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Toxicological information

Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 20, 1993 to February 4, 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Ames test (5 Salmonella strains), GLP. Substance identification: information available from supplier for commercial name Substance analytical certificate available
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From December 20, 1993 to February 4, 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Ames test (5 Salmonella strains), GLP. Substance identification: information available from supplier for commercial name Substance analytical certificate available
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Ames test
Principles of method if other than guideline:
Guideline principles
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Reverse gene mutation assay
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: Stock of S. typhimurium tester strains were obtained from B. N. Ames (University of California Berkeley, USA). Master stocks are held in liquid nitrogen and were aliquots of nutrient broth cultures then stored at -80°C. See below Table 7.6.1/1.
Metabolic activation:
with and without
Metabolic activation system:
The S9 mix was prepared in laboratory from liver of a Sprague-Dawley male rat (IFFA CREDO, France) induced by Aroclor 1254 and stored at -80 °C as aliquots. See below Table 7.6.1/2
Test concentrations with justification for top dose:
HDF 200 was tested as an emulsion in 10% Pluronic F68 aqueous solution (1/1)( v/v) in preliminary test and both main tests.
Doses: 0 (5% Pluronic F68 solution as solvent), 2, 6, 20, 60, 200 µL/plate in Pluronic F68 aqueous solution for all S. typhimurium strains (see below Table 7.6.1/3)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 10% Pluronic aqueous solution
- Justification for choice of solvent/vehicle: the test substance (oil) was insoluble in water and other vehicles (DMSO, acetone)
Untreated negative controls:
yes
Remarks:
Sterile test: plates without the addition of bacteria are prepared in order to assess the sterility of HDF 200, the S9 mix and the medium.
Negative solvent / vehicle controls:
yes
Remarks:
Aqueous Pluronic F68 solution at 5%
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See below Table 7.6.1/4
Remarks:
6 plates for negative control (solvent). 3 plates for positive controls. 3 plates for controls of sterility (S9, solvent, medium).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
After range-finding test, two independent experiments were conducted in the main test by agar plate incorporation with and without S9 mix. The different controls (negative and positive controls, controls for sterility) were tested in the same conditions.

DURATION
- Preincubation period: yes- S9 activation system preincubation at 37°C for 60 min before agar plate incorporation (when negative results in the main first test in the presence of S9 activation)
- Exposure duration: 48h at 37°C

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: three scoring (3 measurements/plate). The mean number and standard deviation of revertants are calculated for all groups. The means for all treatment groups are compared with those obtained for the solvent control groups.


DETERMINATION OF CYTOTOXICITY
- Method: other: a preliminary toxicity assay was conducted in S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 at concentrations between 2 and 200 µL/plate (test substance dilutions in Pluronic F68 solution at 10% (1/1)( v/v)).


OTHER: scoring (3 measurements/plate). The mean number and standard deviation of revertants are calculated for all groups. The means for all treatment groups are compared with those obtained for the solvent control groups: the ratio between test substance revertants and solvent negative control revertants was performed at each dose-concentration
Evaluation criteria:
CRITERIA OF DECISION:
-Biological significance:
a reproducible 2-fold increase in the number of revertants (3 times in the case of TA 1535 and TA 1537 strains) compared with the vehicle controls, in any strain at any dose-level with some evidence of a dose-relationship (3 dose-concentrations) will be considered as a positive result. Reference to historical data may be taken into account in the evaluation of the data obtained.

-Statistical significance:
the test data were subjected to analysis to determine the statistical significance of any increase in revertants according to the Dunnett method.
-Reproductibility:
Positive results should be observed in two independently tests. Positive results observed in one test without reproducibility in two tests independently conducted should not be considered as significant (Brusick ,1980). Complementary test could be performed.

