incubation and cells were harverested onto slides at 72h.
For treatments, chemicals were solubilized in phosphate-buffered saline and added in a total volume of 15 µg/mL of culture 24h after culture initiation. Cells were harvested after 72 h.
* For micronucleus analysis: fixation of slides with absolute methanol, dried, and stained with MG GIEMSA.

* Cell viability was determined by trypan blue dye exclusion.
* A minimum a 1000 binucleated cells per concentration were scored when possible (500/duplicate culture) for micronuclei, 400 cells per concentrations were scored (200/duplicate) for replicative indices.

* Antikinetochore assay was done in parallel: fixation of cells in methanol and application of antikinetochore antibody in 0.1% Tween 20. Application of fluoresceinated rabbit anti-human antibody and nucleus stained with DAPI.
The number of micronucleated cells and those containing kinetochores were scored in 1000 binucleated cells/point.
This test distinguish micronuclei that have high probability of containing a whole chromosome (kinetochore positive) from those containing acentric fragments (kinetochore negative).
Statistics:
Statistical analysis by analysis of variance
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 250 µM
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
All tested concentrations showed increase in micronuclei. Minor increases (2 to 3 fold) were observed up to 100 µM.
Increase 4-5 fold was observed at 200 µM but cell viability decrease at 200 µM (66%) and cytotoxicity was clearly observed at 250 µM (61%).
The modified micronucleus assay using antikinetochore antibody:
Statistical significant increase in micronucleated cells were observed for Catechol.
Significant dose-related increases in kinetochore-positive micronucleated cells were also observed, suggesting that this chemical is likely aneuploidy, inducing agents in human lymphocytes.

Table of results:

Chemical

Conc. (µM)

Total MN

Total MN cells

Total BN cells scored

Standardized per 1000 BN

No. Of cells with indicated no. of micronuclei

Controls

 

 

 

 

MN

MN cells

1

2

3

4

≥ 5

PBS

 

45

37

7800

6

5

33

1

2

1

0

Colchine

0.025

10

5

1000

10

5

3

1

0

0

1

0.05

61

39

1000

61

39

25

9

3

1

1

0.075

106

50

1000

106

50

28

9

2

6

5

0.1

177

81

1241

143

65

41

16

11

5

8

Catechol

0.5

14

12

1000

14

12

10

2

0

0

0

5

14

12

1000

14

12

10

2

0

0

0

50

15

14

1000

15

14

13

1

0

0

0

100

16

16

1000

16

16

16

0

0

0

0

200

28

25

1000

28

25

23

1

1

0

0

250

4

4

290

14

14

4

0

0

0

0

Conclusions:

Positive without metabolic activation in micronucleus test with human lymphocytes.
Executive summary:

In the study of Yager (1990), Human lymphocytes were treated without metabolic activation system with catechol at doses of 0.5, 5, 50, 100, 200 and 250 µM. Cell viability and micronuclei were checked using antikinetochore antibody. Statistical significant increase in micro nucleated cells was observed. Statistical significant dose related increase in kinetochore-positive micronucleated cells were also observed, suggesting that this chemical was likely aneuploidy-inducing agents in Human lymphocytes.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
03 april 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Micronucleus in vitro using micromethod assay. Study not GLP.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
micromethod assay
GLP compliance:
no
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, each new batch of cells
Metabolic activation:
with and without
Metabolic activation system:
liver and kidney S9 fraction
Test concentrations with justification for top dose:
Without S9, 0h: 39.06, 19.53 and 9.77 µg/ml
Without S9, 20h: 19.53, 9.77 and 4.88 µg/ml
With S9, 24h (liver S9-mix): 156.25, 78.12, 39.06 and 19.53 µg/ml
With S9, 24h (kidney S9-mix): 156.25, 78.12, 39.06 and 19.53 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: solvent usually for soluble test substance
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see details
Details on test system and experimental conditions:
Positive controls:
Without S9-mix: Mitomycin C: 0.05 µg/mL at 0h; 0.025 µg/mL at 24h
With liver S9-mix: cyclophosphamide, 10 µg/mL
With kidney S9-mix: streptozotocine: 500 µg/mL

Cell culture:
A deep freeze sample of cell containing 3 to 4*10E6 cells are resuspended in a 50 ml disposable tube with 40 ml of RPMI medium supplemented with 10% of inactivated horse serum (RPMI 10) to eliminate the DMSO added in medium to preserve cells during deep freezing.
After centrifugation (900 rpm for 5 minutes)the supernatant is discarded and replaced by 40 ml RPMI 10. Cells are resuspended using a 10 ml disposable pipette then transferred to a 80 cm² disposable flask then incubated at 37°C with 5% CO2, 95% humidity for 72 hours in order to obtain about 7 to 9*10E5 cells/ml.

METABOLIC ACTIVATION SYSTEM:
The S9 fraction was prepared from Rat male Sprague Dawley OFA of 7 to 8 weeks receieved a single intraperitoneal injection of Aroclor 1254 (origin- Monsanto, Saint-Louis, USA, batch N° KD 06-618) at dose of 500 mg/kg in corn oil solution. 5 days later, animal were sacrified by cervical rupture and exsanguinated. the livers or kidneys are removed and S9 extracted. S9 mix was prepared as follow for 5 mL:
S9: 2 mL
150 mM KCL: 1 mL
25 mg/mL NADP: 1mL
180 mg/mL Glucose-6-phosphate: 1 mL
S9 mix is diluted 10% in RPMI 10.

Treatment:
Four treatment schdules, each coupled to an assessment of cytotoxicity, are carried out:
- Two 4-hour treatment with metabolic activation followed by a 24-hour recovery period (assay +S9): one with rat liver S9-mix and the other with rat kidney,
- Two 24-hour continuous treatments without metabolic activation: one in which cells are harvested immediately after the treatment (assay +/-S9, 0h), and the other treatment is followed by a 20-hour recovery time (+/-S9, 20h).

The choice of sequences of treatment was determined following numerous kinetic studies using well-known direct and indirect acting genotoxins.

Evaluation criteria:
A test compound is considered as genotoxic in case of a concentration related increase in the number of micronucleated cells and a statistically significant increase of the spontaneous level in at least one dose in at least one treatment schedule with at least a doubling number of spontaneous micronuclei.
Statistics:
CHI2 test (Yates correction).
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
up to 19.53 µg/ml, depending of the assay
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
WITHOUT METABOLIC ACTIVATION:
In the treatment without S9-mix and without recovery period (-S9, 0h), Catechol provoked statistically significant increases in the mean number of micronuclei on L5178Y mouse lymphoma cells, at the 3 concentrations tested of 39.06, 19.63 and 9.77 µg/ml, with 17, 10 and 9 micronucleated cells/2000 mononucleated cells, respectively. A clear dose-effect relationship was observed. Furthermore, the examination of the slides revealed a weak cell density at the 3 concentrations studied as well as the presence of unusual large cells at the concentration of 9.77 µg/ml. Hence, the test compound induced a clear genotoxic activity in the assay without metabolic activation without recovery period.

In the treatment without S9-mix followed by a 20-hour recovery period (-S9, 20h), Catechol induced statistically significant increases in the mean number of micronuclei, at the highest and lowest concentrations tested of 19.53 and 4.88 µg/ml, with 18 and 12 micronucleated cells/ 2000 mononucleated cells, respectively. The intermediate concentration of 9.77 µg/ml induced a non-statistically significant increase in the number of micronuclei, with 8 micronucleated cells/ 2000 mononucleated cells, vs. 2 in the negative control.
Moreover, the examination of the slides revealed a weak cell density at the concentrations of 9.77 and 4.88 µg/ml as well as the presence of unusual large cells in the highest concentration studied of 19.53 µg/ml.

WITH METABOLIC ACTIVATION:
In the treatment with S9-mix by means of liver cells (+S9-liver mix, 24h), Catechol provoked statistically significant increases in the mean number of micronuclei on L5178Y mouse lymphoma cells, at the 4 concentrations tested of 156.25, 78.13, 39.06 and 19.53 µg/ml, with 9 micronucleated cells/1750 mononucleated cells, 21, 29 and 24 micronucleated cells/2000 mononucleated cells, respectively. Furthermore, the examination of the slides revealed a weak cell density at the 2 highest concentrations of 156.25 and 78.13 µg/ml and at the concentration of 19.53 µg/ml, as well as the presence of unusual large cells at the concentration of 78.13, 39.06 and 19.53 µg/ml. Finally, apoptotic cells were observed at the concentration of 39.06 µg/ml. Hence, the test compound induced a clear genotoxic activity in the assay with metabolic activation by means of liver S9-mix.

In the treatment with S9-mix by means of kidney cells (+S9-kidney cells, 24h), Catechol provoked statistically significant increases in the mean number of micronuclei, at the 4 concentrations tested of 156.25, 78.13, 39.06 and 19.53 µg/ml, with 18, 21, 18 and 22 micronucleated cells/2000 mononucleated cells, respectively. Furthermore, the examination of the slides revealed a weak cell density as well as the presence of unusual large cells at all the concentrations and cells with deformed nucleus at the concentrations of 39.06 and 19.53 µg/ml. Hence, the test compound induced a clear genotoxic activity in the assay with metabolic activation by means of kidney S9-mix.

Assay WITHOUT metabolic activation

24-hour continuous treatment without recovery period

24-hour continuous treatment with a 20-hour recovery period

Compound

Conc. µg/ml

% relative survival / control (MMT)

Mean number of µnucleated cells per 2000 mononucleated cells

p

Compound

Conc. µg/ml

% relative survival / control (MMT)

Mean number of µnucleated cells per 2000 mononucleated cells

p

Solvent control

0

100

2

-

Solvent control

0

100

2

-

 

Mitomycin C

0.05

122.55

33

<0.001

 

Mitomycin C

0.05

105.64

81

<0.001

Catechol

39.06

51.09

17*

<0.001

Catechol

19.53

50.77

18***

<0.001

19.53

63.04

10*

<0.05

9.77

63.42

8**

N.S.

9.77

80.98

9**

<0.05

4.88

70.01

2**

<0.01

*: Weak cell density; **: Weak cell density as well as presence of large cells.

***: Presence of large cells

Assay WITH metabolic activation

Rat liver S9

Assay WITH metabolic activation

Rat kidney S9

Compound

Conc. µg/ml

% relative survival / control (MMT)

Mean number of µnucleated cells per 2000 mononucleated cells

p

Compound

Conc. µg/ml

% relative survival / control (MMT)

Mean number of µnucleated cells per 2000 mononucleated cells

p

Solvent control

0

100

3

-

Solvent control

0

100

5

-

 

Cyclophosphamide

10

90.45

126a,**

<0.001

Streptozotocine

500

110.6

49

<0.001

Catechol

156.25

65.45

9b*

<0.05

Catechol

156.25

67.66

18**

<0.01

78.13

73.64

21**

<0.001

78.13

74.73

21**

<0.01

39.06

81.36

29***

<0.001

39.06

82.07

18x

<0.01

19.53

102.73

24**

<0.001

19.53

87.23

22x

<0.01

*: Weak cell density; **: Weak cell density as well as presence of large cells.

***: Presence of large cells

a: Mean number of micronucleated cells on 1500 mononucleated cells

b: Mean number of micronucleated cells on 1750 mononucleated cells

Conclusions:

Positive with and without metabolic activation

Under conditions of this test, Catechol induced a clear genotoxic effect on L5178Y mouse lymphoma cells, both without and with metabolic activation by means of liver or kidney S9-mix, that can be revealed by the in vitro micronucleus assay performed in micromethod, without repetition.
No significant difference in the genotoxic activity induced in presence of liver or kidney S9-mix was observed up to concentration of 19.53 µg/ml
Executive summary:

In a study (Watzinger, 2007), Catechol was tested in an in-vitro micronucleus test, without and with metabolic activation by means of liver or kidney S9 -mix on L5178Y mouse lymphoma cells, by the micromethod.

In this study, Catechol induced a clear genotoxic effect on L5178Y mouse lymphoma cells, both without and with metabolic activation by means of liver or kidney S9-mix, that can be revealed by the in vitro micronucleus assay performed in micromethod, without repetition. No significant difference in the genotoxic activity induced in presence of liver or kidney S9-mix was observed up to concentration of 19.53 µg/ml.

The positive controls induced the appropriate responses in the corresponding assays.

Catechol was positive in an in-vitro micronucleous test, with and without metabolic activation (S9 -liver mix or S9-kidney mix), using the micromethod.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well described. Purity of test substance is not mentioned.
Qualifier:
no guideline required
Principles of method if other than guideline:
To test the hypothesis that benzene and its metabolites can intiate hyper-recombination and to investigate the potential of ROS, a Chinese hamster ovary cell line containing a neo direct repeat recombination substrate (CHO 3-6) was used to determine whether benzene or its metabolites phenol, hydroquinone, catechol or benzoquinone initiated increased homologous recombination and whether this increase could be diminished by the coincubation of cells with the antioxidative enzyme catalase.
GLP compliance:
not specified
Type of assay:
other: Induction of homologous recombination
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
other: CHO recombination cell line CHO 3-6
Metabolic activation:
without
Metabolic activation system:
none
Test concentrations with justification for top dose:
1-10-30-100 µM
Vehicle / solvent:
- Vehicle(s) used: alpha-minimum essential media
- Justification for choice of solvent: cells were maintained in α-minimum essential media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in 5% CO2.
Untreated negative controls:
yes
Remarks:
cells not exposed to Catechol (dose = 0µM)
Negative solvent / vehicle controls:
yes
Remarks:
α-minimum essential media
True negative controls:
no
Positive controls:
no
Positive control substance:
no
Details on test system and experimental conditions:
The (CHO 3-6) cells have a single stably integrated tandem repeat neo recombination substrate, which upon homologous recombination confers resistance to the antibiotic Geneticin® G418. They were maintained in α-minimum essential media supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin at 37°C in 5% CO2.

Recombination studies:
Homologous recombination frequency was determined by plating "CHO 3-6" cells at a density of 10(6) per 10-cm culture dish. For the determination of plating efficiency (cell survival), cells were plated at a density of 300 per 10-cm dish. After 5h, cells were treated with benzene or its metabolites (dose levels: 1; 10; 30; or 100µM) or the vehicle control (media) for 24h. Benzene or its metabolites initiated then cell death and/or homologous recombinations.
After drug exposure, cells used in recombination studies were washed with phosphate-buffered saline (PSB) and grown in fresh media containing G418 (500µg/mL). Cells used for plating efficiency studies were treated the same way except these cells were not exposed to G418. Cells were grown for either 1 week (plating efficiency studies) or two weeks (recombination studies) and then stained with crystal violet (1% methanol). Homologous recombination frequency was determined by counting the number of G418-resistant colonies versus the total number of surviving cells plated.

To study the role of ROS in benzene-initiated toxicity, further experiments evaluated whether catalase could reduce homologous recombination initiated by catechol and other benzene metabolites. In these experiments, cells were plated as described above for both plating efficiency and homologous recombination studies; cells were then exposed to catalase (2000 U/mL) just prior to being exposed to catechol or other benzene metabolite (10µM). The catalase concentration of 2000 U/mL was chosen because it has previously been demonstrated to protect against benzoquinones-initiated ROS. Homologous recombination frequency was then determined as described above.
Evaluation criteria:
Homologous recombination frequency was determined by counting the number of G418-resistant colonies versus the total number of surviving cells plated, with or without addition of catalase.
Statistics:
Results were analyzed using a standard, computerized statistical program (GraphPad Prism 3.0). Groups were compared using a one-factor analysis of variance (ANOVA). The minimum level of significance used throughout was p < 0.05.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at dose levels 10, 30, and 100µM
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
not examined
Additional information on results:
Cell survival:
Cell survival in the different treatments groups was determined; it used to calculate the recombination frequency. Catechol increased cell death at concentrations of 10µM and higher.

Homologous recombination:
Exposure of CHO 3 -6 cells to benzene metabolites led to increased homologous recombination: in presence of catechol, dose-dependent increased recombination occurred 1.9 -, 2.5 -, and 3.2 -fold at 1, 10 and 30µM concentrations, respectively, when compared to vehicle controls.

Effects of catalase on metabolite-initiated cell death and homologous recombination:
The exposure to 10µM catechol did not significantly decrease cell survival in CHO 3 -6 cells (cell survival was 65% without catalase and 70% in presence of catalase).
At 10µM,
- in absence of catalase, homologous recombination induced by catechol was 6.2 fold higher than with vehicle control alone.
- in presence of the antioxydant enzyme, whereas, this difference disappeared, homologous recombination levels for vehicle control alone and in presence of catechol are quite the same (1.5 and 1.7 respectively).
Conclusions:
The evidence presented in this study demonstrate that the benzene-metabolite catechol can initiate increased frequencies of homologous DNA recombination in the CHO 3-6 cell line. This increased frequency of recombination can be completely blocked by the activity of the antioxidative enzyme catalase, supporting the hypothesis that increased oxidative stress plays a role in benzene-initiated toxicity.
Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well described, but not according to recognized guidelines.
Principles of method if other than guideline:
Comet assay. The aim of this study was to address the DNA damaging ability of catechol under improved realistic physiological conditions (presence of serum).
GLP compliance:
not specified
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Species / strain / cell type:
mammalian cell line, other: Human Peripheral Blood Mononuclear Cells (PBMC)
Metabolic activation:
without
Metabolic activation system:
none
Test concentrations with justification for top dose:
0 - 1.1 - 5.5 - 11 - 22 - 66 µg/mL (0 - 10 - 50 - 100 - 200 - 600 µM)
Vehicle / solvent:
Catechol was dissolved in PBS (Na-phosphate 10 mM, pH 7.4, containing 120 mM NaCl and 2.7 mM KCl).
Details on test system and experimental conditions:
* PBMC were isolated from leukocyte-enriched human peripheral blood by a density gradient. Two different media were used: PBS (Phosphate Buffer Saline 10 mM, pH 7.4) or RPMI 1640 with or without foetal calf serum (FCS). The PBMC were incubated with catechol dissolved in PBS in the two different media for 2 and 24 hours at concentrations that did not affect cell viability. DNA damage was quantified by a computerised image analysis system.

* The Comet assay (single cell gel electrophoresis assay) was used to evaluate DNA damage. Aliquots of the cell suspension were centrifuged and the pellet obtained was mixed with low-melting agarose 0.7 % in PBS, distributed onto microscopic slides, pre-coated with normal melting agarose. A second layer of normal-melting agarose was applied. The slides were then immersed in the lysis solution (2.5M NaCl, 100 mM Na2EDTA, 10 mM Tris-HCl, pH10, containing freshly added 1% Triton X100 and 10% DMSO) for 1 hr at 4°C and then placed in into a horizontal electrophoresis apparatus filled with buffer (1 mMNa2EDTA, 300 mM NaOH). After 20 min of pre-incubation (unwinding of DNA), the electrophoresis was run for 20 min at a fixed voltage of 25 V and 300 mA. At the end, the slides were washed (x 3) and stained with ethidium bromide. All above steps were carried out under red light. The cells were analysed using a fluorescence microscope.

