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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 June 1985 to 15 july 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Ames test (5 Salmonella strains), GLP. Substance analytical certificate available provided by the manufacturer. Substance identification: commercial name 98.8% purity
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 for cross-reference:
read-across: supporting information
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
5 June 1985 to 15 july 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Ames test (5 Salmonella strains), GLP. Substance analytical certificate available provided by the manufacturer. Substance identification: commercial name 98.8% purity
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 for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
according to Ames test
Principles of method if other than guideline:
Guideline principles
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 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:
valid
Positive controls validity:
valid
Additional information on results:
No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with Petrepar 147 at any dose level, either in the presence or absence of metabolic activation (S9).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 7.6.1/4: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (First test)

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 1538

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

12

0.6

12

3.8

9

1.5

18

3.0

70

4.2

0**

9

0

10

1.2

7

1.0

21

7.0

66

3.5

50

9

2.0

12

3.6

9

1.2

17

1.0

72

6.1

150

8

3.1

7

1.5

9

1.2

15

4.2

69

4.9

500

8

2.0

11

3.2

5

0

15

3.5

72

3.8

1500

10

3.8

10

3.5

9

2.1

17

3.8

63

3.1

5000

9

2.1

11

3.5

12

1.0

18

3.5

63

5.0

Positive control***

65

5.5

1750

89.1

54

3.8

76

6.7

236

21.2

* Solvent control = acetone

** Sterile 0.1 M sodium phosphate buffer (pH 7.4) instead of test substance dilution

*** Mutagens positive controls: see Table 7.6.1/3

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

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 1538

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

11

1.5

20

5.1

11

4.0

21

1.2

78

13

0**

9

2.3

21

2.1

9

1.5

14

1.5

69

4.0

50

7

0.6

11

1.0

9

4.2

16

3.6

72

5.1

150

8

2.0

10

2.1

9

2.3

13

1.5

69

6.0

500

6

2.0

12

3.0

9

3.1

14

3.5

71

4.7

1500

11

3.5

11

3.2

12

3.1

13

3.1

69

4.6

5000

6

1.0

9

3.8

9

1.5

13

2.3

61

2.9

Positive control***

130

9.0

103

17.4

408

47.5

372

52.4

542

15.9

* Solvent control = acetone

** Sterile 0.1 M sodium phosphate buffer (pH 7.4) instead of test substance dilution

*** Mutagens positive controls: see Table 7.6.1/3

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

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 1538

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

14

2.0

14

3.8

5

1.2

14

3.2

63

2.9

0**

7

2.5

17

0.6

7

3.5

17

1.7

67

7..0

50

7

2.3

17

4.0

6

1.5

17

1.0

70

6.1

150

9

1.2

18

1.2

11

4.0

17

1.5

67

3.8

500

6

1.0

15

1.5

7

0

14

3.2

71

3.5

1500

6

2.1

19

2.0

8

2.9

16

2.6

72

4.0

5000

7

1.5

17

4.0

11

5.0

13

2.6

68

9.3

Positive control***

10

9.3

-

-

56

8.2

49

18.2

228

34.6

* Solvent control = acetone

** Sterile 0.1 M sodium phosphate buffer (pH 7.4) instead of test substance dilution

*** Mutagens positive controls: see Table 7.6.1/3

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

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 1538

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

10

2.6

14

3.8

9

2.6

19

2.6

88

17.9

0**

9

2.1

17

0.6

12

2.6

18

3.6

101

11.0

50

9

1.2

17

4.0

12

3.6

18

2.1

81

13.0

150

9

1.0

18

1.2

11

1.5

16

3.2

76

11.3

500

9

1.7

15

1.5

10

4.5

19

2.0

79

6.1

1500

9

3.1

19

2.0

11

4.0

16

2.6

79

9.1

5000

10

2.5

17

4.0

11

2.6

15

2.0

80

16.9

Positive control***

84

5.9

41

9.0

46

12.3

142

46.0

232

7.5

* Solvent control = acetone

** Sterile 0.1 M sodium phosphate buffer (pH 7.4) instead of test substance dilution

*** Mutagens positive controls: see Table 7.6.1/3

Conclusions:
Interpretation of results:
negative

Under the test conditions, Petrepar-147 did not demonstrate any in vitro mutagenic activity in the Salmonella test system up to 5000 µg/plate.
Executive summary:

This data is being read across from the source study that tested Hydrocarbons, C14-C17, n-alkanes, <2% aromatics based on analogue read across.