ACCEPTANCE CRITERIA:- The mean of the solvent control revertants for each strain should lie within or close to the 99% confidence limits of the current historical control range of the laboratory unless otherwise justified by the study director. The positive control compounds must induce an increase in mean revertants of at least twice (3 times in the case of strains TA 1535 and 1537) the concurrent solvent controls.The test substance must be sterile at the highest concentration after agar plate incubation 48h at 37°C.
Statistics:
The test data were subjected to analysis to determine the statistical significance of any increase in revertants according to the Dunnett method.
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 102 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: no

RANGE-FINDING/SCREENING STUDIES: The substance was freely soluble in the vehicle Pluronic F68 . A preliminary toxicity assay was conducted in S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 at concentrations between 2 and 200 µL/plate in 10% Pluronic F68 solution (1/1) (v/v). A slight cytotoxicity (62.4% to 102.2% of the control survival) at the highest test substance was observed in all strains. Considering the slight toxicity this concentration (200 µL/plate) was used in the main test both in the absence and the presence of S9 activation system.

COMPARISON WITH HISTORICAL CONTROL DATA: yes


ADDITIONAL INFORMATION ON CYTOTOXICITY:no
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 7.6.1/5: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (First test)

Test substance concentration
(µL/plate)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

28

6.8

4

2.6

17

4.1

113

7.1

265

35

2**

21

4

5

0.6

13

3.2

119

10.3

263

46.8

6**

29

5.5

5

1.7

16

1.7

99

4.7

259

27

20**

24

5.5

4

1.7

16

5.1

110

6.9

251

45.4

60**

29

9.1

3

1.5

16

8.4

105

12.5

253

32.5

200**

31

5

3

0

16

3.5

116

9.3

205

16.5

Positive control***

461

37.2

543

159

340

19.7

701

88.9

1360

206.1

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Table 7.6.1/6: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (First test)

Test substance concentration
(µL/plate)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

12

3.2

6

1.9

26

5

114

6

347

19.9

2**

11

2.5

7

2.9

21

3

127

5.7

355

35.1

6**

8

1.2

5

1.2

28

3.6

136

11.7

332

47.3

20**

12

3

5

2.3

24

5.6

141

1.5

331

4.6

60**

12

3.6

4

1

24

5.6

124

5.2

375

99.7

200**

9

0.6

7

3.2

29

5.6

137

8

375

19.2

Positive control***

892

104.6

94

10.5

1324

117.2

2653

361.7

1380

87

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Red results: p 0.01

Table 7.6.1/7: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (Second test)

Test substance concentration
(µL/plate)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

24

3.7

4

2.5

15

3.6

106

11.4

235

15.9

2**

28

5.3

4

2.9

15

4

113

10.6

273

33.1

6**

25

12.1

7

2.3

15

3

108

13.1

240

54.1

20**

25

4.7

4

1.2

15

6.4

108

11.7

225

28.3

60**

19

6

7

2.1

18

1.7

92

5

243

32.5

200**

21

4

5

1.2

18

4.6

105

3.2

225

31.9

Positive control***

825

67.1

133

21.4

318

29.4

1151

235.3

1213

41.6

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Table 7.6.1/8: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation with pre-incubation (Second test)

Test substance concentration
(µL/plate)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

9

5.1

4

1.7

18

6.3

138

12.6

395

45.1

2**

9

0.6

7

4.2

26

3.6

170

23

403

47.7

6**

10

2.6

7

0.6

29

10.7

183

19.2

373

25.7

20**

10

1.7

7

1.5

34

2.5

195

7

379

12.2

60**

9

1.7

7

0

33

11.6

213

9.3

384

41.8

200**

11

1

7

0.6

37

3.5

232

12.2

385

47.4

Positive control***

127

18.6

91

6

1457

193.5

1093

142.3

953

97.9

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Red results: p 0.01

Table 7.6.1/9: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation with pre-incubation (Third test)

Test substance concentration
(µL/plate)

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

0*

19

5.2

107

14.6

2**

26

2.6

100

8.5

6**

25

10.3

118

7.6

20**

31

0

153

5.7

60**

32

5.5

124

14.4

200**

30

1.5

135

17.6

Positive control***

1457

193.5

1305

51.4

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Red results: p 0.01

Conclusions:
Interpretation of results:
negative with and without metabolic activation

Under the test conditions, HDF 200 did not demonstrate any in vitro mutagenic activity in the Salmonella test system.
Executive summary:

The mutagenic potential of HDF 200 was assessed in the Salmonella typhimurium microsomal assay according to the Ames test, and in compliance with Good Laboratory Practice. The histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 102, TA 98 and TA 100 were used in the presence and the absence of metabolic activation system from the liver fraction of Aroclor 1254-induced rats (S9-mix). Each strain was exposed to 5 dose levels according to the direct incorporating plate method. After 48 hours of incubation at 37°C, the revertant colonies were scored. A preliminary toxicity assay was performed according to the direct incorporating method to define the 5 dose levels to be used in the main test. The evaluation of toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies. The test substance was tested in the main experiment according to two tests independently performed in the same way as the range-finding test. The test substance was diluted in 10% Pluronic F68 aqueous solution. Dose levels used in the main assay were 0 (solvent), 2, 6, 20, 60 and 200 µL/plate in the main test, with and without S9-mix. All determinations were made in triplicate (3 automatic scoring measurements / plate). Simultaneous negative (solvent, triplicate) and positive controls (triplicate) were used in all experiments. No toxicity was observed in any of the strains in the absence and in the presence of S-9 mix up to the highest dose tested in the main test (62.4% to 102.2% survival). No increase in revertant mean number was observed in any S. typhimurium strain with and without S9-mix in the preliminary test and in the first main test. However, positive results were observed in the second main test conducted with 60-minute S9 pre-incubation before plate incorporation.

Positive controls gave the expected increases in the number of revertants, with and without S-9 mix. Both statistically significant results and biologically significant results (two-fold increase by comparison with solvent) were observed at the highest test substance dose in S. typhimurium TA 98 but without a dose-effect relationship. Reduced but statistically significant positive results were also observed in S. typhimurium TA 100 with a dose-effect relationship. However, no biological significance was observed at any dose. A third test was performed in S. typhimurium TA 98 and TA 100 under the same conditions as the second main test. In this case the positive results obtained in the previous main test were not observed in either S. typhimurium TA 98 or TA 100. In the absence of reproducible results, the test substance was not considered as mutagenic in S. typhimurium according to the decision criteria of Brusick (1980).

Under the conditions of this study, HDF 200 did not demonstrate any in vitro mutagenic activity in this bacterial test system.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Ames test
Principles of method if other than guideline:
Guideline principles
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): HDF 200
- Substance type: insoluble oil, petroleum product, UVCB
- Physical state: clear liquid
- Analytical purity: 100% Commercial product
- Composition of test material, percentage of components: Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, <2% aromatics
- Lot/batch No.: 7/9/93
- Stability under test conditions: expected to be stable during the study
- Storage condition of test material: dry storage at 4°C in the absence of light

Method

Target gene:
Reverse gene mutation assay
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: Stock of S. typhimurium tester strains were obtained from B. N. Ames (University of California Berkeley, USA). Master stocks are held in liquid nitrogen and were aliquots of nutrient broth cultures then stored at -80°C. See below Table 7.6.1/1.
Metabolic activation:
with and without
Metabolic activation system:
The S9 mix was prepared in laboratory from liver of a Sprague-Dawley male rat (IFFA CREDO, France) induced by Aroclor 1254 and stored at -80 °C as aliquots. See below Table 7.6.1/2
Test concentrations with justification for top dose:
HDF 200 was tested as an emulsion in 10% Pluronic F68 aqueous solution (1/1)( v/v) in preliminary test and both main tests.
Doses: 0 (5% Pluronic F68 solution as solvent), 2, 6, 20, 60, 200 µL/plate in Pluronic F68 aqueous solution for all S. typhimurium strains (see below Table 7.6.1/3)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 10% Pluronic aqueous solution
- Justification for choice of solvent/vehicle: the test substance (oil) was insoluble in water and other vehicles (DMSO, acetone)
Controls
Untreated negative controls:
yes
Remarks:
Sterile test: plates without the addition of bacteria are prepared in order to assess the sterility of HDF 200, the S9 mix and the medium.
Negative solvent / vehicle controls:
yes
Remarks:
Aqueous Pluronic F68 solution at 5%
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See below Table 7.6.1/4
Remarks:
6 plates for negative control (solvent). 3 plates for positive controls. 3 plates for controls of sterility (S9, solvent, medium).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
After range-finding test, two independent experiments were conducted in the main test by agar plate incorporation with and without S9 mix. The different controls (negative and positive controls, controls for sterility) were tested in the same conditions.