* In order to assess the role of FCS in increasing or reducing the genotoxicity of the compound, the cells were also incubated for 2 h with 600µM of Catechol in 4 media: PBS, PBS + 5%FCS, RPMI, RPMI + 50% FCS.

* For the comparative evaluation of the damage, the 75th percentile, defined as the value that 75% of the cells do not exceed, was used. The result of each experiment, repeated at least 4 times using different PBMC preparations, were compared using the Students'two-tailed (paired) t-test.

* Cell viability was determined by the trypan blue exclusion test.
Species / strain:
mammalian cell line, other: Human Peripheral Blood Mononuclear Cells (PBMC)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 600 µM
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
- The different concentration tested did not reduce cell viability to  less than 95% (blue dye exclusion method). 
- Catechol did not induce DNA damage at concentrations of 10, 50 and 100  µM.
- Catechol was genotoxic at 200 and 600 µM when the cells were incubated in PBS (in conditions not pertinent for hazard evaluation for humans, as no proteins were present), but had very little effects when the cell were incubated in RPMI + 5% FCS.
- In the presence of 5%  of serum, the cells were completely protected from catechol induced effects in both media.

A positive response was observed when cells were incubated with the highest concentration of catechol in phosphate serum buffer only. No such results were obtained in RPMI medium, and under more physiological conditions , i.e. following the addition of foetal calf serum. Therefore, the positive result obtained in very simplified medium, wihout any proteins, is not relevant.

Remark: The alkaline comet assay does not clearly distinguish between single and double-strand break.
Conclusions:

Negative without metabolic activation in Comet assay with human peripheral blood monoclear cells.
Executive summary:

Human Peripheral Blood Mononuclear Cells (PBMC) was exposed to catechol at following concentrations: 0 - 1.1 - 5.5 - 11 - 22 - 66 µg/mL (0 - 10 - 50 - 100 - 200 - 600 µM) without metabolic activation system.

After lysis of cells and elution in specific comet assay conditions, DNA damage were evaluated in single cell gel electrophoresis by fluorescence. Cell viability was determined by the Trypan blue. The different concentration tested did not reduce cell viability to less than 95%. Catechol did not induce DNA damage at concentrations of 10, 50 and 100 µM. Catechol was genotoxic at 200 and 600 µM when the cells were incubated in PBS (in conditions not pertinent for hazard evaluation for humans, as no proteins were present), but had very little effects when the cell were incubated in RPMI + 5% FCS. In the presence of 5% of serum, the cells were completely protected from catechol induced effects in both media.

The positive response was observed when cells were incubated with the highest concentration of catechol in phosphate serum buffer only. No such results were obtained in RPMI medium, and under more physiological conditions , i.e. following the addition of foetal calf serum. Therefore, the positive result obtained in very simplified medium, wihout any proteins, is not relevant.

 

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The test was performed in a very simplified system, with only DNA fragments. The test was not carried out according to the international recognised guidelines. This test was performed to understand the redox properties of catechol onto DNA damage
Principles of method if other than guideline:
DNA damage
GLP compliance:
not specified
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Species / strain / cell type:
mammalian cell line, other: DNA fragments
Metabolic activation:
without
Metabolic activation system:
none
Test concentrations with justification for top dose:
0 - 0.011 -0.022 - 0.055 - 0.110 - 0.220 - 0.550 - 1.101 - 2.202 µg/mL (0 - 0.1 - 0.2 - 0.5 - 1.0 - 2.0 - 5 - 10 - 20 µM)
Vehicle / solvent:
none
Details on test system and experimental conditions:
* Detection of DNA damage:
- DNA singly 32P-labelled fragments were prepared from the pbcNI plasmid, which contains a 6.6-kb BamHI chromosomal DNA segment with the human c-Ha-ras-1 protooncogene.
- The standard reaction mixture contained the 32P-labelled DNA fragment, catechol, sonicated calf thymus DNA and CuCl2 in sodium phosphate buffer containing 5 µM DTPA. After 60 min incubation at 37°C, the DNA fragments were heated for 20 min at 90°C in 1M piperidine where indicated, and
electrophoresed on a 8% polyacrilamide /8M urea gel (as described in [Kawanishi and Yakamoto (1991) Biochemistry, 30, 3069-3075]). - Preferred cleavage sites were determined by a direct comparison of the chemical reaction products with the position of the nucleotides utilised.
* Spectroscopic measurements: 1H NMR spectra were performed.

* Measurement of O2- generation:
the quantity of O2- generated by the reactions of catechol with Cu2+ was determined by cytochrome c reduction.
Species / strain:
mammalian cell line, other: Human DNA fragments
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Remarks:
Not relevant
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
* DNA damage: 
- In the absence of Catechol, with or without the addition of 20 µM Cu2+  and 100 µM NADH, no DNA cleavage was observed.
- Catechol alone at the concentration of 20 µM (without the addition of  Cu2+ or NADH) did not produce DNA damage.
- Catechol at the concentration of 10 and 20 µM induced a small DNA  damage in the presence of Cu2+ ions, but not at 5 µM. The addition of  NADH to this experimental condition (5 µM catechol/20 µM Cu2+) induced a  strong DNA damaging effect.

- Lower concentrations were tested in presence of both 20 µM CuCl2 and  100 µM NADH. According to the autoradiogram reproduction, 0.1 and 0.2 µM  catechol produced a barely detectable effect, and 0.5, 1.0 and 2.0 µM  catechol induced a similar moderate DNA cleavage.
- According to the authors, the treatment of damaged DNA with piperidine  enhanced DNA cleavage, suggesting that the DNA damage resulted from base  modification in conjugation with strand breakage. All the results above  related to effects obtained with piperidine. No results are provided for  experimental condition without piperidine. 

* The DNA damage induced by catechol was inhibited by catalase and bathocuprine, a specific chelator of Cu2+. Neither OH* scavenger nor SOD could inhibit this DNA damage, suggesting the induction of DNA damage mediated cooperatively by H2O2 and Cu+.
Conclusions:
This experiment is a mechanistic test using different test conditions to demonstrate that redox properties act on DNA-damage. Catechol could induced DNA damage in specific conditions: presence of NADH and Cu2 +. The DNA clivage was observed at guanine and Thymine , and was due to reactive oxygen species.
Executive summary:

Human DNA fragments were exposed to catechol at doses of 0 - 0.011 -0.022 - 0.055 - 0.110 - 0.220 - 0.550 - 1.101 - 2.202 µg/mL (0 - 0.1 - 0.2 - 0.5 - 1.0 - 2.0 - 5 - 10 - 20 µM) without metabolic activation system. DNA damage and measurement of O2- generation were performed. Catechol, was tested with or without the addition of Cu2+ and/or NADH.

Catechol alone at the concentration of 20 µM (without the addition of Cu2+ or NADH) did not produce DNA damage. Catechol could induced DNA damage in specific conditions: presence of NADH and Cu2 +. 

The DNA damage induced by catechol was inhibited by catalase and bathocuprine, a specific chelator of Cu2+. Neither OH* scavenger nor SOD could inhibit this DNA damage, suggesting the induction of DNA damage mediated cooperatively by H2O2 and Cu+.

The DNA clivage was observed at Guanine and Thymine , and was due to reactive oxygen species.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Results are difficult to analyse; purity is not mentioned;
Qualifier:
no guideline required
Principles of method if other than guideline:
In this assay, the relative total growth of Saccharomyces cerevisiae was assessed by comparing the extent of proliferation of treated and untreated cells. The measurement of total growth was performed by fluorescence collection.
Induction of the RAD54 promoter due to DNA damage results in production of the extremely stable green fluorescent protein (GFP), which is fluorescent in the green spectrum when illuminated by blue light.
In this assay relative total growth is assessed by comparing the extent of proliferation of treated cells with that of untreated cells. Growth data are primarily used to normalize fluorescence data collected in the assay, as it is necessary to distinguish a large concentration of weakly fluorescent cells from a small concentration of highly fluorescent cells.
GLP compliance:
not specified
Type of assay:
other: A yeast (Saccharomyces cerevisiae) DNA repair reporter assay termed the GreenScreen assay (GSA) is described
Target gene:
Since the cell's own DNA damage assessment apparatus is being monitored, the entire genome is used as the target for DNA damage.
Species / strain / cell type:
Saccharomyces cerevisiae
Details on mammalian cell type (if applicable):
The 2 strains of Saccharomyces cerevisiae used were:
The reporter strain GENT01 containing a nuclear, episomally replicating, multiple copy plasmid bearing the entire upstream non-coding DNA sequence of the S. cerevisiae RAD54 gene fused to a yeast codon-optimized derivative of the Aequorea Victoria GFP gene.
The control strain GENC01 containing an identical plasmid except that 2 bp have been removed at the start of the GFP gene, such that no GFP was made. The plasmids were maintained during cell growth and division by selection of uracil prototrophy, conferred by the presence on both plasmids of the yeast URA 3 marker gene.
Metabolic activation:
without
Metabolic activation system:
none
Test concentrations with justification for top dose:
A 1 mM stock of the test chemical was prepared in 2% (v/v) aqueous DMSO and used to make two identical dilution series across the microplate and a `control'. ). To achieve this, 150 µl of the test chemical solution were put into two microplate wells. Each sample was serially diluted by transferring 75 µl into 75 µl of 2% DMSO, mixing and then taking 75 µl out and into the next well. This produced nine serial dilutions of 75 µl each.
Nine concentration for catechol:
- from 177 µg/mL to 880 µg/mL (7.99 mM) were tested for strain GEN C01
- 599 µg/mL to 880 µg/mL (7.99 M) were tested for strain GEN T01
Vehicle / solvent:
Controls were added as follows:
(i)     Compound alone, to provide information on compound absorbance/fluorescence;
(ii)   Yeast cultures diluted with 2% DMSO alone, to give a measure of maximum proliferative potential;
(iii) MMS as a genotoxicity control: `high' = 0.00125% (v/v), `low' = 0.0001875% (v/v);
(iv)  Methanol as a cytotoxicity control: `high' = 3.5% (v/v), `low' = 1.5% (v/v);
(v)    Growth medium alone, to confirm sterility/lack of contamination.
Untreated negative controls:
yes
Remarks:
Yeast cultures diluted with 2% DMSO alone, to give a measure of maximum proliferative potential
Negative solvent / vehicle controls:
yes
Remarks:
Growth medium alone, to confirm sterility/lack of contamination
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: MMS as a genotoxicity control: `high' = 0.00125% (v/v), `low' = 0.0001875% (v/v); Methanol as a cytotoxicity control: `high' = 3.5% (v/v), `low' = 1.5% (v/v)
Remarks:
An other control has been established as a "reference": the compound alone, to provide information on compound absorbance/fluorescence.
Details on test system and experimental conditions:
Strains and plasmids
The Saccharomyces cerevisiae strain FF18984 (MATa, leu2-3,112 ura3-52 lys2-1 his7-1) was obtained from Francis Fabre (French Atomic Energy Commission, Fontenay-aux-Roses, France). The strain has been neither modified to increase permeability nor sensitized to DNA damage by mutations.
The reporter strain (GenT01) is FF18984 containing a nuclear, episomally replicating, multiple copy plasmid bearing the entire upstream non-coding DNA sequence of the S.cerevisiae RAD54 gene fused to a yeast codonoptimized derivative of the Aequorea victoria (jelly®sh) GFP gene (Cormack et al., 1997). The control strain (GenC01) is FF18984 containing an identical plasmid except that 2 bp have been removed at the start of the GFP gene, such that no GFP is made. The plasmids are maintained during cell growth and division by selection of uracil prototrophy, conferred by the presence on both plasmids of the yeast URA3 marker gene.

Microplate preparation
Assays were carried out in 96-well, black, clear-bottomed microplates (Matrix ScreenMates, catalogue no. 4929; Matrix Technologies, Hudson, NH). A number of alternative microplates were assessed, although the variable background absorbance and fluorescence both within and between plates from individual manufacturers were generally unacceptable, leading to the conclusion that only Matrix or Corning (catalogue no. 3651; New York, NY) plates were appropriate for the assay at the time of writing. The assays were performed using a liquid handling robot (MicroLabS single probe; Hamilton GB Ltd, Birmingham, UK) in a protocol designed to test up to four compounds on a single 96-well microplate. Set-up takes 30 min per plate. Results for a subset of compounds have been reproduced using a Genesis 8-probe robot (Tecan UK Ltd, Theale, UK), which can set up a similar microplate in 5 min. Microplates can also be rapidly and effectively filled using a multi-channel pipette. The arrangement of samples in the microplate is also well suited to manual filling.
A 1 mM stock of the test chemical was prepared in 2% (v/v) aqueous DMSO and used to make two identical dilution series across the microplate and a `control' (see below). To achieve this, 150 ml of the test chemical solution were put into two microplate wells. Each sample was serially diluted by transferring 75 ml into 75 ml of 2% DMSO, mixing and then taking 75 ml out and into the next well. This produced nine serial dilutions of 75 ml each.
Controls were added as follows:
(i)     Compound alone, to provide information on compound absorbance/fluorescence;
(ii)   Yeast cultures diluted with 2% DMSO alone, to give a measure of maximum proliferative potential;
(iii) MMS as a genotoxicity control: `high' = 0.00125% (v/v), `low' = 0.0001875% (v/v);
(iv)  Methanol as a cytotoxicity control: `high' = 3.5% (v/v), `low' = 1.5% (v/v);
(v)    Growth medium alone, to confirm sterility/lack of contamination.
Stationary phase cultures of GenT01 and GenC01 were diluted to an optical density (OD600 nm) = 0.2 in double strength F1 medium. An aliquot of 75 ml of the yeast suspension was added to each well of the diluted chemical: GenT01 to one series and GenC01 to the second series of each compound, and to appropriate standards and controls (i.e. GenT01 to MMS-containing wells and GenC01 to methanol-containing wells). After the plates were filled, they were sealed using either a gas-permeable membrane (Breath- Easy; Diversified Biotech, USA) or a plastic lid and then incubated, without shaking, overnight (16±20 h) at 25°C.

Data collection and handling
Following overnight incubation, GFP reporter fluorescence and yeast culture absorbance data were collected from the microplates. Three different microplate readers which combine fluorescence and absorbance functionality have been used, and comparable data were obtained. These were: a Tecan Ultra-384 (Tecan UK Ltd, Theale, UK), excitation 485 nm, emission 535 nm with an additional dichroic mirror (reflectance 320±500 nm, transmission 520±800 nm); a BMG PolarStar (BMG Labtechnologies, Offenburg, Germany), excitation 485 nm, emission 520 nm; a Wallac 1420 Victor2 (Perkin Elmer Life Sciences, Wellesley, MA), excitation 485 nm, emission 535 nm. Absorbance was measured through a 620 nm filter in both the Tecan and BMG instruments and through a 600 nm filter in the Wallac. The data were inserted into a Microsoft Excel spreadsheet and converted to graphical format. Data processing requires only simple mathematical manipulations. Absorbance data give an indication of reduction in proliferative potential and these data were normalized to the untreated control (= 100% growth). Fluorescence data were divided by absorbance data to give `brightness units', the measure of average GFP induction per cell. These data were normalized to the untreated control (= 1). In order to correct for induced cellular auto-fluorescence and intrinsic compound fluorescence, the brightness values for the GenC01 strain were subtracted from those of GenT01. This makes visual assessment of the data more reliable. All the data were checked with and without this correction and the decision on whether or not a compound was classified as being genotoxic was not affected.
Evaluation criteria:
Two thresholds were set from the absorbance data:
The first is the threshold at which there is a statistically significant reduction in the proliferative potential or relative total growth (RTG). This threshold is not used in data handling but is provided to give an indication to the user that the compound is causing some growth inhibition. It is set at 80% of the maximum extent of yeast cell proliferation on each microplate (i.e. the cell density reached by the untreated control cells). This is greater than 3 times the standard deviation of the background. Mortality is not measured in this assay: 80% RTG does not mean 20% of the cells are dead.
The second threshold is set at 30% RTG. This is a rejection threshold for genotoxicity data and reflects two properties of the system. Firstly, this threshold recognizes the limits imposed by instrumentation: at cell densities lower than 30% RTG, interference in the optical measurements becomes significant due to variation in the background reflectance and absorbance of the microplate. Secondly, this level of growth means that the culture has been unable to complete even one doubling and as such is a toxicity threshold. A breakdown in cell integrity can lead to non-specific DNA damage, although if a cell is dead or dying this is clearly of little genetic consequence: apoptosis in mammalian cells is itself a deliberate manifestation of this.

The genotoxic threshold reflects a statistically significant increase in brightness compared with the untreated control. It is set at 1.3 (i.e. a 30% increase) and this is greater than 3 times the standard deviation of the background.

Compounds that tested negative for genotoxicity in the first assay were retested up to the 30% RTG threshold or the limit of solubility. Compounds that tested positive for genotoxicity were re-tested at the same compound concentrations to corroborate the initial result.
Species / strain:
Saccharomyces cerevisiae
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
at a very high concentration (> 599 µg/mL)
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
After treatement with catechol, a clear positive response was measured in growth inhibition rate with strain GENC01 at 177 µg/mL, and clear genotoxicity with GFP induction with strain GEN T01 at 599 µg/mL.
Cell proliferation and genotoxicity reporter controls:
The cytotoxicity controls indicate that the yeast is behaving normally; the genotoxicity controls demonstrate that the strains are responding normally to DNA damage; the growth inhibition and genotoxicity controls are in place for qualitative reasons to demonstrate that the assay is responding in a dose-dependent manner; controls are included on each microplate tested; considering the results from 24 tests conducted over a period of 7 months, all controls `passed' by the criteria prescribed.