The mutagenic potential of Petrepar 147 was assessed in the Salmonella typhimurium microsomal assay according to the Ames test in compliance with Good Laboratory Practice.

The histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 1538, TA 98 and TA 100 mutants 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 72 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 test substance was then tested in another experiment performed in the same way as the range-finding 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 dissolved in acetone. Dose levels used in the main assay were 0, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix. All determinations were made in triplicate (3 automatic scoring measurements / plate). Two independent main tests were performed. Simultaneous negative (solvent, triplicate) and positive controls (triplicate) were used in all experiments and compared.

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. No increase in the mean number of revertant colonies for any S. typhimurium strains with and without S9-mix in both tests (pre-test and main test).
Positive controls gave the expected increases in the number of revertants, with and without S-9 mix.

Under the conditions of this study, Petrepar 147 did not demonstrate any in vitro mutagenic activity in this bacterial test system.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report date:
1985

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Hydrocarbons, C14-C17, n-alkanes, <2% aromatics
EC Number:
917-828-1
Molecular formula:
none available - not a single isomer - see remarks
IUPAC Name:
Hydrocarbons, C14-C17, n-alkanes, <2% aromatics
Constituent 2
Reference substance name:
Petrepar-147
IUPAC Name:
Petrepar-147
Details on test material:
- Name of test material (as cited in study report): Petrepar -147
- Substance type: petroleum product, UVCB
- Physical state: colourless liquid
- Analytical purity: 100% Commercial product
- Storage condition of test material: room temperature

Method

Target gene:
Reverse gene mutation assay
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: S. typhimurium TA 1538 (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 CD rat (Sprague-Dawley derived from Charles River Ltd, UK) induced by Aroclor 1254 and stored at -80 °C as aliquots.
Test concentrations with justification for top dose:
5, 50, 500, 5000 µg/plate in dose range-finding test (see below Table 7.6.1/2)
50, 50, 500, 1500, 5000 µg/plate in the main mutation tests
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: no data
Controls
Untreated negative controls:
yes
Remarks:
Sterile test: plates without the addition of bacteria are prepared in order to assess the sterility of test substance, the S9 mix and the vehicle
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
yes
Remarks:
Sterile 0.1 M sodium phosphate buffer (pH 7.4)
Positive controls:
yes
Positive control substance:
other: See below Table 7.6.1/3
Remarks:
See freetext "Any other information on materials and methods"
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) Two independent experiments (range-finding test as first and main test as second test) in agar by direct plate incorporation for both experiments with and without S9 mix.
Pre-incubation test substance activation was not performed in presence of S9-mix.

DURATION
- Preincubation period: no
- Exposure duration: 72 h

SELECTION AGENT (mutation assays): histidine deficient agar

NUMBER OF REPLICATIONS: 3 measurements/plate) with a Biotran Automatic colony counter.


DETERMINATION OF CYTOTOXICITY
- Method: revertant colony counts during the dose-range finding assay


OTHER: scoring (3 measurements/plate) with a Domino automated counter
Evaluation criteria:
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 negative (solvent) and positive control groups. The effect of metabolic activation is assessed by comparing the results obtained both in the presence and absence of the liver microsomal fraction for each treatment group.
Statistics:
A compound is deemed to provide evidence of mutagenic potential if:
- a statistically significant dose-related increase in the number of revertant colonies is obtained in two separate experiments, and
- the increase in the number of revertant colonies is at least twice the concurrent solvent control value.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 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:
valid
Positive controls validity:
valid
Additional information on results:
No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with Petrepar 147 at any dose level, either in the presence or absence of metabolic activation (S9).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/4: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (First test)