DURATION
- Preincubation period: yes- S9 activation system preincubation at 37°C for 60 min before agar plate incorporation (when negative results in the main first test in the presence of S9 activation)
- Exposure duration: 48h at 37°C

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: three scoring (3 measurements/plate). The mean number and standard deviation of revertants are calculated for all groups. The means for all treatment groups are compared with those obtained for the solvent control groups.


DETERMINATION OF CYTOTOXICITY
- Method: other: a preliminary toxicity assay was conducted in S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 at concentrations between 2 and 200 µL/plate (test substance dilutions in Pluronic F68 solution at 10% (1/1)( v/v)).


OTHER: scoring (3 measurements/plate). The mean number and standard deviation of revertants are calculated for all groups. The means for all treatment groups are compared with those obtained for the solvent control groups: the ratio between test substance revertants and solvent negative control revertants was performed at each dose-concentration
Evaluation criteria:
CRITERIA OF DECISION:
-Biological significance:
a reproducible 2-fold increase in the number of revertants (3 times in the case of TA 1535 and TA 1537 strains) compared with the vehicle controls, in any strain at any dose-level with some evidence of a dose-relationship (3 dose-concentrations) will be considered as a positive result. Reference to historical data may be taken into account in the evaluation of the data obtained.

-Statistical significance:
the test data were subjected to analysis to determine the statistical significance of any increase in revertants according to the Dunnett method.
-Reproductibility:
Positive results should be observed in two independently tests. Positive results observed in one test without reproducibility in two tests independently conducted should not be considered as significant (Brusick ,1980). Complementary test could be performed.

ACCEPTANCE CRITERIA:- The mean of the solvent control revertants for each strain should lie within or close to the 99% confidence limits of the current historical control range of the laboratory unless otherwise justified by the study director. The positive control compounds must induce an increase in mean revertants of at least twice (3 times in the case of strains TA 1535 and 1537) the concurrent solvent controls.The test substance must be sterile at the highest concentration after agar plate incubation 48h at 37°C.
Statistics:
The test data were subjected to analysis to determine the statistical significance of any increase in revertants according to the Dunnett method.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 102 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: no

RANGE-FINDING/SCREENING STUDIES: The substance was freely soluble in the vehicle Pluronic F68 . A preliminary toxicity assay was conducted in S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 at concentrations between 2 and 200 µL/plate in 10% Pluronic F68 solution (1/1) (v/v). A slight cytotoxicity (62.4% to 102.2% of the control survival) at the highest test substance was observed in all strains. Considering the slight toxicity this concentration (200 µL/plate) was used in the main test both in the absence and the presence of S9 activation system.

COMPARISON WITH HISTORICAL CONTROL DATA: yes


ADDITIONAL INFORMATION ON CYTOTOXICITY:no
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/5: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (First test)

Test substance concentration
(µL/plate)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

28

6.8

4

2.6

17

4.1

113

7.1

265

35

2**

21

4

5

0.6

13

3.2

119

10.3

263

46.8

6**

29

5.5

5

1.7

16

1.7

99

4.7

259

27

20**

24

5.5

4

1.7

16

5.1

110

6.9

251

45.4

60**

29

9.1

3

1.5

16

8.4

105

12.5

253

32.5

200**

31

5

3

0

16

3.5

116

9.3

205

16.5

Positive control***

461

37.2

543

159

340

19.7

701

88.9

1360

206.1

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Table 7.6.1/6: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (First test)

Test substance concentration
(µL/plate)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

12

3.2

6

1.9

26

5

114

6

347

19.9

2**

11

2.5

7

2.9

21

3

127

5.7

355

35.1

6**

8

1.2

5

1.2

28

3.6

136

11.7

332

47.3

20**

12

3

5

2.3

24

5.6

141

1.5

331

4.6

60**

12

3.6

4

1

24

5.6

124

5.2

375

99.7

200**

9

0.6

7

3.2

29

5.6

137

8

375

19.2

Positive control***

892

104.6

94

10.5

1324

117.2

2653

361.7

1380

87

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Red results: p 0.01

Table 7.6.1/7: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (Second test)