Test compound controls:
The compound absorbance control allows a warning to be generated if a test compound is significantly absorbing.
The compound fluorescence control allows a warning to be generated when a compound is highly auto-fluorescent. The routine subtraction of GenC01 from GenT01 data removes this interference from the data.
Conclusions:

positive without metabolic activation
Executive summary:

DNA damage and repair was checked in microplates method with Saccharomyces cerevisiae. The relative total growth of Saccharomyces cerevisiae was assessed by comparing the extent of proliferation of treated and untreated cells. The measurement of total growth was performed by fluorescence collection. Two strains were tested (GENC01 and GEN T01) at concentration of catechol from 177 µg/mL to 880 µg/mL without metabolic activation system. A clear positive response was measured in growth inhibition rate with strain GENC01 at 177 µg/mL, and clear genotoxicity with GFP induction with strain GEN T01 at 599 µg/mL.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well described, no details about controls.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mammalian cell line, other: Syrian Hamster Embryo (SHE) cells
Metabolic activation:
without
Metabolic activation system:
none
Test concentrations with justification for top dose:
1 - 3 - 10 - 30 µM
Vehicle / solvent:
none
Details on test system and experimental conditions:
- SHE cell cultures were established from 13-day-gestation foetuses collected from inbred Syrian hamster, strain LSH/ss LAK. The cell culture medium (complete medium) used was IBR Dulbecco's modified Eagle's reinforced medium supplemented with 10 % foetal bovine serum.
- Catechol was first dissolved in serum free culture medium to give the concentration of 10 mM. Aliquots of this freshly made solution were added to the culture to give the appropriate final concentration.
- Cell growth: Cells in logarithmic phase were plated in triplicate, and after overnight incubation, treated at 1 - 30 µM catechol for 48 h.
- Mutation experiment: Cells were plated, and after overnight incubation, treated at 1 -30 µM catechol for 48 h.
The cells were grown for an expression time of 4 days, and then 10E5 cells were plated on each of ten dishes per group with medium containing 3.3 µg/ml TG (6-thioguanine) or 1.1 mM Oua (ouabain). They were incubated for 7 days for colony formation. The mutation frequency was calculated as
described by Barrett et al., 1978.
Evaluation criteria:
Achromatic lesions greater than the width of the chromatid were scored as gaps unless there was displacement of the broken piece of chromatid. If there was displacement, these were recorded as breaks.
Statistics:
Statistical analysis was performed by x² test.
Species / strain:
mammalian cell line, other: Syrian Hamster Embryo (SHE) cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 10 µM
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
- Catechol induced gene mutation at the two loci in SHE cells: both ouabain-resistant (Na+/K+-ATPase) and 6-thioguanine resistant (hprt) mutant frequencies were increased.
- Catechol at the concentration of 1, 3, 10 and 30 µM decreased cell survival to 85.5%, 70.2%, 28.8 % and 1.4% of untreated cells, respectively.

Table of results:

Doses µM

Relative cell survival by treatment of SHE cells

Gene mutation in SHE cells

Percent of survival

Mutation frequency (x10E6)

TG

Oua

1

85.2 (± 6.5)

<1

1.4

3

70.2 (± 5.2)

6.3

1.4

10

28.8 (± 3.8)

15.4

21.1

30

1.4 (± 0.5)

Not studied

112

Executive summary:

Syrian Hamster Embryo (SHE) cells were treated with catechol for 48 h at doses: 1 - 3 - 10 - 30 µM without metabolic activation system, and 4 hours expression time. 10E5 cells were plated on each of ten dishes per group with medium containing 3.3 µg/ml TG (6-thioguanine) or1.1 mMOua (ouabain). They were incubated for 7 days for colony formation.Achromatic lesions were scored, gaps and breaks were recorded. Catechol induced gene mutation at the two loci in SHE cells: both ouabain-resistant (Na+/K+-ATPase) and 6-thioguanine resistant (hprt) mutant frequencies were increased in dose dependent manner for TG.

Catechol at the concentration of 1, 3, 10 and 30 µM decreased cell survival to 85.5%, 70.2%, 28.8 % and 1.4% of untreated cells, respectively.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well described, no data about controls. The results obtained could not be compared with controls and completely validated.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Species / strain / cell type:
mammalian cell line, other: Syrian Hamster Embryo (SHE) cells
Metabolic activation:
without
Metabolic activation system:
none
Test concentrations with justification for top dose:
1, 3, 10, 30 and 100 µM
Vehicle / solvent:
none
Details on test system and experimental conditions:
* SHE cell cultures were established from 13-day-gestation foetuses collected from inbred Syrian hamster, strain LSH/ss LAK. The cell culture medium (complete medium) used was IBR Dulbecco's modified Eagle's reinforced medium supplemented with 10 % foetal bovine serum (FBS).
* Catechol was first dissolved in serum free culture medium to give the concentration of 10 mM. Aliquots of this freshly made solution were added to the culture to give the appropriate final concentration.
* Cell growth: Cells in logarithmic phase were plated in triplicates, and after overnight incubation, treated at 1 -100 µM catechol for 48 h.
* UDS experiment:
Cells in logarithmic phase were plated in triplicate on Thermanox coverslips in 16mm tissue culture cluster dishes in complete medium. After overnight incubation, the medium was replaced with medium containing 1 % FBS and the cultures were incubated for 2 days. The cells were then treated with catechol for 1 hour in FBS medium containing 10 mM HU (hydroxyurea). The cells were labelled with [3H]dThd for 6 h. The uptake was stopped and the coverslips were placed in scintillation vials for determination.
* Similar experiments were performed 2 or 3 times, and the results obtained were reproducible.
Statistics:
Statistical analysis was performed by x² test.
Species / strain:
mammalian cell line, other: Syrian Hamster Embryo (SHE) cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 10 µM
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
- Catechol induced UDS in SHE cells (1 to 100 µM). The [3H]thymidine cpm/culture-well increased in a dose-dependent manner from about 500 at 1 and 3 µM, to about 620 at 10 µM and 800 at 30 µM. The levels of UDS were comparatively decreased to 450 cpm/well in cells treated at the highest dose of 100 µM, probably due to toxicity. No results are available for negative or positive controls.
Conclusions:


Positive without metabolic activation in UDS test with SHE cells.
Executive summary:

SHE cells were treated with catechol for 1 hour at doses of 1, 3, 10, 30 and 100 µM in FBS medium containing10 mMHU (hydroxyurea). The cells were labelled with [3H]dThd for6 h.Catechol induced UDS in SHE cells (1 to 100 µM). The [3H]thymidine cpm/culture-well increased in a dose-dependent manner from about 500at 1and 3 µM, to about 620at 10µM and 800 at 30 µM. The levels of UDS were comparatively decreased to 450 cpm/well in cells treated at the highest dose of 100 µM, probably due to toxicity.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The test was not performed according to international guidelines.
Principles of method if other than guideline:
Method: DNA cleavage/relaxation assays
GLP compliance:
not specified
Type of assay:
other: Topoisomerase II assay
Species / strain / cell type:
other: Plasmid pBR322 DNA
Metabolic activation:
without
Test concentrations with justification for top dose:
0, 1, 10, 30, 100 300 µM (0, 33 µg/mL (300 µM))
Details on test system and experimental conditions:
*Topo II-mediated DNA cleavage: The effect of catechol on stabilisation of Topo II-DNA complexes were evaluated using the method of Gantchev and Hunting (1998) Mol Pharmacol, 53, 422-428. Reactions were modified to use 300 ng pBR322 plasmid DNA and 5 µg bovine serum albumin (BSA).
The pBR322 plasmid DNA (300 ng) was combined with topo II (6U) following 10-minute incubation with compound at indictaed concentrations in the absence or presence of 100 µM etoposide. The dose-dependent loss of the relaxed band is indicative of inhibition of overall topo II catalytic activity.

* Electrophoretic mobility shift assay: as described in Kurz et al. (2000) J Biol Chem, 275, 13948-13954.
* HRP activation: An HRP/H2O2 protocols that models the bone marrow myeloperoxidase metabolic system was performed as described in Eastmond et al. (1986) Mol Pharmacol, 30, 674-679. Catechol was activated for 60 min at 22 °C in a 15 µL reaction volume containing 2.0 mM H2O2 and 0.0075 unit HPR.
Evaluation criteria:
analysis of elepctophoretic migration
Species / strain:
other: Plasmid pBR322 DNA
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified

- In a DNA cleavage /relaxation assay, catechol had no effect on the inhibition of topo II at concentrations up to 300 µM. - On peroxidase activation, inhibition was seen with catechol at 30 µM. However, cleavable complex stabilisation was not observed.

- Etoposide alone stabilizes enzyme-linked DNA complexes (indicated by the linear band), no antagonize effect were observed in presence of catechol. catechol

Conclusions:
Catechol did not exert toxicity against topoisomerase II in this in vitro toxicity study.
Executive summary:

DNA cleavage/relaxation assays was studied at concentration of catechol of 0, 1, 10, 30,100 300µM (0, 33 µg/mL (300 µM)). Catechol had no effect on the inhibition of topoisomerase II at concentrations up to 300 µM. On peroxidase activation, inhibition was seen with catechol at 30 µM. However, cleavable complex stabilisation was not observed.

Endpoint:
genetic toxicity in vitro, other
Remarks:
Type of genotoxicity: other: in vitro topoisomerase inhibition
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test not performed according to standardised international guidelines.
Principles of method if other than guideline:
Method: inhibition topoisomerase I and II assays according to protocols provided by TopoGen
GLP compliance:
not specified
Type of assay:
other: Topoisomerase I and II assays
Target gene:
supercoiled plasmid substrat DNA, kinetoplast (kDNA) and teniposide (VM26)
Species / strain / cell type:
other: Human topoisomerases I and II
Metabolic activation:
without
Test concentrations with justification for top dose:
Concentration of Catechol for Topoisomerase assay I: 110 µg/mL (1000 µM)
Concentration of Catechol for Topoisomerase assay II: 55 - 110 µg/mL (500 - 1000 µM)
Vehicle / solvent:
water
Species / strain:
other: Human topoisomerases I and II
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
other: not relevant
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified

*Topoisomerase I inhibition assay: no inhibition was seen.
*Topoisomerase II inhibition assay: no inhibitory effects were observed at concentrations as high as 500 µM. 
Inhibition was observed at the higher 1000 µM concentration of catech
ol.

Conclusions:
In topoisomerase assay I, no inhibition effect of catechol was observed at the only high dose tested 1000 µM.
In topoisomerase assay II, no inhibition effect was observed at 500 µM but inhibition effect was observed at 1000 µM.
Executive summary:

The inhibitory effect of catechol on the activity of purified human topoisomerases I and II was tested with different catechol concentrations for Topoisomerase assay I: 110 µg/mL (1000 µM) and for Topoisomerase assay II: 55 - 110 µg/mL (500 - 1000 µM). The results indicated in topoisomerase assay I, no inhibition effect of catechol was observed at the only high dose tested 1000 µM, and in topoisomerase assay II, no inhibition effect at 500 µM but inhibition effect was observed at 1000 µM. 

Endpoint:
genetic toxicity in vitro, other
Remarks:
Type of genotoxicity: other: in vitro topoisomerase inhibition
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test not performed according to standardised international guidelines.
Reason / purpose for cross-reference:
reference to other study
Principles of method if other than guideline:
Method: topoisomerase II assays according to protocols provided by TopoGen
GLP compliance:
not specified
Type of assay:
other: Topoisomerase II assay
Target gene:
supercoiled plasmid substrat DNA, kinetoplast (kDNA)
Species / strain / cell type:
other: Human topoisomerases II
Metabolic activation:
without
Test concentrations with justification for top dose:
Concentration of Catechol for Topoisomerase assay II: 0, 1, 10, 100 µM
Vehicle / solvent:
distilled deionized water
Evaluation criteria:
Topoisomerase II activity was determined by assaying the decatenation of kDNA. After electrophoresis, the appearnace of either open circular or linearized kDNA indicated an active and functional enzyme.
If inhibition occured, the kDNA remained in the catenated form and did not migrate from the well.
Statistics:
no
Species / strain:
other: Human topoisomerasesII
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
controls consisted of horseradish peroxidase, H2O2 and water

Catechol, like all the other benzene metabolites tested inhibited the ability of topoisomerase II to decatenate kDNA.

The results permit to distinguish the substance which acted (inhibition of the enzyme) when added directly to the enzyme to the substance that required bioactivation by peroxidase enzymes to inhibit the enzyme.

Catechol required bioactivation by peroxidase in the presence of hydrogen peroxide to inhibit topoisomerase II at low molecular concentrations (10µM and 100 µM).

Direct addition of the metabolic products to topoisomerase II did not substantially alter the inhibitory concentrations.

Conclusions:
Catechol required bioactivation by peroxidase in the presence of hydrogen peroxide to inhibit topoisomerase II at low molecular concentrations (10µM and 100 µM).
Direct addition of the metabolic products to topoisomerase II did not substantially alter the inhibitory concentrations.
Executive summary:

The inhibitory effect of Catechol was studied without metabolic activation system on Topoisomerase II: 0, 1, 10, 100 µM. The Topoisomerase II activity was determined by assaying the decatenation of kDNA. After electrophoresis, the appearnace of either open circular or linearized kDNA indicated an active and functional enzyme. Catechol required bioactivation by peroxidase in the presence of hydrogen peroxide to inhibit topoisomerase II at low molecular concentrations (10µM and 100 µM).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

11 reliable in vivo tests are available for Catechol and are the following: 3 studies equivalent to OECD 474 TG with reliability 2, 1 study equivalent to OECD 484 TG with reliability 2, 1 in vivo comet assay with reliability 1, 4 studies similar to OECD 486 TG with reliability 2 and 2 other studies on DNA with reliability 2.

All these studies indicated a genotoxic effect of Catechol.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian germ cell study: gene mutation
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Only one dose was tested instead of at least 2. Statistical analysis is limited. There is no data about the signs of toxicity for the selected concentration.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 484 (Genetic Toxicology: Mouse Spot Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
mouse spot test
Species:
mouse
Strain:
C57BL
Sex:
female
Details on test animals or test system and environmental conditions:
The study was also performed with Limonene and results compared with those obtained with catechol.
A complementary test was also performed in this study with Saccharomyces cerevisiae model to determine if the substance induced recombination or gene mutation.
Route of administration:
intraperitoneal
Vehicle:
Phosphate buffer
Details on exposure:
Mouse embryos, which are heterozygous for different recessive coat-color genes, are treated in utero between days 9 and 11 postconception by injection of a mutagen into the peritoneal cavity of the mother animal, or by other appropriate routes of administration. If this treatment in a pigment precursor cell leads to an alteration of loss of the wild-type allele of one of the genes under study, a color spot in the coat of the adult animal may be seen. The occurenced of ventral white spots (containing no pigment cell killing and not to the expression of a recessive gene.
* The embryos treated were of the cross C57BL x T, i.e. homozygous for nonagouti (a/a), heterozygous for brown (b/+), pink eyed dilution and chinchilla (p cch/++), dilute and short ear (d se/++).
* Catechol was injected at the dose of 22 mg/kg on days 9, 10 and 11 post-conception. 122 females with vaginal plug were treated, 33  of them produced litters.
* In another group, 30 mg/kg of the carcinogen ethylnitrosourea (ENU) was injected i.p. simultaneously with catechol on the 9th day postconception. In addition, this animals were given catechol alone on days 10 and 11 postconception.
* Negative controls received 0.2 ml phosphate buffer per mouse on days 9, 10 and 11 post-conception.
Duration of treatment / exposure:
1 injection (catechol) on days 9, 10 and 11 postconception.
Frequency of treatment:
- administration of catechol alone: On days 9, 10 and 11 post-conception.
- coadministration: 30 mg/kg ENU (ethylnitrosourea) a known carcinogen was injected by i.p. simultaneously with catechol on the 9th day postconception.
Catechol was given also alone on days 10 and 11 postconception.
Post exposure period:
no data
Remarks:
Doses / Concentrations:
22 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
122 females treated with catechol alone and 237 females treated with catechol + ENU.
Control animals:
yes, concurrent vehicle
Positive control(s):
Yes, treatment with Ethylnitrosourea (ENU).
Evaluation criteria:
If gene mutations were the main reason for appearance of color spots, then the comutagen Catechol enhance the frequency of color spots.
If recombination were the main reason for appearance of color spots, the effect of Catechol should reduce the frequency of color spots.
If both recombinations and mutations occur with similar frequencies, the yield of color spots should not be altered.
Statistics:
Statistical analysis was performed only for ENU+catechol vs ENU+limonene using t-test.
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Catechol is inactive when given alone. The mutation rate in the catechol treated animals (2%), was the same as the control mutation rate (2%).
Catechol enhances the effects of ENU.
The authors concluded that not only mutations but also recombination plays a role in the appearance of color spots.

Effects of ethylnitrosourea (ENU), Catechol in the mammalian spot test:

Treatment

 

Day post- conception

Exp n°

with vaginal plug

F1 animals

Animals with color spots

Substance

Dose (mg/kg)

treated

Without litter

Surviving 4w of age

Midventral white

Of genetic relevance

ENU

30

9

1

59

35

164

3

25 (15%)

30

9

2

49

40

51

1

8 (16%)

30

9

3

66

50

110

3

21 (19%)

 

 

Total

174

125

325

7

54 (19%)

Catechol

22

9

1

122

89

216

-

2 (1%)

22

10

22

11

ENU +

Catechol+

Catechol+

Catechol

30

9

1

62

46

83

1

21 (25%)

22

9

2

72

41

183

8

42 (23%)

22

10

3

103

72

203

4

37 (18%)

22

11

 

 

 

 

 

 

 

 

Total

237

159

469

13

100 (21%)

Control (0.2 ml PBS per mouse

-

9

1

120

95

162

-

2 (1%)

-

10

2

60

37

126

-

1 (1%)

-

11

 

 

 

 

 

 

 

 

Total

180

132

288

-

3 (1%)

ENU + Catechol vs.ENU + limonene: P < 0.025 (t-test)

Conclusions:
Negative
Executive summary:

Female mouse received i.p. injections of catechol at concentration of 22 mg/kg bw on days 9, 10 and 11 postconception with or without co-treatment with ENU. Catechol  given alone did not modified the apparition of color spots 2/216 (1%) compared to negative controls, so same mutation rate.

When catechol was co-administred with ENU, the effects of ENU.ethylnitrosourea was slightly but not satistically significant enhanced.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Only male mice were treated. Test substance purity was not specified. Only one dose tested.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, MA.
- Age at study initiation: 7-10 weeks old
- Weight at study initiation: 25-29 g
- Assigned to test groups randomly: yes
- Diet: Formulab chow 5008 (Ralston Purina Co., St. Louis, MO) ad libitum
- Water: ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS: sterile environment
- Temperature (°C): 25°C
- Humidity (%): controlled
- Air changes (per hr): artificial ventilation
- Photoperiod (hrs dark / hrs light): 12 hours of light per day

No more data
Route of administration:
oral: gavage
Vehicle:
- vehicle used: olive oil
Details on exposure:
One dose of 150 mg/kg by oral route was administered to mice.
Animals were sacrificed 30 hours after dosing by cervical dislocation. One femur was immediately used for the micronucleus test.
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Only one time
Post exposure period:
no data
Remarks:
Doses / Concentrations:
150 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
3 to 5 animals in treatment groups, and 5 animals in control group.
Control animals:
yes, concurrent vehicle
Positive control(s):
3-methylcholanthrene; beta-naphtoflavone; sodium phenobarbital
- Route of administration: i.p.
- Doses / concentrations: 80 mg/kg
Tissues and cell types examined:
Bone marrow cells.
Details of tissue and slide preparation:
The bone marrow from one femur was flushed into a centrifuge tube containing a minimal amount of fetal calf serum (Gibco Laboratories).
Two slides were prepared from each cell button, and stained with Wright and Giemsa stains (Fisher scientific Co.). The latter was diluted with pH 6 phosphate buffer.
1000 polychromatic erythrocytes (PCE) were scored and the number of micronucleated ones (MPCE) recorded as per 1000 of PCEs counted.
Evaluation criteria:
Values less than 0.05 were considered to be statistically significant.
Statistics:
The Mann-Whitney U-test was used to analyse the data.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Catechol did not induce micronclei in the bone marrow. The average (sample mean) value of micronucleated PCE/1000 PCE was 1.2 +/- 1.1 in  males CD-1 mice treated with a single dose of 150 mg catechol/kg. 
This  value was not statistically different with data from the control group (olive oil) (0.6 +/- 0.9 micronucleated PCE/1000 PCE). The range of  micronucleated cells in the control and treated groups was 0-2 per 1000 PCE.
One animal died from the convulsive seizures which immediately followed dosing.