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 1538

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

12

0.6

12

3.8

9

1.5

18

3.0

70

4.2

0**

9

0

10

1.2

7

1.0

21

7.0

66

3.5

50

9

2.0

12

3.6

9

1.2

17

1.0

72

6.1

150

8

3.1

7

1.5

9

1.2

15

4.2

69

4.9

500

8

2.0

11

3.2

5

0

15

3.5

72

3.8

1500

10

3.8

10

3.5

9

2.1

17

3.8

63

3.1

5000

9

2.1

11

3.5

12

1.0

18

3.5

63

5.0

Positive control***

65

5.5

1750

89.1

54

3.8

76

6.7

236

21.2

* Solvent control = acetone

** Sterile 0.1 M sodium phosphate buffer (pH 7.4) instead of test substance dilution

*** Mutagens positive controls: see Table 7.6.1/3

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

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 1538

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

11

1.5

20

5.1

11

4.0

21

1.2

78

13

0**

9

2.3

21

2.1

9

1.5

14

1.5

69

4.0

50

7

0.6

11

1.0

9

4.2

16

3.6

72

5.1

150

8

2.0

10

2.1

9

2.3

13

1.5

69

6.0

500

6

2.0

12

3.0

9

3.1

14

3.5

71

4.7

1500

11

3.5

11

3.2

12

3.1

13

3.1

69

4.6

5000

6

1.0

9

3.8

9

1.5

13

2.3

61

2.9

Positive control***

130

9.0

103

17.4

408

47.5

372

52.4

542

15.9

* Solvent control = acetone

** Sterile 0.1 M sodium phosphate buffer (pH 7.4) instead of test substance dilution

*** Mutagens positive controls: see Table 7.6.1/3

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

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 1538

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

14

2.0

14

3.8

5

1.2

14

3.2

63

2.9

0**

7

2.5

17

0.6

7

3.5

17

1.7

67

7..0

50

7

2.3

17

4.0

6

1.5

17

1.0

70

6.1

150

9

1.2

18

1.2

11

4.0

17

1.5

67

3.8

500

6

1.0

15

1.5

7

0

14

3.2

71

3.5

1500

6

2.1

19

2.0

8

2.9

16

2.6

72

4.0

5000

7

1.5

17

4.0

11

5.0

13

2.6

68

9.3

Positive control***

10

9.3

-

-

56

8.2

49

18.2

228

34.6

* Solvent control = acetone

** Sterile 0.1 M sodium phosphate buffer (pH 7.4) instead of test substance dilution

*** Mutagens positive controls: see Table 7.6.1/3

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

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 1538

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

10

2.6

14

3.8

9

2.6

19

2.6

88

17.9

0**

9

2.1

17

0.6

12

2.6

18

3.6

101

11.0

50

9

1.2

17

4.0

12

3.6

18

2.1

81

13.0

150

9

1.0

18

1.2

11

1.5

16

3.2

76

11.3

500

9

1.7

15

1.5

10

4.5

19

2.0

79

6.1

1500

9

3.1

19

2.0

11

4.0

16

2.6

79

9.1

5000

10

2.5

17

4.0

11

2.6

15

2.0

80

16.9

Positive control***

84

5.9

41

9.0

46

12.3

142

46.0

232

7.5

* Solvent control = acetone

** Sterile 0.1 M sodium phosphate buffer (pH 7.4) instead of test substance dilution

*** Mutagens positive controls: see Table 7.6.1/3

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative

Under the test conditions, Petrepar-147 did not demonstrate any in vitro mutagenic activity in the Salmonella test system up to 5000 µg/plate.
Executive summary:

The mutagenic potential of Petrepar 147 was assessed in the Salmonella typhimurium microsomal assay according to the Ames test in compliance with Good Laboratory Practice.

The histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 1538, TA 98 and TA 100 mutants 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 72 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 test substance was then tested in another experiment performed in the same way as the range-finding 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 dissolved in acetone. Dose levels used in the main assay were 0, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix. All determinations were made in triplicate (3 automatic scoring measurements / plate). Two independent main tests were performed. Simultaneous negative (solvent, triplicate) and positive controls (triplicate) were used in all experiments and compared.

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. No increase in the mean number of revertant colonies for any S. typhimurium strains with and without S9-mix in both tests (pre-test and main test).
Positive controls gave the expected increases in the number of revertants, with and without S-9 mix.

Under the conditions of this study, Petrepar 147 did not demonstrate any in vitro mutagenic activity in this bacterial test system.