Test substance concentration
(µL/plate)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

24

3.7

4

2.5

15

3.6

106

11.4

235

15.9

2**

28

5.3

4

2.9

15

4

113

10.6

273

33.1

6**

25

12.1

7

2.3

15

3

108

13.1

240

54.1

20**

25

4.7

4

1.2

15

6.4

108

11.7

225

28.3

60**

19

6

7

2.1

18

1.7

92

5

243

32.5

200**

21

4

5

1.2

18

4.6

105

3.2

225

31.9

Positive control***

825

67.1

133

21.4

318

29.4

1151

235.3

1213

41.6

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Table 7.6.1/8: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation with pre-incubation (Second test)

Test substance concentration
(µL/plate)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

9

5.1

4

1.7

18

6.3

138

12.6

395

45.1

2**

9

0.6

7

4.2

26

3.6

170

23

403

47.7

6**

10

2.6

7

0.6

29

10.7

183

19.2

373

25.7

20**

10

1.7

7

1.5

34

2.5

195

7

379

12.2

60**

9

1.7

7

0

33

11.6

213

9.3

384

41.8

200**

11

1

7

0.6

37

3.5

232

12.2

385

47.4

Positive control***

127

18.6

91

6

1457

193.5

1093

142.3

953

97.9

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Red results: p 0.01

Table 7.6.1/9: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation with pre-incubation (Third test)

Test substance concentration
(µL/plate)

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

0*

19

5.2

107

14.6

2**

26

2.6

100

8.5

6**

25

10.3

118

7.6

20**

31

0

153

5.7

60**

32

5.5

124

14.4

200**

30

1.5

135

17.6

Positive control***

1457

193.5

1305

51.4

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Red results: p 0.01

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative with and without metabolic activation

Under the test conditions, HDF 200 did not demonstrate any in vitro mutagenic activity in the Salmonella test system.
Executive summary:

The mutagenic potential of HDF 200 was assessed in the Salmonella typhimurium microsomal assay according to the Ames test, and in compliance with Good Laboratory Practice. The histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 102, TA 98 and TA 100 were used in the presence and the absence of metabolic activation system from the liver fraction of Aroclor 1254-induced rats (S9-mix). Each strain was exposed to 5 dose levels according to the direct incorporating plate method. After 48 hours of incubation at 37°C, the revertant colonies were scored. A preliminary toxicity assay was performed according to the direct incorporating method to define the 5 dose levels to be used in the main test. The evaluation of toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies. The test substance was tested in the main experiment according to two tests independently performed in the same way as the range-finding test. The test substance was diluted in 10% Pluronic F68 aqueous solution. Dose levels used in the main assay were 0 (solvent), 2, 6, 20, 60 and 200 µL/plate in the main test, with and without S9-mix. All determinations were made in triplicate (3 automatic scoring measurements / plate). Simultaneous negative (solvent, triplicate) and positive controls (triplicate) were used in all experiments. No toxicity was observed in any of the strains in the absence and in the presence of S-9 mix up to the highest dose tested in the main test (62.4% to 102.2% survival). No increase in revertant mean number was observed in any S. typhimurium strain with and without S9-mix in the preliminary test and in the first main test. However, positive results were observed in the second main test conducted with 60-minute S9 pre-incubation before plate incorporation.

Positive controls gave the expected increases in the number of revertants, with and without S-9 mix. Both statistically significant results and biologically significant results (two-fold increase by comparison with solvent) were observed at the highest test substance dose in S. typhimurium TA 98 but without a dose-effect relationship. Reduced but statistically significant positive results were also observed in S. typhimurium TA 100 with a dose-effect relationship. However, no biological significance was observed at any dose. A third test was performed in S. typhimurium TA 98 and TA 100 under the same conditions as the second main test. In this case the positive results obtained in the previous main test were not observed in either S. typhimurium TA 98 or TA 100. In the absence of reproducible results, the test substance was not considered as mutagenic in S. typhimurium according to the decision criteria of Brusick (1980).

Under the conditions of this study, HDF 200 did not demonstrate any in vitro mutagenic activity in this bacterial test system.