Effect of β-Naphtoflavone pretreatment on benzene Myeloclastogenicity and effects on phenol, Catechol and Hydroquinone administration in male CD-1 mice :

Dose (p.o.)

mg/kg

X ± S.D of MPCE/ 1000 PCE

G.X ± S.D of MPCE/ 1000 PCE

Range of MPCE / 1000 PCE

Control (olive oil)

0.6 ± 0.9

1.2 ± 1.4

0 – 2

BNF + benzene (440)

85.4 ± 17.7

83.9 ± 1.2

63 – 104

 Control (distilled water)

0.6 ± 1.4

1.2 ± 0.9

0 – 2

 Phenol (250)

1 ± 0.7

1.2 ± 1.4

0 – 1

 Catechol (150)

1.2 ± 1.1

1.5 ± 1.5

0 – 2

 Hydroquinone (200)

10 ± 2.2

9.8 ± 1.3

7 - 13

Conclusions:
Negative
Executive summary:

3 to 5 male mice received by oral route 0 or 150 mg/kg bw of catechol.  30 hours after dosing the animals were sacrificed and bone marrow from femur were  used for the micronucleus test. The statistical analysis did not revealed difference between treated and negative controls animals in micronucleated PCE/1000PCE. In this study catechol at 150 mg/kg bw was not considered to induce micronucleus.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Only 4 animals per group were used, only males. There is no data about the signs of toxicity for the selected concentration. No positive controls used.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (Calco, Italy)
- Age at study initiation: 6-8 weeks old
No more data.
Route of administration:
other: i.p. or oral
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water.
Details on exposure:
The aim of this study was to compare the induction of micronuclei after oral administration or intraperitoneal injection.
Swiss CD-1 mice about 6-8 weeks old were randomized and treated in groups of 4 with a vehicle (water) or test substance.
Animals were killed by cervical dislocation and bone marrow smears were prepared at various time 0-48 h after administration of chemical. Smears were fixed in methanol (5 min) and stained with May-Grünwald and Giemsa in Sörensen's phophate buffer. All slides were coded and scored blind. The proportion of polychromatic erythrocytes (PCE) was calculated by counting both normochromatic erythrocytes (NCE) and PCE until 3000 PCE had been scored for the presence of micronuclei. 
Duration of treatment / exposure:
Single
Frequency of treatment:
Only one time
Post exposure period:
No data
Remarks:
Doses / Concentrations:
40 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
4 animals per dose
Control animals:
not specified
Evaluation criteria:
A decrease in the PCE/NCE ratio compared to the control was considered as a consequence of the toxic activity of catechol (bone marrow depression).
Statistics:
The significance level of induced micronuclei was assessed by the use of Kastenbaum-Bowman tables, while the significance of induced cell toxicity (PCE/NCE ratio) was evaluated with the t-test after arc-sin transformation.
Sex:
male
Genotoxicity:
positive
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
- Oral: catechol produced a significant increase (P< 0.05) of micronuclei only at 24 h with evident bone marrow depression.

- Intraperitoneal injection gave significant genotoxic effects (P< 0.01) at 24 h, and evident bone marrow depression from 18 h after treatment. This compound produced weak genotoxicity regardless the route of administration.

Frequencies of micronuclei (MN) in the PCEs from bone marrow of pregnant females after a single treatment with Catechol:

 

Time (h)

MN PCEs

(% ± SD)

PCE :NCE ratio

(± SD)

Catechol (40 mg/kg)

0

0.2 ± 0.04

1.2 ± 0.07

15

0.29± 0.14

1.92 ± 0.74

18

0.32± 0.15

1.49± 0.06

24

0.46± 0.07**

1.68 ± 0.26

30

0.24± 0.02

1.59± 0.2

36

0.36± 0.04*

1.23± 0.4

40

0.47± 0.01**

2.07± 0.28

Significantly different from the negative control group: *p<0.05; **p<0.01

Frequencies of micronucleated MN PCEs in the fetal liver after a single treatment with Catechol:

 

Time (h)

MN PCEs

(% ± SD)

PCE :NCE ratio

(± SD)

Catechol (40 mg/kg)

0

0.37± 0.06

6.21± 0.91

9

0.33± 0.02

3.99 ± 0.77*

12

0.28± 0.03

4.2 ± 2.7**

15

0.52± 0.04

5.37± 0.23

18

0.57± 0.01*

8.73 ± 3.5

21

0.5 ± 0.02

5.05± 0.16

24

0.36± 0.06

6.52± 2.12

Significantly different from the negative control group: *p<0.05; **p<0.01

Conclusions:

Positive results were obtained after oral and i.p. administration of catechol.
The effect by i.p. were more evident than by oral route.
The bon marrow depression is the sign of cytotoxicity. The result should be taken with caution because only one dose has been tested.
Executive summary:

4 mice per group were administered with vehicle or catechol at 40 mg/kg bw in single time by oral or intraperitoneal route. Animals were killed 0-48 h after. Bone marrows smears were evaluated for micronuleus. The proportion of polychromatic erythrocytes (PCE) was calculated by counting both normochromatic erythrocytes (NCE) and PCE. Until 3000 PCE had been scored for the presence of micronuclei after 18, 24, 42 and 48 h. By oral route, the catechol produced significant increase of micronuclei at 24h with evident bone marrow depression. After intraperitoneal injection also significant genotoxic effects more pronounced than by oral route at 24 h was observed, and with evident bone marrow depression from 18 h after treatment.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Only male animals were studied. OECD Guidelines are provided for mammalian liver cells, whereas cells from the pyloric mucosa of the stomach were examined in this study, which is by liquid scintillation counting (which is susceptible to interfere with S-phase cells), instead of autoradiography. No data about the positive control.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
yes
Remarks:
cells examined (pyloric mucosa of stomach instead liver cells)
GLP compliance:
not specified
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc., Kanagawa)
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation: approximately 200 g
- Diet: rats were given a limited amount of diet (4 g of commercial pellet diet, Nihon Clea, Tokyo, per rat of 200 g body weight) overnight to reduce their dietay stomach contents.
No more data
Route of administration:
oral: gavage
Vehicle:
Vehicle used: distilled, deionized water
Details on exposure:
Male Fischer rats were given limited amount of diet overnight to reduce their dietary stomach contents. The following day they were given 1.0 mL of an aqueous solution of catechol by gastric intubation.
After administration of catechol in vivo, RDS and UDS in the pyloric mucosa of the stomach were determined in in vitro organ culture in the  presence of tritiated thymidine with or without 10 mM hydroxiurea (HU),  an inhibitor of RDS. The DNA fraction was extracted from the tissue, dissolved in ACS II and the incorporation of tritiated thymidine was determined in a Beckman liquid scintillation counter. 
The DNA content of the DNA fraction was determined with 3,5-diaminobenzoic acid as standard.
Values for 5 individual rats were determined in each experiment.
Duration of treatment / exposure:
Single
Frequency of treatment:
Only one time
Post exposure period:
no data
Remarks:
Doses / Concentrations:
0, 10, 20, 37.5, 75, 90 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5 animals per dose.
Control animals:
yes, concurrent vehicle
Tissues and cell types examined:
Pyloric mucosa of the stomach.
Evaluation criteria:
The unsheduled DNA synthesis (UDS) was evaluated in absence or presence of hydroxyurea which inibits the Replicative DNA synthesis (RDS) by measurement of the incorpration of 3H thymindine by µg of DNA.
Statistics:
Student's t test (P<0.001)
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
The increase of DNA synthesis in absence of hydroxyurea was observed in the pyloric mucosa of rat stomach after administration of catechol at a dose of 75 mg/kg bw after 24h.
The UDS was not induced in the pyloric mucosa of rat stomach at either time after administration of catechol at a dose of 37.5 or 75 mg/kg bw.

Administration of catechol at doses of 37.5 to 90 mg/kg bw did not induce UDS in the pyloric mucosa of the stomach after 2 and 12 h.

Stimulation of RDS:

The increase of DNA synthesis in absence of hydroxyurea was observed in the pyloric mucosa of rat stomach after administration of catechol at a dose of 75 mg/kg bw with mean value of 1300 dpm3HdThd/µg DNA after 24h. After 48h, this value decrease for a half.

In normal stomach, RDS is always observed in cells of the proliferative zone of the pyloric mucosa where cells are renewed. The negative control had a DNA synthesis value in pyloric region = 153 +/- 75 dpm3HdThd/µg DNA. At 24h after catechol administration, the DNA synthesis increase about 8 times comparing to the negative control.

It exist a dose dependent stimulation of RDS in the pyloric mucosa20hafter administration of catechol at doses of 10 to 90 mg/kg bw.

Dose-dependent stimulation of replicative DNA synthesis in the pyloric mucosa of the stomach of rats treated with catechol

dose (mg/kgbw) 

DNA synthesis (dpm3HdThd/µg DNA) - mean for 5 rats at 20h after administration of catechol

P value by Student's t test 

163 +/-61 

 

10 

315 +/-162 

 

 20

 628 +/-124

 <0.01

 90

 745 +/-336

 <0.05

 

Non-induction of UDS:

DNA synthesis in the pyloric mucosa of rat stomach was measured in the presence of hydroxyurea, an inhibitor of RDS, in in vitro cultures 2 and6hafter administration of catechol to rats. The results showed that the UDS was not induced in the pyloric mucosa of rat stomach at either time after administration of catechol at a dose of 37.5 or 75 mg/kg bw.

Addition of10 mMof hydroxyurea to the culture medium inhibited RDS in the pyloric mucosa about 95%.

Absence of induction of unscheduled DNA synthesis in the pyloric mucosa of the stomach of rats treated with catechol.

Experiment

Dose (mg/kg bw)

Time after treatment (h) 

DNA synthesis (dpm3HdThd/µg DNA) - means in five rats 2h or 6h after admnistration of catechol 

0 (= no inhibition by10 mMhydroxyurea)

2

21.7 +/-4.0 

 

 37.5

28.1 +/- 9.9

II 

0  (= no inhibition by10 mMhydroxyurea)

 25.8 +/- 3.9

 

75 

35.2 +/- 9.1 

 

III

0  (= no inhibition by10 mMhydroxyurea)

6

 15.6 +/- 5.44

 

37.5 

 12.7 +/- 3.55

 

75 

 6

 14.0 +/- 2.60

 

Conclusions:

On pyloric mucosa Catcehol induced stimulation of RDS after oral adminitration, in dose-dependent manner.
The UDS was not induced in the pyloric mucosa of rat stomach at either time after administration of catechol at a dose of 37.5 or 75 mg/kg bw.
Executive summary:

5 rats per groups received by gavage a single administration of catechol at concentration of 0, 10, 20, 37.5, 75, 90 mg/kg bw.

RDS and UDS in the pyloric mucosa of the stomach were determined in in vitro organ culture in the presence of tritiated thymidine with or without10 mMhydroxiurea (HU), an inhibitor of RDS. The DNA fraction was extracted from the tissue, dissolved in ACS II and the incorporation of tritiated thymidine was determined in a Beckman liquid scintillation counter.

The DNA content of the DNA fraction was determined with 3,5-diaminobenzoic acid as standard.

The results indicated a dose-dependent stimulation of replicative DNA synthesis, and an absence of induction of unscheduled DNA synthesis in the pyloric mucosa of the stomach of rats treated with catechol.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The test was not performed according to international standardised guidelines. Only male animals were treated. No data about the positive control.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Induction of ornithine decarboxylase
GLP compliance:
not specified
Type of assay:
other: Induction of ornithine decarboxylase
Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc., Kanagawa)
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation: approximately 200 g
- Diet: rats were given a limited amount of diet (4 g of commercial pellet diet, Nihon Clea, Tokyo, per rat of 200 g body weight) overnight to reduce their dietay stomach contents.
No more data
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: distilled, deionized water
Details on exposure:
Male Fischer rats were given limited amount of diet overnight to reduce their dietary stomach contents. The following day they were given 1.0 mL of an aqueous solution of catechol by gastric intubation.
ODC (ornithine decarboxylase) activity in extracts of the pyloric mucosa of the stomach was determined with L-[1-14C]ornithine as a substrate , as described previously by the author in cross reference study. The protein content of the extracts was determined by micro-assay as described with bovine serum albumin as a standard.
Results are shown as means for duplicate assays on pooled materials from four rats.
Duration of treatment / exposure:
Single
Frequency of treatment:
Only one time
Post exposure period:
no data
Remarks:
Doses / Concentrations:
0 - 10 - 40 - 80 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
4 animals per dose
Control animals:
yes, concurrent vehicle
Tissues and cell types examined:
ODC activity in extracts of the pyloric mucosa of the stomach was determined with L-[1-14C]ornithine as a substrate, as described in [Furita et al. (1985) Carcinogenesis, 6, 91-94], 0, 4, 8, 16, 24 and 48 hr after administration. The protein content of the extract was determined by micro-assay with bovine serum albumine as a standard.
Evaluation criteria:
Results are shown as means for duplicate assays on pooled material from four rats.
Sex:
male
Genotoxicity:
positive
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Administration of catechol at doses of 10, 40 and 80 mg/kg bw induced up to 19-fold increase (at 40 mg/kg bw) in ornithine decarboxylase activity with a maximum after 8 h in the pyloric mucosa of the stomach (75 mg/kg bw).
The  induction of ornithine decarboxylase (ODC) activity in the pyloric mucosa of rat stomach was observed after administration of catechol at a dose of 75 mg/kg bw . The ODC activity in the pyloric mucosa of control rat stomach was about the lowest level detectable by the assay methods used (3.7 ± 4.0 pmol CO2/30 min/mg protein. The activity was increased between 4 and 24 h after administration of catechol with a maximum after 8 hours ( 70 pmol CO2/30 min/mg protein) and returned to the control level within 48 hours. 
- Dose-dependence of induction of ODC activity in the pyloric mucosa 8 h after administration of catechol at doses of 10 to 80 mg/kg bw was observed in 18 experiments with 4 animals:
Dose = 0 mg/kg bw, ODC activity = 3.72 pmol CO2/30 min/mg protein;
Dose = 10 mg/kg bw, ODC activity = 16.6 pmol CO2/30 min/mg protein;
Dose = 40 mg/kg bw, ODC activity = 71.2 pmol CO2/30 min/mg protein;
Dose = 80 mg/kg bw, ODC activity = 59.7 pmol CO2/30 min/mg protein.
Conclusions:

The results of this work showed the dose-dependent induction of ODC activity after oral administration of catechol on pyloric mucosa stomach. This result indicated that catechol may have tumor-promoting activity.
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The test was not performed according to international standardised guidelines. Only male animals were treated. The origin of the data about positive control is not clear. Although they are plotted on the same raph than data on catechol, they seem to come from previous work of the same authors.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Alkaline Elution Method of Sina et al, (1983) modified.
GLP compliance:
not specified
Type of assay:
other: alkaline elution of DNA
Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc., Kanagawa)
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation: approximately 200 g
- Diet: rats were given a limited amount of diet (4 g of commercial pellet diet, Nihon Clea, Tokyo, per rat of 200 g body weight) overnight to reduce their dietay stomach contents.
No more data
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: distilled, deionized water
Details on exposure:
Male Fischer rats were given limited amount of diet overnight to reduce their dietary stomach contents. The following day they were given 1.0 mL of an aqueous solution of catechol by gastric intubation.
A sample of 5 mg of pyloric mucosa was lysed on a filter with 3.0 mL of lysis solution pH 9.7, in the presence of proteinase K (1mg/mL) for 1 hour in the dark, and then DNA was eluted in the dark at 20-25 °C with 30 mL of solution of pH 12.1 at a flow rate of 0.05 mL/min. Fractions of 3.0 mL were collected and the DNA contents of the fractions and on the filters were detremined with 3,5-diamino-benzoic acid. The elution rate constant (mL -1) of DNA was aclculated from a plot (log scale) from the start to the 5th fraction. the results were analysed by means of Student's t test.
Duration of treatment / exposure:
Single
Frequency of treatment:
Only one time
Post exposure period:
no data
Remarks:
Doses / Concentrations:
37.5 to 90 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
4 animals per dose.
Control animals:
yes, concurrent vehicle
Positive control(s):
Yes: N-ethyl-N-nitro-N-nitrosoguanidine (MNNG)
- Route of administration: oral, gavage
- Doses / concentrations: 10 mg/kg bw
Tissues and cell types examined:
Pyloric mucosa of stomach.
Details of tissue and slide preparation:
Alkaline elution: 
A sample of pyloric mucosa was lysed on a filter in the presence of proteinase K for 1 h in the dark, and then, DNA was eluted in the dark at 20-25 °C.The DNA contents of the fractions collected on the filters were determined with 3,5-diamino-benzoic acid. The elution rate of DNA was calculated from a plot from the stract of the 5th fraction.
Statistics:
The results were analysed by means of student's t-test.
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified

Administration of catechol at doses of 37.5 to 90 mg/kg bw did not induce DNA single strand scission in the pyloric mucosa of the stomach as determined by the alkaline elution method after 2 and 12 h. The fraction of DNA remaining on filter 2 and 12h after administration of  catechol at the dose of 75 mg/kg remains in the same range than distilled  water (0.8 to 1.0). The following mean elution rate constant were observed (ml-1 x 10E3):

 Dose (mg/kg bw)

Time after treatment (h) 

N° of experiments 

Elution rate constant

(ml-1 x 10E3) 

2/12 

2.07 +/- 0.80

37.5 

 2.78 +/- 1.93 

75 

1.30 +/- 1.07

75 

12 

2.55 +/- 1.93

90 

1.71 +/- 0.19

The elution rate constant did not increase after administration of  catechol suggesting that catechol did not induce single break scission of  DNA in the pyloric mucosa.

The positive control MNNG increased the elution rate constant 2h after the administration at 10 mg/kg bw.

Conclusions:

The elution rate constant did not increase after administration of catechol at doses of 37.5, 75 or 90 mg/kg bw, suggesting that catechol did not induce single break scission of  DNA in the pyloric mucosa.
Executive summary:

The DNA damage and repair by Alkaline elution was studied on pyloric mucosa. The administration of catechol at doses of 37.5 to 90 mg/kg bw did not induce DNA single strand scission in the pyloric mucosa of the stomach as determined by the alkaline elution method after 2 and12 h.The fraction of DNA remaining on filter 2 and12hafter administration of catechol at the dose of 75 mg/kg remains in the same range than distilled water (0.8 to 1.0). The elution rate constant did not increase after administration of catechol suggesting that catechol did not induce single break scission of DNA in the pyloric mucosa.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Only one dose tested. The route of exposure is not detailed (oral route), the exact vehicule tested unknown.
Reason / purpose for cross-reference:
reference to same study
Principles of method if other than guideline:
Evaluation of the E. coli K-12 DNA repair host-mediated assay in a short term in vivo genotoxicity test. The test substance was administered to mice by oral route, after intravenous administration of a mixture of DNA repair deficient and profeicient derivatives of bacteria strain Escherichia coli K-12.
After an incubation period the relative survival of two bacteria strains was determined in blood, liver, lungs, kidneys and testes of the host (Mice) just sacrified.
The methods used was as described by Kerkalaan et al, 1985, Induction of repairable DNA damage in E. coli cells recovered from liver, spleen, kidneys and the blood stream of mice with methylating carcinogens, Mutation Res., 148, 1-12.
GLP compliance:
not specified
Type of assay:
other: E.coli K-12 DNA repair host-mediated assay
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
Male NMRI mice weighing 25-35 g were purcchased from an authorized breeder, randomly distributed over the cages upon arrival and acclimatized for approximately 7 days in a controlled environment.
The bacteria strains were derived from the archetype E. coli K-12 343/113. The reference strain was 343/636 with the genotype uvrB+-/recA+/lac- and the DNA repair deficient strain was 343/591 uvrB-/recA-/lac+.
The bacterial strains, growth and suspension media were described in cross reference study: Hellmer L. and G. Bolcsfoldi, 1992, An evaluation of the E. coli K-12 uvr B/recA DNA repair host mediated assay. I. In vitro sensitivity of the bacteria to 61 compounds, Mutation Res., 241, 145-160.
Route of administration:
oral: unspecified
Vehicle:
sterile distilled water, corn oil or methocel. For oral administration ethanol or dimethyl sulfoxide were also used.
The solvent used by substance tested is not detailed in the publication.
Details on exposure:
The bacterial mix was prepared by adding 7 mL of the overnight culture of strain 343/636 to 100 mL of strain 343/591, and then centrifuging the mixture in 8 mL phosphate buffer saline (PBS) and kept at room temperature in the dark until administration to the animals.
0.2 mL of the concentrated bacterial mix was injected in the lateral tail vein of the mouse. The test substance was administered by oral route shortly after batecrial injection.
Duration of treatment / exposure:
The duration of exposure: 2 hours. This duration was chosen since the relative survival of the DNA repair deficient strain showed a slight decrease after 2h in a test with untreated animals.
Frequency of treatment:
once
Post exposure period:
The mice were anesthetized with carbon dioxide /air and 100 µL blood were removed from heart cavity and diluted in 0.9 mL PBS.
The liver, lungs, kidneys and testes were removed and placed in 5 ml PBS.
The organs were then homogenized and appropriate dilutions were made.
Duplicate aliquots from each organ were spread on NR agar petri plates, which were incubated for 1 day at 37°C.
Remarks:
Doses / Concentrations:
200 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
Seven animals per dose and negative control were used.
Control animals:
yes, concurrent vehicle
Tissues and cell types examined:
The number of bacteria colonies was counted by eye against a light background. The individual survival of each strain can be determined because the strains differ in their ability to ferment lactose.
The DNA repair deficient strain forms red colonies while the DNA repair proficient strain forms white colonies when neutral red is included as a pH indicator in the medium.
Evaluation criteria:
The differential killing is expressed as the fraction:
number of colonies of the DNA repair deficient strain/number of colonies of the DNA repair proficeint strain
Statistics:
The fraction is statistaclly evaluate by one-sided Student's t-test with confidence interval of 95%.
The low and high dose group for each organ was compared separately against the control group.
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
not specified

Results:

 

Dose (mg/kg)

N° of colonies of DNA repair deficient strain /N° of colonies of DNA repair proficient strain +/- standard deviation

 Catechol

 

Blood

Liver

Lungs

Kidneys 

Testes

 -

1.33+/- 0.36

0.80 +/- 0.24

0.54+/- 0.13

1.02+/- 0.13

0.60 +/- 0.23

 200

1.15+/- 0.40

0.78 +/- 0.17

0.91 +/- 0.23

1.60 +/- 0.38

0.57+/- 0.21

Conclusions:
Negative
Executive summary:

In this Host mediated DNA repair assay with mouse, The animals had been exposed for 2 hours with bacteria mix and then catechol administered by oral route at concentration of 200 mg/kg.

Different organs were examined for the colonies types they contained.

The results indicated no difference between treated and control for the ratio in N° of colonies of DNA repair deficient strain/N° of colonies of DNA repair proficient strain.

Catechol was considered not to have genotoxic effect in this study against E. coli K-12 uvr B/recA DNA repair in the Mice organs evaluated.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The route of administration is of low relevance. Only male animals were treated. There is no data about the signs of toxicity for the selected concentrations. 4 different animal stocks were used. 3 of each stock were included in the control group, but the origin of the mice in the catechol groups is unknown. The relevance of the statistical analysis performed to compare these groups is therefore questionable.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
route of administration
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (Calco-Como, Italy)
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: 25-30 g
- Assigned to test groups randomly: yes
No more data.
Route of administration:
intraperitoneal
Vehicle:
- vehicle used: desionised and bidistilled water
Details on exposure:
- Animals were randomised and treated by i.p. Administration in group of 3 with the solvent alone (desionised and bidistilled water) or catechol.
- Animals were killed by cervical dislocation 18 h after treatment and bone marrow cells were obtained and processed according to the standard Mn test (Schmid, 1975).
Duration of treatment / exposure:
Single
Frequency of treatment:
Only one time
Post exposure period:
no data
Remarks:
Doses / Concentrations:
10 - 20 - 30 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
3 animals per dose.
Control animals:
yes, concurrent vehicle
Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
- For each mouse, at least 3000 PCEs were scored. To minimise influences due to 4 different animal stocks employed in total for all experiment of the study, animals were randomised throughout the study and 12 animals were used as control (3 control for each stock). The number of normochromatic erythrocytes (NCEs) was also counted, to evaluate the PCEs/NCEs ratio as an index of bone marrow cell toxicity.
1)-Measurement of: - the induction of micronucleated polychromatic erythrocytes (MnPCEs)
- micronucleated monochromatic erythrocytes (MnNCEs)

2)- Evaluation of ratio: PCEs/NCEs
Statistics:
1- The Mann-Whitney U-test was utilized to evaluate the statistical significance of the induction of micronucleated polychromatic erythrocytes (MnPCEs) and micronucleated normochromatic erythtocytes (MnNCEs) after a single administration of the 3 compounds.

2- Student's t-test was used to investigate the presence of cell toxicity by evaluating the statistical significance of the PCEs/NCEs ratio variations after arcsin transformation.
Sex:
male
Genotoxicity:
positive
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
- Catechol statistically (p< 0.05) induced micronuclei, with a  dose-dependant increase (no statistical assessment), with no variation of  the PCEs/NCEs ratio. The frequency of micronucleated cells was 0.163 +/-  0.042 % in the control group, 0.285 +/- 0.018 in the mice treated with 10  mg/kg catechol, 0.327 +/- 0.035% in the mice treated with 20 mg/kg  catechol and 0.519 +/- 0.025% in the mice treated with 30 mg/kg catechol.  
The PCEs/NCEs ratio for the groups treated with 0, 10, 20 and 30 mg/kg  catechol were 0.849 +/- 0.062, 0.880 +/- 0.025, 0.884 +/- 0.026 and 0.795  +/- 0.009.
- The interaction of catechol with other mixtures is not described here.

Micronuclei induction after a single i.p. administration of Catechol:

Dose (mg/kg)

MnPCEs/PCEs

(per animal)

MnPCEs

(% ± SD)

MnNCEs/NCEs

(per animal)

MnNCEs (% ± SD)

PCEs/NCEs

(± SD)

Control

7/3021

0.163 ± 0.042

4/3172

0.138 ± 0.036

0.849 ± 0.062

6/3034

4/3191

7/3103

6/3302

5/3082

4/3814

4/3032

5/3780

5/3073

5/3804

4/3133

3/3769

3/3110

6/3604

4/3056

4/3699

5/3030

6/3805

6/3072

8/3789

4/3047

5/3794

Catechol

10

9/3028

0.285 ± 0.018*

7/3337

0.184 ± 0.034

0.88 ± 0.025

9/3053

7/3556

8/3029

5/3456

20

9/3047

0.327 ± 0.035*

8/3339

0.184 ± 0.05

0.884 ± 0.026

10/3103

6/3600

11/3023

5/3447

30

17/3118

0.519 ± 0.025*

7/3890

0.163 ± 0.014

0.795 ± 0.009

16/3109

6/3894

15/3025

6/3853

 *p<0.05; **p<0.01

Conclusions:
Positive in PCEs but negative in NCEs. No modification in PCEs/NCEs ratios in comparison to negative controls.

After administration of single i.p. of Catechol, a statistically significant dose-dependent increase in micronucleus in ploychromatic erythrocytes was observed.
None of the dose tested led to significant variation in the PCEs/NCEs ratio, which is an index of bone marrow cell toxicity.
Executive summary:

3 male mice per group received single administration of catechol by intraperitoneal route at concentration of 0-10 - 20 - 30 mg/kg bw.18hafter the animals were sacrificed and bone marrow of femur were analysed for micronucleus. Catechol statistically induced micronuclei in a dose-dependant manner, with no variation of the PCEs/NCEs ratio.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29/05/2007 - 27/11/2008
Reliability:
1 (reliable without restriction)
Reason / purpose for cross-reference:
reference to other study
Principles of method if other than guideline:
The single cell gel electrophoresis (SCGE) assay, also known as the "comet assay", is a rapid, simple, visual and sensitive technique for measuring and analysing DNA breakage in mammalian cells. The purpose of the in vivo Comet assay following the alkaline version (pH > 13) developed by Singh et al. (1988), is to identify those agents, which induce DNA damage such as single or double DNA strand breaks (SSB or DSB), alkali-labile sites, DNA-DNA / DNA-protein cross-linking and SSB associated with incomplete excision repair sites. The advantages of the Comet assay include its demonstrated sensitivity for detecting low levels of DNA damage. The purpose of this study is to assess the genotoxic activity of the test compound in one or several target organs under these experimental conditions.
GLP compliance:
yes
Type of assay:
other: comet assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River France origin, saint-Germain-sur-l'Arbresle; France
- Age at study initiation: 5 to 10 week old
- Weight at study initiation: 156 to 192 g
- Housing: animals were housed in polypropylene cages measuring 42.5 x 26.6 x 15 cm, covered by stainless steel netted lid, in which they were placed in groups of 3 or 2 by random distribution. The animals are identified by numbered ear rings.
- Diet (e.g. ad libitum): the animals were not fasted at the treatment time.
- Water (e.g. ad libitum): no data
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 55 +/- 15%
- Air changes (per hr): 20times per hour
- Photoperiod (hrs dark / hrs light): light 12h a day
Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: distilled water
Details on exposure:
- Rats were treated with cathecol by gavage with standard volume of 10 mL/kg
- Catechol was administered in solution in distilled water, which were prepared before use. The test compound was administerd using two treatments at 24 hours interval.
- The negative control rats received distilled  water.
- the positive control rats received MNNG or dimethylhydrazine
- 3 to 6 hours after second treatment animals were sacrified and cells of the target organs were isolated.
Duration of treatment / exposure:
Two treatments at 24 hour interval and sacrifice 3 to 6 hours after the second treatment.
Frequency of treatment:
twice
Post exposure period:
no
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
200 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
400 mg/kg/day
Basis:
nominal in water
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- For stomach : MNNG, 20 mg/kg/day
- For duodenum : Dimethylhydrazine, 20 mg/kg/day
Tissues and cell types examined:
Stomach and duodenum
Details of tissue and slide preparation:
1) ISOALTION OF CELLS:
The abdominal surface of the animal is rinsed with 70% (v/v) ethanol and a 'V' shaped incision is made from the centre of the lower abdomen to the rib cage. The skin and muscles are removed to reveal the abdominal cavity.

STOMACH:
The stomach is removed, opened and rinsed with calcium- and magnesium-free phosphate buffer saline (PBS). The forestomach is discarded and the cells of gastric mucosa are isolated by enzymatic digestion following Burlinson et al. (1989) and Brault et al. (1999) as described hereafter:

The gastric mucosa is incubated in calcium- and magnesium-free Hank’s balanced salt solution (HBSS) containing 50 U/mL protease at 37 °C for 30 minutes in the oven. After the first incubation period, the mucosa is then flushed with the incubation medium to remove cells. The cell suspensions are centrifuged at 350 g for 5 minutes. The cell pellets are resuspended in HBSS containing 0.25 % dispase II and the suspensions are incubated at 37 °C for 15 minutes in a hot-bath.
After the second incubation period, 0.5 mL of fetal calf serum is added and the cell suspensions are filtered through a 150 µm nylon filter. The cell suspensions are then centrifuged at 350 g for 5 minutes and the cells are resuspended in HBSS.

DUODENUM:
The totality of the duodenum is removed and rinsed with calcium- and magnesium-free phosphate buffer saline (PBS). Cells of duodenum are isolated by enzymatic digestion following Evans et al. (1992) as described hereafter.

Firstly, a ligature is tightened at one end of each duodenum sample. Then, a mix of collagenase XI and dispase I (300 U/ml, 0.02 %) is directly injected into the sample of duodenum using a syringe and a second ligature is placed on the other end of the sample of duodenum. The sample of duodenum filled with the enzymatic mix is then incubated for 30 minutes at 37 °C in a hot-bath, in 10 mL of HBSS. After the incubation period, around 0.2 mL of fetal calf serum is added. The sample of duodenum is then opened and carefully scrapped in order to facilitate the cell dissociation. After that, the cell suspension is centrifuged 5 minutes at 150 g. The cell pellets are resuspended in HBSS.

For both tissues: the proportion of viable cells was determined with the help of a Malassez haemocytometer using Trypan blue technique before preparing slides to be assessed for DNA fragmentation.

2) COMET ASSAY

The Comet assay is performed under alkaline conditions essentially following the procedure of Singh et al. (1988). At least three slides are prepared for each animal and there was 4 animals per group, i.e. at least 12 slides per type of treatment (negative or positive control and two dose levels). There was 50 cells per slide that are randomly scored, i.e. 150 cells per animal.

The essential steps of comet assay are successively, layering of cells mixed with low melting point agarose (over coated glass microscope slides), lysis (to lyse the cell and nuclear membranes and other proteins), unwinding of DNA, electrophoresis, neutralization, staining and scoring.

2.1) Dried slides preparation (pre-layering)

Conventional slides are dipped in a 1.5 % normal melting point agarose in PBS while it was hot. After gently remove, underside of slides was wiped in order to remove excess agarose. The slides were then laid in a tray on a flat surface to dry.

2.2) Slide preparation

Before use, a volume of 85 µl of 0.8% of Normal Agarose (NA) was added on microscope slide pre-layered with 1.5% of NA and cover with a glass coverslip. Slides were placed on a slide tray resting on the ice packs until the agarose layer hardens (3 to 5 minutes). Around 3 x 104 cells of the different concentrations tested were mixed with 75 µl of 0.5% of Low Melting Point Agarose (LMPA) kept at 37 °C and added on microscope slide after gentle slide off the coverslip. They were then covered with a new glass coverslip. Slides were placed on a slide tray on ice packs for 3 to 5 minutes.

2.3) Lysis at pH=10

After the top layer of agarose has solidified, the glass coverslips are removed and the slides are immersed for at least 1 hour at + 4 °C in the dark in a lysing solution consisting of 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10, to which 1% Triton X-100 and 10% DMSO are freshly added (pH adjusted to 10 with NaOH).

2.4) Unwinding, electrophoresis and staining

The slides were then removed and placed on a horizontal gel electrophoresis unit and the unit filled with freshly prepared alkaline buffer (1 mM EDTA and 300 mM NaOH, pH > 13) to around 0.25 cm above the slides. In order to avoid excessive variation across the groups for each electrophoretic run, for each animal, only one of the quadriplicate slides is precessed in each run. The cells were exposed to the alkali for 20 minutes to allow the DNA unwinding, and expression of single-strand breaks and alkali-labile sites. Next, electrophoresis was conducted for 20 minutes at 0-4°C by applying an electric current of 0.7 V / cm (25 V / 300 mA). All these steps were conducted sheltered from the daylight to prevent the occurrence of additional DNA damage. After electrophoresis at pH >13, the slides were neutralized twice for 5 minutes with 0.4 M tris (pH 7.5) and the DNA was exposed for 5 minutes to absolute ethanol in order to preserve all the comet assay slides. Subsequently, the slides were airdried and then stored at room temperature until they were scored for DNA migration.
Just prior to scoring, the DNA was stained using Propidium Iodide (20 µg/mL distilled water; 30 µL/slide).

2.5) Image analysis

Slides were examined at 250 x magnification using a fluorescent microscope (Leica Microscopy and Scientific Instruments Group, Heerbrugg, Switzerland) equipped with an excitation filter of 515-560 nm and a barrier filter of 590 nm, connected through a gated CCD camera to Comet Image Analysis System, version 4.0 software (Kinetic Imaging Ltd, Liverpool, UK).

At least three slides were prepared for each animal and there was 4 animals per group, i.e. at least 12 slides per type of treatment (negative or positive control and two dose levels).

2.6) Tail parameters

Olive Tail Moment (OTM) preconised by Olive (1993) was used to evaluate DNA damage. The OTM, expressed in arbitrary units, was calculated by multiplying the percentage of DNA (fluorescence) in the tail by the length of the tail in µm (B. Hellman et al., 1995; E. Rojas et al., 1999). The tail length was measured between the edge of comet head and the end of the comet tail.

A major advantage of using the OTM as an index of DNA damage was that both the amount of damaged DNA and the distance of migration of the genetic material in the tail were represented by a single number (J. Ashby et al., 1995).


Evaluation criteria:
Cytotoxicity:
Cytotoxicity was determined on a small sample of each isolated cell suspension following the Trypan blue vital dye exclusion technique.
In accordance with a recognized group of scientists, the decrease in the viability should not be more than 30 % when compared to the concurent control. Cell viability in the target tissue that is below 70 % of that in the control animals may thus be considered excessive.
Therefore, according to the data obtained from the cytotoxicity assessment, doses are actually selected for genotoxicity assessment (two dose levels are chosen for each structured organ).
At least 1.2 x 105 viable cells are required for proceeding to slides preparation (4 slides with 3 x 104 viable cells per slide).

Acceptance criteria for result:
A study is accepted if both following criteria are fulfilled:

- In the solvent control group, the OTM median must be lower than 8.
- In the positive control groups, the OTM median must be statistically increased compared to the control group.

A compound is found to demonstrate genotoxic properties against the target organ if it results in a statistically significant increase in the OTM median compared with the negative control group and if the genotoxicity detected shows a dose-effect relationship.

A compound is found to have no genotoxic effect on the target organ if it does not comply with any of the criteria listed above.

If neither situation occurs, the results are discussed case by case and another independent study may be implemented after modifying the dose range taking into account all available relevant data. Any complementary assay will be the subject of a new study plan.

The criteria are not absolute but do constitute an aid to decision, which will make it possible to reach a conclusion in most cases.
Statistics:
Statistical analysis using non-parametric tests: Since the OTM frequencies and other tail parameters do not follow a gaussian distribution (E. Bauer et al., 1998), the non-parametric Kruskall-Wallis test was used to display a possible dose-effect relationship. Moreover the statistical significance of differences in the median values between each group versus the control group was determined with the non-parametric Mann-Whitney U-test.

Statistical analysis using parametric tests: First, for each assay, a box plot on Olive Tail Moment over group was realized in order to show the distribution per slide per animal. The statistical test for treatment effect was a trend-test using linear contrast in the treatment group: an estimation of the differences between each treated group and the negative control group was provided with its associated 95 % confidence interval. This statistical method was particularly well adapted for interpretation of in vivo data with possible inter-individual variations.

Sex:
male
Genotoxicity:
positive
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The in vivo comet assay performed under alkaline conditions, i.e. pH > 13 (Alkaline Single Cell Gel Electrophoresis) in the OFA Sprague Dawley male rats, stomach and duodenum, after two treatments by oral route at 3 dose levels (the maximum tolerated dose, MTD, 50 and 25% MTD), followed by one expression time of 3 to 6 hours after the last treatment. The test CATECHOL was dissolved in distilled water up to a maximum concentration of 80 mg/mL (toxicity assay) or 40 mg/mL (genotoxicity assay) and administered at a dose volume of 10 mL/kg, giving final doses of 800 and 400 mg/kg, respectively.
The different inferior dilutions were also performed with distilled water. Regarding the positive reference substance, both MNNG and dimethylhydrazine were dissolved in distilled water and administered under a dose volume of 10 mL/kg by oral route.

PRELIMINARY TEST RESULT

Results of the toxicity test by the oral route in OFA Sprague Dawley male rats (preliminary and confirmatory assays):
In this toxicity assay, two groups of 4 male rats were dosed orally twice at 800 and 400 mg/kg/day.
The maximum tolerated dose of CATECHOL was set at 400 mg/kg/day (x2) by oral route in OFA Sprague Dawley male rats. Indeed, the dose of 800 mg/kg/day (x2) elicited strong clinical signs, as described below and at the dose of 500 mg/kg/day (x2), 3 animals out of 4 died 25 min after the first
treatment. In return, the dose of 400 mg/kg/day (x2) induced no clinical signs.
DOSE OF 400 MG/KG/DAY (X2)
The dose level of 400 mg/kg/day (x2) induced no death or clinical signs in the four male rats after the 1st or the 2nd treatment.
DOSE OF 800 MG/KG/DAY (X2)
The first treatment at the high dose level of 800 mg/kg/day (x2) induced very strong clonic convulsions in the four male rats, 15 min to 30 min after the first treatment. For ethical reasons, the animals were euthanasied and no second treatment was performed.
In order to confirm that the dose of 400 mg/kg/day (x2) is the actual maximum tolerated dose in OFA Sprague Dawley male rats, a 2nd toxicity assay with a third intermediary dose of 500 mg/kg/day (x2) was implemented.
DOSE OF 500 MG/KG/DAY (X2)
The first treatment at the dose level of 500 mg/kg/day (x2) induced strong clonic convulsions in the four male rats, 15 min to 30 min after the first treatment. Three animals out of 4 died and for ethical reasons, the 4th animal was euthanasied and no second treatment was performed.
Under these conditions, the dose of 400 mg/kg/day (x2) was retained as the maximum dose to be tested in the comet assay. Two inferior doses were also tested, i.e. 200 and 100 mg/kg/day (x2).

MAIN EXPERIMENT
At least 5 male rats per dose were treated orally twice with 400, 200 and 100 mg/kg/day (x2) CATECHOL for the in vivo comet assay on stomach and duodenum. The 2 highest doses of 400 and 200 mg/kg/day (x2), giving acceptable cell viability using the Trypan blue vital dye exclusion technique, i.e. >70%, were analysed first. At sponsor’s request, the 3rd dose of 100 mg/kg/day (x2), also presenting a cell viability above 70%, was analyzed and the results presented in the Final report. The test results are summarized in Tables.
The historical data for negative control and positive control were constituted with an assay after two treatments followed by one sampling time, 3 to 6 hours after the last treatment.
Significant increases in the mean OTM median values were noted in the groups treated with MNNG and dimethylhydrazine, demonstrating the sensitivity of the animal strain used to specific clastogenic agents on stomach and duodenum. Thus, the validity criteria for the test were fulfilled and the test was valid. The weight homogeneity of the animals used in this test after random-distribution was verified by comparing the weight mean of the treatment groups with that of the control group (Student t-test). There was no statistically significant difference between the weights of animals treated with the test item and those of control rats.

The results of the cellular viability determination upon the Trypan blue exclusion method are presented: the calculated relative viabilities for stomach and duodenum cells were superior to 70 %.

However, it is to be noted that very strong clonic convulsions were observed 5 minutes after oral administration of Catechol at the dose of 400 mg/kg/day (x2), leading to one death between 30 minutes and 2 hours after the 1st treatment. Furthermore, the 4 animals were trembling after the 2nd treatment at 400 mg/kg/day. The animals treated with the two inferior doses of 200 and 100 mg/kg/day elicited no clinical signs.
Furthermore, the observation of slides during image analysis of stomach and duodenum cells, showed a very low cell density at the two highest doses analysed of 400 and 200 mg/kg/day (x2). This low cell density indicates the presence of cell lysis that could not be identified with the Trypan blue vital dye exclusion technique nor with the measurement of ghost cells.

GENOTOXICITY IN RAT STOMACH CELLS
No statistically significant increase in the OTM medians was observed in rat stomach cells treated with the three doses of Catechol, i.e. 400 – 200 and 100 mg/kg/day (x2). Indeed, the values of median OTM for 600 cells were of 0.81 – 0.35 and 1.13 at 400 – 200 and 100 mg/kg/day (x2), respectively, vs. 2.26 in the negative control group. The OTM medians at the three tested doses were under the value of OTM median for the control group.
The F test (F of Snedecor) showed a statistically significant difference between group OTM variance (p<0.0001). Due to this non-homogeneity of variance between OTM groups, statistical analysis was performed using non-parametric tests.
The non-parametric statistical assessment allowed to display a significant dose-response relationship (Kruskall-Wallis, p<0.0001).
The pair wise analysis using Mann-Whitney showed statistically significant decreases in the median OTM of the three tested doses of 400 – 200 and 100 mg/kg/day (x2), vs. the negative control (Mann-Whitney, p<0.0001). Nevertheless, this decrease had no meaning in terms of genotoxicity.
Regarding the percentages of ghost cells, no increase compared to the negative control was observed.
Under these conditions, Catéchol was not considered as a DNA strand breaks and/or alkali-labile sites inducer on stomach cells in the rat.

GENOTOXICITY IN RAT DUODENUM CELLS
Statistically significant increases in the OTM medians were observed in duodenum cells from rats, treated with the doses of 200 and 100 mg/kg/day (x2) of Catéchol. Indeed, the values of median OTM for 600 cells (4 animals tested per group) reached 3.38 and 4.64 at the two doses of 200 and 100 mg/kg/day (x2), respectively, vs. 1.48 in the negative control group (Tables 3 and 10). At these two dose-levels of 200 and 100 mg/kg/day (x2), two animals out of the four treated, presented particularly strong increases in the median OTM, with values reaching 6.07 and 4.21 at the dose of 200 mg/kg/day (x2) and 9.73 and 7.29 at the dose of 100 mg/kg/day (x2) (for 150 cells observed by animal, Table 10). In return, the dose of 400 mg/kg/day (x2) did not induce a statistically significant increase in the median OTM, with a value of 1.28. This value was under the value of OTM median for the control group.
It is noteworthy that the highest median OTM was observed at the lowest dose tested of 100 mg/kg/day (x2). The decrease in the values of median OTM at the two upper doses of 400 and 200 mg/kg/day (x2), compared to the dose of 100 mg/kg/day (x2), is most probably related to toxicity of the test item Catechol, consistent with the clinical signs described above, i.e. strong clonic convulsions were observed 5 minutes after oral administration of Catechol at the dose of 400 mg/kg/day (x2), leading to one death between 30 minutes and 2 hours after the 1st treatment and the 4 animals were trembling after the 2nd treatment at 400 mg/kg/day. In return, no clinical signs were observed after the treatments at 200 and 100 mg/kg.
Furthermore, the cytotoxicity evaluated with the Trypan blue vital exclusion method and the measurement of the percentage of ghost cells during image analysis, did not permit to evaluate the potential cytotoxicity at the three doses tested.
The bell-shaped curve effect observed is considered as an indicator of the genotoxic activity of Catechol. No dose without genotoxic effect could be determined at a concentration lower than 100 mg/kg/day (x2). Therefore, it would be useful to implement a complementary assay in order to investigate if Catechol induces a genotoxic effect at doses lower than 100 mg/kg/day (x2) and to determine the highest dose under 100 mg/kg/day (x2) without genotoxic activity.
Regarding the statistical assessment, the F test (F of Snedecor) showed a statistically significant difference between group OTM variance (p<0.0001). Due to this non-homogeneity of variance between OTM groups, statistical analysis was performed using non-parametric tests.
The non-parametric statistical assessment allowed to display a significant dose-response relationship
(Kruskall-Wallis, p<0.0001). It is noteworthy that this is a reverse dose-effect relationship, i.e. there is a decrease in the median OTM when increasing the doses of Catechol.
The pair wise analysis using Mann-Whitney showed statistically significant increases in the median OTM of the two tested doses of 200 and 100 mg/kg/day (x2), vs. the negative control. In return, the dose of 400 mg/kg/day (x2) did not induce a statistically significant increase in the median OTM.
Regarding the percentages of ghost cells, no increase compared to the negative control was observed.
Under these conditions, Catechol was considered as a DNA strand breaks and/or alkali-labile sites inducer on duodenum cells in the rat.
HYPOTHESIS ON THE GENOTOXICITY OF CATECHOL
The results obtained in the in vivo comet assay on stomach in presence of CATECHOL, i.e.:
• no statistically significant increases in DNA strand breaks at non-lethal dose on rat stomach cells after oral administration.
• statistically significant decreases in the median OTM of the three tested doses and displaying significant reverse dose-response relationship
• absence of signs of cytotoxicity at the three doses tested with the Trypan blue vital exclusion method
• no significant increase in the measurement of the percentage of ghost cells on the slides
• very low cell density observed during image analysis indicating a probable cell lysis due to cytotoxicity and /or highly damaged cells with loss of information and in duodenum, i.e. :
• statistically significant increases in DNA strand breaks, with the highest increase of median OTM the lowest dose tested, decrease in the values of median OTM at the two upper doses and statistically significant increases at the highest dose tested
• displaying a significant reverse dose-response relationship, decrease in the median OTM when increasing the doses of Catechol, i.e. a bell-shaped curve response.
• absence of signs of cytotoxicity at the three doses tested with the Trypan blue vital exclusion method
• no significant increase in the measurement of the percentage of ghost cells on the slides
• very low cell density observed during image analysis indicating a probable cell lysis due to cytotoxicity and /or highly damaged cells with loss of information lead to different hypothesis.

1/ The test item Catechol seems to induce excessive fragmentation of DNA. This extreme DNA fragmentation may be caused by:
- DNA strand breaks, single or double DNA strand breaks (SSB or DSB).
- Another possible mechanism could be DNA fragilization through the formation of alkali-labile sites. In such cases, the formation of apurinic or apyrimidic sites by excision of damaged bases by a DNA glycosylase, may alter and fragilize DNA. The alkali-labile sites are stable up to a pH of 12.5 but are
eliminated at a pH of 13, as in the in vivo rodent alkaline assay, causing DNA strand breaks (Eastman & Barry, 1992).
The difference of genotoxic activity observable in stomach and in duodenum cells may be related to the fact that the concentration of Catechol in contact with the tested organ is higher in the stomach than in the duodenum.
The observation of the slides at the two highest doses tested of 400 and 200 mg/kg/day (x2) during image analysis showed a total absence of ghost cells, as well as a very low cell density, that may be related to the total lysis of the cells after DNA fragmentation. The strong pulverization of DNA hindered correct staining of the DNA and thus observation and scoring of comet cells. The limit of detection has been trespassed.
Furthermore, the lowest dose tested of 100 mg/kg/day (x2), inducing a genotoxic activity in the comet
assay, presented a normal cell density on the slides during image analysis.
The absence of signs of cytotoxicity at the three doses tested using techniques such as Trypan blue vital exclusion method and the measurement of the percentage of ghost cells on the slides may be due to the fact that some ghost cells are not visible on the slides. Indeed, as stated by Burlinson (2007), for certain chemicals, the enhanced DNA migration is not observed in the in vivo rodent alkaline comet assay, despite the presence of necrosis or apoptosis in the target organs. In that case, the cytotoxicity is directly exerted by Catechol.

2/ The test item Catechol is most probably an oxidizing agent, producing free radicals damaging
DNA. In order to show that reactive oxygen species may be involved in the genotoxic activity of Catechol, an in vivo rodent alkaline (pH>13) comet assay adding a specific endonuclease enzyme, e.g. formamidopyrimidine-DNA glycosylase (Fpg), could be performed at lower doses than the ones tested in the current study.
The Fpg enzyme presents a DNA glycosylase activity, associated with an AP lyase activity leading to the formation of AP sites in DNA followed by the formation of single strand DNA breaks. In a normal cell, DNA repair is achieved by the action of other enzymes (DNA polymerase and DNA ligase). Under the conditions of the Comet assay, these enzymes are present until the sacrifice of animals but are absent during the Unwinding, Electrophoresis and staining steps of the Comet Assay. The adjunction of Fpg during the Comet assay allows to increase the number of breaks in DNA induced through an oxidizing mechanism and leads to an increase in comet response in case of DNA oxidative damage induced by Catechol. The expected results after treatment with Catechol and addition of Fpg, would be an increase in median OTM values, compared to the OTM values of cells exposed to Catechol alone, without addition of Fpg enzyme.
Nevertheless, performing this new comet assay will not modify the results and conclusions of the current study.
The decrease in the median OTM in stomach and duodenum cells when increasing the doses of Catechol and the reverse dose-response relationship indicate the possible existence of a bell-shape curved. As described by Burlinson et Al (2007), in some cases it is also possible to detect a decrease in DNA migration, as observed with Catechol in stomach and duodenum cells, due to:
- the loss of heavily damaged or dying cells during sample processing or electrophoresis, and /or secondary toxicity induced by Catechol.
- The downturn phenomenon in the dose-response curve may also be attributed to an altered bioavailability at higher dose levels.
Due to the bell-shape curve effect of the genotoxic activity of Catechol, it is not excluded that Catechol may display genotoxic activity both in stomach and duodenum cells at dose-levels lower than 100 mg/kg/day (x2).

CONTROL OF CONCENTRATION IN DOSING FORMULATIONS
A satisfactory agreement was observed between the actual and nominal concentrations of CATECHOL in treatment solutions used in the in vivo comet assay performed on stomach and duodenum. Indeed, the deviations from nominal concentrations were within an acceptable range of ±10%. Furthermore, solutions of CATECHOL can be considered as stable at –18°C, during two months.

Table: In vivo comet assay isolated rat stomach cells recapitulative table

 

In vivo Comet assay in isolated rat stomach cells

groups

compound

doses in mg/kg/day (x2)

OTM Median for 600 cells (1)

F Snedecor (homogeneity of variances)

NON PARAMETRIC statistical evaluation

NON PARAMETRIC statistical evaluation

Relative ratio of ghost cells (4)

 

 

 

 

 

p kruskall-wallis (2)

P Mann-whitney (3)

 

solvent control

distilled water

0

2.26

p< 0.0001

p< 0.0001

-

-

treated

catechol

400

0.81

p< 0.0001

p< 0.0001

p< 0.0001

0.54

treated

catechol

200

0.35

p< 0.0001

p< 0.0001

p< 0.0001

0.59

treated

catechol

100

1.13

p< 0.0001

p< 0.0001

p< 0.0001

0.96

Positive control

MNNG

20 mg/kg/day (x1)

5.37

-

-

p< 0.0001

0.95

 

A statistically significant linear trend in nuclear fragmentation, excluding positive control was revealed

by the Kruskall-Wallis test. The analysis showed a statistically significant dose-related decrease in the

median OTM at the three tested doses of 400 – 200 and 100 mg/kg/day (x2). The trend had hence no

meaning in terms of genotoxicity.

 

Table: In vivo comet assay isolated rat duodenum cells recapitulative table

 

In vivo Comet assay in isolated rat duodenum cells

groups

compound

doses in mg/kg/day (x2)

OTM Median for 600 cells (1)

F Snedecor (homogeneity of variances)

NON PARAMETRIC statistical evaluation

NON PARAMETRIC statistical evaluation

Relative ratio of ghost cells (4)

 

 

 

 

 

p kruskall-wallis (2)

P Mann-whitney (3)

 

solvent control

distilled water

0

1.48

p< 0.0001

p< 0.0001

-

-

treated

catechol

400

1.28

p< 0.0001

p< 0.0001

N.S.

0.79

treated

catechol

200

3.38

p< 0.0001

p< 0.0001

p< 0.0001

0.94

treated

catechol

100

4.64

p< 0.0001

p< 0.0001

p< 0.0001

1.10

Positive control

Dimethylhydrazine

20 mg/kg/day (x1)

8.94

-

-

p< 0.0001

0.87

 

(1) for 450 cells in positive control

(2) total group without positive control

(3) OTM values obtained in treated group compared to OTM values obtained in solvent control group

(4) corresponds to the percentage of ghost cells per treated group/ percentage of ghost cell in negative control group

 A statistically significant linear trend in nuclear fragmentation, excluding positive control was revealed by the Kruskall-Wallis test.

Conclusions:
Positive
Executive summary:

The test item CATECHOL (batch FPC0619301) provided by RHODIA was investigated by the means of the in vivo comet assay on stomach and duodenum, under alkaline conditions (SCGE) in the male OFA Sprague Dawley rats treated orally twice with 400, 200 and 100 mg/kg/day, with one sampling time 3 to 6 hours after the last treatment. Following the results of the toxicity assay, the maximum tolerated dose (MTD) determined was of 400 mg/kg/day. This dose was retained as the maximum dose to be tested, as well as two lower doses corresponding to MTD/2 and MTD/4. These two doses were not toxic, indeed, no clinical signs were observed. Under these experimental conditions, CATECHOL induced no statistically significant increases in DNA strand breaks at non-lethal dose on rat stomach cells after oral administration. CATECHOL is hence devoid of genotoxic activity on the stomach.

In return, CATECHOL induced statistically significant increases in DNA strand breaks at non-lethal doses on rat duodenum cells after oral administration, with the highest increase of median OTM at the lowest dose tested of 100 mg/kg/day (x2). Furthermore, the very low cell density observed at the two highest doses tested during image analysis, indicates a probable cell lysis due to cytotoxicity

and /or highly damaged cells with loss of information. Regarding the criteria described in paragraph 9, atest item is found to demonstrate genotoxic properties against the target organ if it results in a statistically significant increase in the OTM median compared with the negative control group and if the genotoxicity detected shows a dose-effect relationship. In fact the results showed a statistically significant increase in the median OTM, but with an inverse dose-effect relation, with a bell-shaped curve response. The test item Catechol was thus considered as genotoxic on rat duodenum cells. It would be useful to implement a complementary assay under the same experimental conditions on duodenum cells only, but using doses lower than 100 mg/kg/day (x2), in order to determine if there is a high dose without genotoxic effect under the dose level of 100 mg/kg/day (x2). Under these conditions, CATECHOL was considered as a DNA strand breaks and/or alkali-labile sites inducer on duodenum cells. A satisfactory agreement was observed between the actual and nominal concentrations of CATECHOL in treatment solutions used in the in vivo comet assay performed on stomach and duodenum. Indeed, the deviations from nominal concentrations were within an acceptable range of

±10%.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Only male animals were studied. OECD Guidelines are provided for mammalian liver cells, whereas oesophageal epithelial cells were examined in this study, which is acceptable. During the main study, the uptake of tritiated-thymidine was determined by liquid scintillation counting (which is susceptible to interfere with S-phase cells), instead of autoradiography. Other compounds were tested simultaneously, but none of them was a proved positive control. It seems that the results included the data from different studies, are not in accordance with the described test conditions.
Principles of method if other than guideline:
uptake of tritiated-thymidine
GLP compliance:
not specified
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Eppley Institute breeding colony
- Age at study initiation: 4-5 week old
- Weight at study initiation: about 100 g.
- Housing: 5 per cage on granular cellulose bedding which was changed 3 times weekly, maintained on Wayne Lab Blox (Allied Mills, Inc., Chicago, IL)
- Diet (e.g. ad libitum): the semi-purified diet consisted of 46.5% corn-starch, 20% vitamin-free casein, 15% dextrose, 8% Alphacel cellulose fiber, 6% corn oil, 3.5% mineral mix (Williams-Briggs modified), and 1% vitamin fortification mix, all from Teklad, Inc., Madison, WI.
- Water (e.g. ad libitum): tap water
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/-2°C
- Humidity (%): 40 +/-5%
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h light/12h dark
Route of administration:
other: drinking water and/or diet
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water
Details on exposure:
- Rats were treated with cathecol in drinking water or in semipurified diet.
- Catechol was administered in solution in distilled water, which were freshly prepared each day and were given in brown bottles as drinking water for 7 days. The rats received semi-purified diet during this period. Each experiment contained 18 rats, including 6 control and 3 groups of 4 rats.
- Semipurified diet: solutions of catechol (20 g/ml distilled water) were mixed with the appropriate amount of semi-purified diet every second day and were fed to rats together with tap water for drinking. 
- The control rats received a semi-purified diet and distilled or tap water, as appropriate, for drinking.
- Body weight were followed, and levels of catechol were chosen that allowed the rats to gain at least 10 g/week. The control gained 50-60 g/week).
Duration of treatment / exposure:
7 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
Drinking water: 1, 2, 4 and 8 g/L water; Diet: 4 g/kg diet
Basis:
nominal conc.
No. of animals per sex per dose:
4 animals per dose, and 6 for control group.
Control animals:
yes
Tissues and cell types examined:
[3H]dThd-I into the oesophageal epithelial DNA was measured by ip injection 1 hour before the rats were sacrificed. The oesophageal epithelial DNA was isolated from each rat and then applied to glass fiber disk, counted for radioactivity in a toluene based scintillator, and assayed colometrically.
Sex:
male
Genotoxicity:
positive
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not examined
Additional information on results:
 Catechol at concentration of 1-8 g/l significantly enhanced the uptake of tritium-labelled thymidine relative to that in untreated rats.
In another experiment, autoradiography was used to show that the increased 3H-thymidine due to treatment with catechol was associated with an increased number of cells undergoing replicative DNA synthesis. One group of four 50 g male rats was given 0.1 % catechol in the drinking water for 7 days, and another group was given tap water. All rats received then 3H-Thymidine and were killed one hour later. Autoradiography showed labeling indices in the oesophageal epithelial basal cells of 23.9 for the treated rats and 14.0 for the control (percent labeled cells)). These results were significantly different, with p < 0.001.
When Catechol was administered in a semipurified diet, [3H]dThd-I was significantly enhanced, and the effect seemed more consistent than that for an equal concentration of catechol given in drinking water at the same time.

[3H]dThd-I into esophageal epithelial DNA of rats treated with Catechol in drinking water or semipurified diet

 

 

Conc. g/L water or g/kg diet

[3H]dThd-I, mean ± SE (No. of rats)

 

Vehicle

Treated rats

Control rats

P-value*

Catechol

water

1

140±7 (26)

100± 4(26)

<0.001

water

2

130±8 (12)

100± 6(17)

<0.01

water

4

147± 8(11)

100± 4(17)

<0.001

water

8

165± 18(3)

100± 9(5)

<0.02

Catechol

diet

4

172± 18(7)

100± 5(10)

<0.001

* Comparison between treated and control groups

 

Conclusions:

After oral administration of Catechol to rats, and incorporation of [3H]dThd into esophageal epithelial cells, the authors measured the radioactivity incorporated in esophageal epithelial DNA and showed a dose-dependent increase, indicated a DNA synthesis stimulated by catechol.
Executive summary:

4 rats per group were treated with cathecol in drinking water  for 7 days at doses of 0, 1, 2, 4 and 8 g/L water. The rats received semi-purified diet during this period. Tritium-labelled thymidine was injected 1 hour before sacrifice of the rats into the oesophageal epithelial and the oesophageal epithelial DNA was isolated to measure the radioactivity. Catechol at concentration of 1-8 g/l significantly enhanced the uptake of tritium-labelled thymidine relative to that in untreated rats in a dose dependent manner. Catechol was able to stimulate the DNA synthesis.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

In vitro genotoxicity studies:

A lot of in vitro genotoxicity studies exist on catechol: 60 were reported in the IUCLID 5 dossier, only data considered as validity 2 according to Klimish scale are summarized below.  

MUTAGENICITY

Mutagenicity studies on bacteria strains according to or similar to OECD 471:

-18 Ames tests had been reported in this IUCLID 5 dossier, 5 were attributed validity 2 according to Klimish scale. None of the tests was performed according to current OECD guideline 471. Some weakly positive results were noted in limited and poorly number of assays. In the 3 studies considered as valid, the results indicated no mutagenic activity with and without S9 mix from liver Rat induced with Aroclor in all strains of Salmonella Typhimurium tested (TA 98,TA 100,TA 1535,TA 1537,TA 1538).  In the study of Martinez (2000), a plate mutation reverse assay was performed with Escherichia coli WP2uvrA/pKM101; IC203 and ICI 188. The bacteria were exposed to at least 5 doses of catechol, but only highest doses are reported (with no observable toxicity):1000 - 2000 - 3000 µg/plate with and without metabolic activation system from rat liver induced. Catechol was found to be an oxidative mutagen, the mutagenicity response being positive in the IC203 strain and negative in the IC188 strain. Mutagenesis by catechol is completely inhibited by S9. Cytotoxic effects: IC203 is more sensitive to the toxic effect than IC188 and the presence of S9 had a protective effect. In this study (Watzinger, 2007), Catechol was tested in a Ames test using a screening micromethod assay, with Salmonella typhimurium strains TA1537,TA98, TA100 and TA102, with and without metabolic activation system from rat livers or kidneys induced by Aroclor 1254. Doses tested ranging from 0 to 5000 µg/plate. Catechol induced a clear mutagenic activity in strain TA102 without metabolic activation and in presence of metabolic activation by kidney S9-mix, in this screening micromethod assay of the Ames's test performed without repetition. No mutagenic activity was observed in the Salmonella typhimurium strains TA1537, TA98 or TA100 tested in absence or in presence of metabolic activation system.The positive controls induced the appropriate responses in the corresponding strains. No repetition was performed in the test. Salmonella typhimurium TA 102 (like Escherichia coli) is revert mutant with AT base pairs. The positive response of strain TA 102 is probably due to a substitution of AT to GC by oxidative mechanism.

 

Mutagenicity studies on mammalian cells similar to OECD 476: 4 studies were available and 3 were validity 2 and were used to evaluate the mutagenicity effect of catechol on mammalian cells.

- 2 references of Mouse lymphoma assays with mouse lymphoma L5178Y cells were tested without metabolic activation system and were considered as validity 2.

 In the study of Wangenheim (1988), cells were treated at doses of 0- 1.145 - 2.874 - 5.516 - 11.450 - 28.736 µg/mL for 4 hours, Catechol was found to increase the mutation frequency in a non dose dependent manner.

 

In the study of McGregor (1988), cells were treated at doses of 2.5 - 4 - 5.5 - 7 - 8.5 µg/mL. The results indicated positive effects on cells treated without metabolic activation system. The lowest effective dose producing cytotoxic effect in the absence of metabolic activation was the lowest dose tested, i.e. 2.5 µg/ml. The mutagenic effects of catechol were not dose dependent. The mutagenic potential of catechol was completely negated by coincubation with SOD. It was noticeable that SOD had little effect upon toxicity. 

 

- Another reference on In vitro Mammalian Cell Gene Mutation Test was performed by Tsutsui (1997) on Syrian Hamster Embryo (SHE) cell. The cells were treated for 48 h at doses: 1 - 3 - 10 - 30 µM without metabolic activation system, and 4 hours expression time. 10E5 cells were plated on each of ten dishes per group with medium containing 3.3 µg/ml TG (6-thioguanine) or 1.1 mMOua (ouabain). They were incubated for 7 days for colony formation. Achromatic lesions were scored, gaps and breaks were recorded. Catechol induced gene mutation at the two loci in SHE cells: both ouabain-resistant (Na+/K+-ATPase) and 6-thioguanine resistant (hprt) mutant frequencies were increased in dose dependent manner for TG.

Catechol at the concentration of 1, 3, 10 and 30 µM decreased cell survival to 85.5%, 70.2%, 28.8 % and 1.4% of untreated cells, respectively.

 

 

CONCLUSION FOR MUTAGENICITY:

Mutagenicity effects of catechol in the different in vitro models (bacteria or mammalian cells), studied without metabolic activation system, indicated positive effects sometimes linked with oxidation property of catechol. In Ames tests where the metabolic activation system was tested negative results were observed. Except with Salmonella typhimurium strain TA 102 with kidney metabolic activation system in the micromethod assay (Watsinger, 2007). The positive response of strain TA 102 is probably due to a substitution of AT to GC by oxidative mechanism.

 

CLASTOGENICITY

In vitro mammalian chromosome aberration test similar according to or similar toOECD guideline 473: 5 studies are available. 3 had validity 2 according to Klimish scale.

In the study of Stich (1981), the capacity of catechol to induce chromatid breaks and exchanges in CHO cells were evaluated after treatment with 50 µg/mL (454 µM) of catechol with and without metabolic activation system induced by Aroclor 1254-pre-treated Fisher male rats. In addition, the modulating effect of Cu2+ and Mn2+ was examined.

Catechol exhibited a chromosome-damaging potential (chromatid break or exchange). The addition of an S9 mixture reduced the clastogenic activity of catechol.

 

In the study of Tsutui (1997), Syrian Hamster Embryo (SHE) cells were treated with catechol at doses: 1 - 3 - 10 - 30 µM for 6, 24 or 48 h, for chromosome aberration or aneuploidy without metabolic activation system. After 24 h, a dose-dependant increase in the frequencies of chromosomal aberrations (mainly gaps and breaks) in SHE cells was induced by catechol at doses of 3, 10, and 30 µM. Slight aneuploidy in the near diploid range of SHE cells was significantly induced by catechol (30 µM, 48 h). No significant increase was observedat 3 and 10 µM.

 

In the study of Do Ceu Silva (2003), the induction of chromosomal aberrations by catchol in V79 cells was studied at different concentration 0 - 20 - 40 - 60 - 80 µM, pH values (6.0, 7.4 and 8.0 with and without metabolic activation system from Wistar rat liver induced with Aroclor 1254 at pH 7.4. At the same pH values, the production of hydroxyl radicals was assessed by measuring the degradation of deoxyribose. The clastogenic effect of catechol is dependent on the pH: non-significant increase in chromosomal aberrations at pH 6.0, at any dose-level; at pH values 7.4 and 8.0, catechol induced significant levels of chromosomal aberrations at lower doses (< 80 µM); at higher doses and in a pH-dependent manner, it showed a significant induction of multi-aberrant cells (with more than 10 chromosomal aberrations), which represent in some cases more than 50% of the aberrant cells.

S9 mix, catalase + SOD, and catalase or SOD alone do not lead to a significant reduction on the level of chromosomal aberration induced by the catechol.

The addition of S9mix and SOD+catalase lead to a reduction in the number of multi-aberrant cells and it was observed that it can produce OH° radicals, so a participation of a radical-type mechanism can not be excluded in the genotoxicity of catechol.

 

 

In vitro mammalian cell micronucleus test according to or similar to OECD 487: 2 studies were available and considered as validity 2 according to Klimish scale. 

In the study of Yager (1990), Human lymphocytes were treated without metabolic activation system with catechol at doses of 0.5, 5, 50, 100, 200 and 250 µM. Cell viability and micronuclei were checked using antikinetochore antibody. Statistical significant increase in micro nucleated cells was observed. Statistical significant dose related increase in kinetochore-positive micronucleated cells were also observed, suggesting that catechol was likely aneuploidy-inducing agents in Human lymphocytes.

 

In a study (Watzinger, 2007), Catechol was tested in an in-vitro micronucleus test, without and with metabolic activation by means of liver or kidney S9 -mix on L5178Y mouse lymphoma cells, by the micromethod.

In this study, Catechol induced a clear genotoxic effect on L5178Y mouse lymphoma cells, both without and with metabolic activation by means of liver or kidney S9-mix, that can be revealed by the in vitro micronucleus assay performed in micromethod, without repetition. No significant difference in the genotoxic activity induced in presence of liver or kidney S9-mix was observed up to concentration of 19.53µg/ml.

The positive controls induced the appropriate responses in the corresponding assays.

Catechol was positive in an in-vitro micronucleous test, with and without metabolic activation (S9 -liver mix or S9-kidney mix), using the micromethod.

 

CONCLUSION FOR CLASTOGENICITY:

In in vitro mammalian chromosome aberration test, Catechol exhibited a chromosome-damaging potential (chromatid break or exchange). The addition of an S9 mixture reduced the clastogenic activity of catechol.

The clastogenic effect of catechol was shown to be dependent on the pH (increase with pH increase). The addition of S9 mix and SOD+ catalase lead to a reduction in the number of multi-aberrant cells and it was observed that it can produce OH° radicals, so a participation of a radical-type mechanism can not be excluded in the genotoxicity of catechol.

In vitro micronucleus assay presented clear positive effect with Catechol with and without metabolic activation system.

 

DNA DAMAGE

24 studies were available, 12 had validity 2 according to Klimish scale.

 

In Vitro Sister Chromatid Exchange Assay in Mammalian Cells according to or similar to OECD guideline 479:

In the study of Tsutsui (1997), Syrian Hamster Embryo (SHE) cells were treated without metabolic activation system with catechol at doses of 0.11- 0.33- 1.1 - 3.3 - 11 µg/L (1 - 3 - 10 - 30 - 100 µM) in presence of BrdU. Three hours before the end of treatment, colcemid was added and metaphase chromosomes were prepared. The diploid number of chromosomes were analysed for SCE frequency.

Similar experiments were performed 2 or 3 times, and the results obtained were reproducible. 

Negative results were obtainedat 1and 3 µM. SCE in SHE cells occured with catecholat 10µM (11.06 SCEs/cell) and 30 µM (15.40 SCEs/cell). At 100 µM, catechol was overly toxic to obtain SCEs data. Catechol was also cytotoxic in dose dependent mannerat 10and 30 µM.

 

Unscheduled DNA synthesis (UDS) or DNA damage and/or repair according to or similar to OECD 482):

In the study of Tsutsui (1997), SHE cells without metabolic activation system were treated with catechol for 1 hour at doses of 1, 3, 10, 30 and 100 µM in FBS medium containing10 mMHU (hydroxyurea). The cells were labelled with [3H]dThd for6 h.Catechol induced UDS in SHE cells (1 to 100 µM). The [3H]thymidine cpm/culture-well increased in a dose-dependent manner from about 500at 1and 3 µM, to about 620at 10µM and 800 at 30 µM. The levels of UDS were comparatively decreased to 450 cpm/well in cells treated at the highest dose of 100 µM, probably due to toxicity.

 

In the study of Cahill (2004), DNA damage and repair was checked in microplates method with Saccharomyces cerevisiae. The relative total growth of Saccharomyces cerevisiae was assessed by comparing the extent of proliferation of treated and untreated cells. The measurement of total growth was performed by fluorescence collection. Two strains were tested (GENC01 and GEN T01) at concentration of catechol from 177 µg/mL to 880 µg/mL without metabolic activation system. A clear positive response was measured in growth inhibition rate with strain GENC01 at 177 µg/mL, and clear genotoxicity with GFP induction with strain GEN T01 at 599 µg/mL.

 

In the study of Solveig Walles (1992), rat hepatocytes were treated with 0 - 110 - 220 - 330 µg/mL (0 - 1000 - 2000 - 3000 µM) of catechol for 1 hour without metabolic activation system. DNA content was determined after alkaline treatment of the cells and neutralisation. DNA was quantitated using a fluorochrome. The results indicated and increase in the rate of elution of DNA, indicating the formation of single strand breaks (SSB) in DNA. The dose-response curve showed a threshold value of1 mMafter which the DNA damage increased.

The viability was about 75 % and unchanged after treatment. DNA damage increased slightly with the period of exposure (0, 20, 40 and 60 min) at 3000 µM catechol.

When the hepatocytes were pre-treated for 30 min with the Ca2+-chelator Quin-2 AM, there was a decrease of the DNA damage, indicating probable oxidative damage. The mechanism for repairing the DNA damage induced was challenged by post-treatment of the hepatocytes with an inhibitor of poly(ADP-ribose) polymerase (3-aminobenzamide - 3AB). Upon such treatment, the level of DNA damage by catechol was increased.

 

In the study of Pellack-Walker (1985), Mouse L5178YS cells radiolabelled-thymidine were exposed at concentrations of0.11to 110 µg/mL (0.1 to 1000 µM, 10 concentrations including 0.1, 1, 10, 100, 1000 µM) of catechol without metabolic activation system.

Cells were exposed to catechol for 30 min.Then cells were washed.

Immediately, and at 30, 60 and 90 min after chemical washout, an aliquot of control or treated cells was pulsed with 3H-thymidine and then filtered. The radioactivity content on filters was then determined. Cell viability and protein synthesis were determined. Effects were designated as "DNA specific" when DNA synthesis was inhibited in the absence of discernable effects on cell membrane integrity and protein synthesis.

The DNA synthesis inhibition was evaluated: After treatment for 30 min at 1000 µM Catechol, DNA synthesis inhibition was 65 %, 60 min after washing (maximum inhibitory effect). Beyond 60 min, a small recovery was observed. However, when the dose of catechol was increased further, an irreversible inhibition of DNA synthesis was observed at >1.0 mM. A specific dose-dependent inhibition of DNA synthesis was shown following 30 min of exposure to catechol and 60 min washout which was correlated to the oxidative potential of catechol. ED50 dose value was 250 µM.

Cell viability: Concentrations as high as1.0 mMhad no effect on protein synthesis. Similarly, treatment with1.0 mMcatechol had no effect on membrane integrity (trypan blue dye exclusion).

Catechol effects on inhibition of DNA synthesis occurred only at concentrations which were > 250 times greater than that of benzoquinone.

 

In another study of Pellack-Walker (1986), Mouse lymphoma cells line L5178YS was tested to determine the ability of catechol to induce DNA strand breaks or alkaline-labile sites in LY-S cells at concentration of0.11to 110 µg/mL (1 µM to 1000 µM) without metabolic activation system. The percentage of single stranded DNA was expressed by comparison of treated cells and controls. No DNA damage test was observed.

 

In the study of Lee (1989), Mouse Bone marrow cells was incubated with catechol at concentrations of 0 - 0.66 - 1.321 - 1.982 - 2.642 µg/mL (0 - 6 - 12 - 18 - 24 µM) and with the reaction components (DNA polymerase alpha from calf thymus or DNA polymerase I from E. coli, maximally activated calf thymus DNA, nucleotide triphophates, magnesium acetate glycerol and bovine serum albumine (BSA) in Tris buffer) without metabolic activation system for 30 min prior the addition of [3H]thymidine, and the reaction was terminated 30 min later.

The precipitated DNAs were filtered and analysed by scintillation.

Catechol was able to inhibit 52 % of the nuclear synthetic activity at 24 µM (IC50 = 23 µM).

In a cell-free DNA synthetic system, catechol did not inhibit the incorporation of 3H-thymidine triphosphate up to 24 µM.

The viability of the cells was not modified at all test concentrations.

 

In the study of Hirakawa (2002) Human DNA fragments were exposed to catechol at doses of 0 - 0.011 -0.0 22 - 0.055 - 0.110 - 0.220 - 0.550 - 1.101 - 2.202 µg/mL (0 - 0.1 - 0.2 - 0.5 - 1.0 - 2.0 - 5 - 10 - 20 µM) without metabolic activation system. DNA damage and measurement of O2- generation were performed. Catechol, was tested with or without the addition of Cu2+ and/or NADH.

Catechol alone at the concentration of 20 µM (without the addition of Cu2+ or NADH) did not produce DNA damage. Catechol could induced DNA damage in specific conditions: presence of NADH and Cu2 +. 

The DNA damage induced by catechol was inhibited by catalase and bathocuprine, a specific chelator of Cu2+. Neither OH* scavenger nor SOD could inhibit this DNA damage, suggesting the induction of DNA damage mediated cooperatively by H2O2 and Cu+.

The DNA clivage was observed at Guanine and Thymine , and was due to reactive oxygen species.

 

In the study of Sze (1996), Chinese Hamster Ovary cells (CHO-K1) were exposed to catechol at concentration of 0 - 5.5 - 11 - 16.5 - 22 - 27.5 µg/mL (0 - 50 - 100 - 150 - 200 - 250 µM), without metabolic activation system. Cytotoxicity was determined by the cell survival or clonogenicity assay, DNA strand breaks evaluated by Fluorimetric analysis of DNA unwinding. Catechol did not produce DNA strand breaks.

 

In the study of Oikawa (2001), Human Leukaemia cell line HL-60 and HP 100 was exposed to concentration of catechol: 0 - 1.1 - 2.2 - 5.5 µg/mL (0 - 10 - 20 - 50 µM) without metabolic activation system.

The formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and its hydrogen peroxide (H2O2)-resistant clone HP-100 was investigated by using an electrochemical detector coupled to HPLC. HP100 cells, which have a higher level of catalase activity, were used to assess whether H2O2 participates in catechol-induced DNA lesion.

- Catechol treatment resulted in increased 8-oxodG content, indicative of oxidative base damage, in a dose dependant manner in HL-60 cells, but not in HP-100 cells.

- DNA ladder, associated with apoptosis, were observed at (20, just visible, and) 50 µM in HL-60 cells, but not in HP-100 cells.

- Catechol caused DNA damage depending of its concentration in presence of Cu2+.  DNA damage was enhanced in presence of 100 µM NADH.

- Catechol frequently induced piperidine labile sited at thymine residues.

- Catechol increased 8-oxodG content in calf thymus DNA in presence of Cu2+, but not in absence of Cu2+ or in the presence of other metal ions (Fe3+, Co2+, Ni2+, Mn2+ or Mg2+).

 

In the study of Garberg (1988), Mouse lymphoma cells L5178Y (TK+/TK-) were exposed to catechol concentration of 0 - 55 - 165 - 550.5 - 1651.5 µg/mL (0 - 500 - 1500 - 5000 -15000µM) with and without metabolic activation system from rat liver induced with Aroclor 1254.

The proportion of single- to double-stranded DNA was measured by alkaline unwinding elution and chromatography on hydroxyapatite. 

Results are equivocal in the absence of S9 mix, and positive in presence of S9 mix.

 

In vitro comet assay: 2 studies were available and 1 study performed on Human cells was considered as validity 2 according to Klimish scale.

In the study of Fabiani (2001), Human Peripheral Blood Mononuclear Cells (PBMC) was exposed to catechol at following concentrations: 0 - 1.1 - 5.5 - 11 - 22 - 66 µg/mL (0 - 10 - 50 - 100 - 200 - 600 µM) for 4 hours without metabolic activation system.

After lysis of cells and elution in specific comet assay conditions, DNA damage were evaluated in single cell gel electrophoresis by fluorescence. Cell viability was determined by the Trypan blue. The different concentration tested did not reduce cell viability to less than 95%. Catechol did not induce DNA damage at concentrations of 10, 50 and 100 µM. Catechol was genotoxic at 200 and 600 µM when the cells were incubated in PBS (in conditions not pertinent for hazard evaluation for humans, as no proteins were present), but had very little effects when the cell were incubated in RPMI + 5% FCS. In the presence of 5% of serum, the cells were completely protected from catechol induced effects in both media.

The positive response was observed when cells were incubated with the highest concentration of catechol in phosphate serum buffer only. No such results were obtained in RPMI medium, and under more physiological conditions, i.e. following the addition of foetal calf serum. Therefore, the positive result obtained in very simplified medium, without any proteins, is not relevant.

 

CONCLUSION FOR DNA DAMAGE:

In different in vitro studies on DNA damage, UDS, comet assay…direct effect of catechol under DNA was studied without metabolic activation system. It was noted that Catechol exerted cytotoxicity at high doses tested. Positive effects were also noted indicating genotoxicity:

- single strand break

- inhibition of DNA synthesis

The DNA damage induced by catechol was shown in the study of Hirakawa (2002) to be dependent on specific conditions: presence of NADH and Cu2+, suggesting that the DNA damage was mediated by H2O2 and Cu2+. The oxidative base damage of Catechol seems to be dose-dependent.

OTHER IN VITRO STUDIES considered as validity 2 according to Klimish scale.

In vitro topoisomerase inhibition

In the study of Chen (1995), theinhibitory effect of catechol on the activity of purified human
topoisomerases I and II was determined without metabolic activation system. The Concentrations tested were for Topoisomerase assay I: 110 µg/mL (1000 µM) and for Topoisomerase assay II: 55 - 110 µg/mL (500 - 1000 µM). The results indicated in topoisomerase assay I, no inhibition effect of catechol was observed at the only high dose tested 1000 µM, and in topoisomerase assay II, no inhibition effect at 500 µM but inhibition effect was observed at 1000 µM. 

In the study of Frantz (1996), the inhibitory effect of Catechol was studied without metabolic activation systemon Topoisomerase II: 0, 1, 10, 100 µM. The Topoisomerase II activity was determined by assaying the decatenation of kDNA. After electrophoresis, the appearance of either open circular or linearized kDNA indicated an active and functional enzyme. Catechol required bioactivation by peroxidase in the presence of hydrogen peroxide to inhibit topoisomerase II at low molecular concentrations (10µM and 100 µM).

 

In the study of Baker (2001), DNA cleavage/relaxation assays was studied at concentration of catechol of 0, 1, 10, 30,100 300µM (0, 33 µg/mL (300 µM)). Catechol had no effect on the inhibition of topoisomerase II at concentrations up to 300 µM. On peroxidase activation, inhibition was seen with catechol at 30 µM. However, cleavable complex stabilisation was not observed.

 

CONCLUSION FOR INHIBITION OF TOPOISOMERASE:

In different in vitro studies, the inhibition effect on Catcehol on Topoisomeras I or II was studied and in specific conditions, the inhibitory effect was observed.

 

In vitro cell transformation

In the study of Tsutsui (1997), Syrian Hamster Embryo (SHE) cells were treated with catechol concentration of: 0.11-0.33- 1.1 - 3.3 µg/mL (1 - 3 - 10 - 30 µM) without metabolic activation system.The cells were fixed with absolute methanol and stained with 10 % Giemsa solution. The number of surviving colony and morphologically transformed colonies were scored. Catechol induced morphological transformations of SHE cells at concentrations of 1-30 µM. The frequency of transformation increased with increasing dose of catechol. Catechol at the concentration of 10 µM decreased cell survival to 28.8 % of untreated cells and the percentage of surviving cell was 1.4% of untreated cells at 30 µM catechol.

 

In vivo genotoxicity studies:

 

In vivo Mammalian Erythrocyte Micronucleus Test equivalent or similar to OECD Guideline 474

5 studies were available, 3 of them were considered as validity 2 according to Klimish scale.

In the study of Gad-El-Karim (1985), 3 to 5 males mice received by oral route 0 or 150 mg/kg bw of catechol.  30 hours after dosing the animals were sacrificed and bone marrow from femur was used for the micronucleus test. The statistical analysis did not revealed difference between treated and negative controls animals in micronucleated PCE/1000PCE. In this study catechol at 150 mg/kg bw was not considered to induce micronucleus.

In the study of Ciranni (1988) 4 mice per group were administered with vehicle or catechol at 40 mg/kg bw in single time by oral or intraperitoneal route. Animals were killed 0-48 h after. Bone marrows smears were evaluated for micronuleus. The proportion of polychromatic erythrocytes (PCE) was calculated by counting both normochromatic erythrocytes (NCE) and PCE. Until 3000 PCE had been scored for the presence of micronuclei after 18, 24, 42 and 48 h. By oral route, the catechol produced significant increase of micronuclei at 24h with evident bone marrow depression. After intraperitoneal injection also significant genotoxic effects more pronounced than by oral route at 24 h was observed, and with evident bone marrow depression from 18 h after treatment.

In the study of Marrazzini (1994), 3 males mice per group received single administration of catechol by intraperitoneal route at concentration of 0-10 - 20 - 30 mg/kg bw.18hafter the animals were sacrificed and bone marrow of femur were analysed for micronucleus. Catechol statistically induced micronuclei in a dose-dependant manner, with no variation of the PCEs/NCEs ratio.

Mouse Spot Test equivalent or similar to OECD Guideline 484

In the study of Fahrig (1984), female mouse received i.p. injections of catechol at concentration of 22 mg/kg bw on days 9, 10 and 11 postconception with or without co-treatment with ENU. Catechol given alone did not modified the apparition of color spots 2/216 (1%) compared to negative controls, so same mutation rate.

When catechol was co-administered with ENU, the effects of ENU (ethylnitrosourea) was slightly but not statistically significant enhanced.

CONCLUSION FOR IN VIVO MICRONUCLEUS ASSAY:

In the different in vivo Mammalian Erythrocytes micronucleus tests, contradictory results were observed. Catechol induced micronuclei formation after oral and intraperitoneal administration in a dose-dependent manner.

Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo equivalent or similar to OECD Guideline 486 - ornithine decarboxylase activity - DNA damage and repair by alkaline elution

In the study of Furihata (1989), 5 rats per groups received by gavage a single administration of catechol at concentration of 0, 10, 20, 37.5, 75, 90 mg/kg bw. RDS and UDS in the pyloric mucosa of the stomach were determined inin vitroorgan culture in the presence of tritiated thymidine with or without10 mMhydroxiurea (HU), an inhibitor of RDS. The DNA fraction was extracted from the tissue, dissolved in ACS II and the incorporation of tritiated thymidine was determined in a Beckman liquid scintillation counter. The DNA content of the DNA fraction was determined with 3,5-diaminobenzoic acid as standard. The results indicated a dose-dependent stimulation of replicative DNA synthesis, and an absence of induction of unscheduled DNA synthesis in the pyloric mucosa of the stomach of rats treated with catechol.

In the same study, the authors studied the ODC (ornithine decarboxylase) activity in extracts of the pyloric mucosa of the stomach after oral adminitration by gavage to 4 rats per group of 0 - 10 - 40 - 80 mg/kg bw. Catechol presented a dose-dependent induction of ODC activity on pyloric mucosa stomach. This result indicated that catechol may have tumor-promoting activity. They also studied the DNA damage and repair by alkaline elution. The administration of catechol at doses of 37.5 to 90 mg/kg bw did not induce DNA single strand scission in the pyloric mucosa of the stomach as determined by the alkaline elution method after 2 and12 h.The fraction of DNA remaining on filter 2 and12hafter administration of catechol at the dose of 75 mg/kg remains in the same range than distilled water (0.8 to 1.0). The elution rate constant did not increase after administration of catechol suggesting that catechol did not induce single break scission of DNA in the pyloric mucosa.

In vivo comet assay

In the in vivo comet assay of Watzinger (2008) the male OFA Sprague Dawley rats were treated orally twice with 400, 200 and 100 mg/kg/day, with one sampling time 3 to 6 hours after the last treatment. Stomach and duodenum tissues, were studied under alkaline conditions (SCGE). Under these experimental conditions, CATECHOL induced no statistically significant increases in DNA strand breaks at non-lethal dose on rat stomach cells after oral administration. CATECHOL is hence devoid of genotoxic activity on the stomach. In return, CATECHOL induced statistically significant increases in DNA strand breaks at non-lethal doses on rat duodenum cells after oral administration, with the highest increase of median OTM at the lowest dose tested of 100 mg/kg/day (x2). Furthermore, the very low cell density observed at the two highest doses tested during image analysis, indicated a probable cell lysis due to cytotoxicity and /or highly damaged cells with loss of information. A test item is found to demonstrate genotoxic properties against the target organ if it results in a statistically significant increase in the OTM median compared with the negative control group and if the genotoxicity detected shows a dose-effect relationship. In fact the results showed a statistically significant increase in the median OTM, but with an inverse dose-effect relation, with a bell-shaped curve response. The test item Catechol was thus considered as genotoxic on rat duodenum cells. It would be useful to implement a complementary assay under the same experimental conditions on duodenum cells only, but using doses lower than 100 mg/kg/day (x2), in order to determine if there is a high dose without genotoxic effect under the dose level of 100 mg/kg/day (x2). Under these conditions, CATECHOL was considered as a DNA strand breaks and/or alkali-labile sites inducer on duodenum cells.

Other In vivo DNA studies

In the study of Mirvish (1985), 4 rats per group were treated with cathecol in drinking water  for 7 days at doses of 0, 1, 2, 4 and 8 g/L water. The rats received semi-purified diet during this period. Tritium-labelled thymidine was injected 1 hour before sacrifice of the rats into the oesophageal epithelial and the oesophageal epithelial DNA was isolated to measure the radioactivity. Catechol at concentration of 1-8 g/l significantly enhanced the uptake of tritium-labelled thymidine relative to that in untreated rats in a dose dependent manner. Catechol was able to stimulate the DNA synthesis.

In the study of Hellmer (1992) an Host mediated DNA repair assay with mouse, The animals had been exposed for 2 hours with bacteria mix and then catechol administered by oral route at concentration of 200 mg/kg. Different organs were examined for the colonies types they contained. The results indicated no difference between treated and control for the ratio in N° of colonies of DNA repair deficient strain/N° of colonies of DNA repair proficient strain.Catechol was considered not to have genotoxic effect in this study against E. coli K-12 uvr B/recA DNA repair in the Mice organs evaluated.)

CONCLUSION FOR IN VIVO DNA DAMAGE

In vivo studies on DNA damage indicated a dose-dependent stimulation of replicative DNA synthesis, and an absence of induction of unscheduled DNA synthesis in the pyloric mucosa of the stomach of rats treated with catechol. Furthermore, Catchol may have tumor-promoting activity.Catechol was genotoxic under in vivo comet assay on rat duodenum after oral administration.


Justification for classification or non-classification

All the in vitro and in vivo studies available indicated a genotoxic effect of Catechol on different animals somatic cells line studied. This effect seems to be dose- dependent and link to specific mechanism of oxidative property. It had not been clearly demonstrate whether or not this genotoxic effect had a threshold.

According to classification criteria of EC regulation 1272/2008 the catechol should be classified in Category 2 mutagen (H341: Suspected of causing genetic defects).

This classification proposed based on the data is consistent with the harmonized classification according to the 13th ATP of the CLP Regulation. This ATP was published in the EU official journal on 4 October 2018 and was enter into force 20 days after publication. This ATP has to be applied from 1 May 2020.