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Genetic toxicity: in vitro

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Administrative data

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:
Between 29 July 2009 and 25 September 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of GLP inspection: 19 August 2009 Date of Signature on GLP certificate: 04 March 2009
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
E0286P/040A
IUPAC Name:
E0286P/040A
Details on test material:
Sponsor's identification: E0286P/040A
Description : Amber coloured viscous liquid
Chemical name: A complex mixture of titanium salts
Purity : 80%
Batch number : E0286P/040A
Date received : 20 July 2009
Storage conditions: Room temperature in the dark

Method

Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
Species / strainopen allclose all
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:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: Salmonella strains TA100 & TA1537 and E.coli strain WP2uvrA- (with and without S9): 50, 150, 500, 1500, 5000 µg/plate.
Salmonella strain TA1535 (without S9): 1.5, 5, 15, 50, 150, 500, 5000 µg/plate.
Salmonella strain TA98 (without S9): 15, 50, 150, 500, 1500, 5000 µg/plate.
Salmonella strains TA1535 & TA98 (with S9): 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water, dimethyl sulphoxide and acetone at 50 mg/ml but was fully miscible in tetrahydrofuran at 200 mg/ml in solubility checks performed in-house. Tetrahydrofuran was therefore selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate of TA100
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix

Migrated to IUCLID6: at 3 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous muation rate of TA100
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 1 µg/plate
Remarks:
with S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA1535
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix

Migrated to IUCLID6: at 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA1535
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 2 µg/plate
Remarks:
with S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix

Migrated to IUCLID6: at 2 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 10 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA98
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix

Migrated to IUCLID6: at 0.2 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA98
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 mix

Migrated to IUCLID6: at 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA1537
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix

Migrated to IUCLID6: at 80 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA1537
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 2 µg/plate
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation (experiment 1) and pre-incubation(experiment 2))

DURATION
- Culture Preincubation period: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): 20 minutes
- Fixation time (start of exposure up to fixation or harvest of cells): 48 -72 hrs


SELECTION AGENT (mutation assays): Not applicable.


NUMBER OF REPLICATIONS: Triplicate plating.


NUMBER OF CELLS EVALUATED: Not applicable.


DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.


OTHER EXAMINATIONS: None
Evaluation criteria:
Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviation

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 micro.g/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 micro.g/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
EXAMPLE
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test material was was fully miscible in tetrahydrofuran at 200 mg/ml in solubility checks performed in-house.
- Precipitation: A test material precipitate (greasy in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

111

110

112

97

112

119

120

112

117

111P

101P

+

TA100

113

106

111

92

109

98

112

106

89

84P

80P

-

WP2uvrA-

23

29

32

27

27

36

34

33

35

26P

27P

+

WP2uvrA-

35

35

30

36

25

36

37

27

26

32P

36P

P         precipitate

In the range-finding test (plate incorporation method) the test material caused no visible reduction in the growth of the bacterial background lawns at any dose level either in the presence or absence of S9. In the main test (pre-incubation method) the test material induced toxicity to the bacterial background lawns of Salmonella strains TA100, TA1535 and TA1537 (absence of S9) and TA1535 (presence of S9) at 5000 µg/plate. No toxicity was noted to any of the remaining bacterial strains. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. A precipitate (greasy in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation for the Main test are presented in the tables below:

Table1 Spontaneous Mutation Rates (Concurrent Negative Controls)

Range-finding Test (Experiment 1)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

86

 

10

 

15

 

18

 

10

 

95

(86)

12

(14)

10

(15)

18

(19)

11

(8)

76

 

20

 

21

 

22

 

3

 

 

7

 

21

(12)†

9

 

Main Test

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

109

 

18

 

30

 

23

 

13

 

103

(103)

13

(16)†

26

(24)†

23

(22)†

10

(11)

97

 

16

 

16

 

20

 

9

 

 

19

 

 

27

(23)†

22

 

Confirmatory Test

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

TA1535

13

 

13

(13)

12

 


         Experintal procedure perford at later dates with and/or without S9 due to a number of factors (see relevant data tables)

Table 2 Test Results: Range-Finding Test– Without Metabolic Activation

Test Period

From: 16 August 2009

21 August 2009†

To: 19 August 2009

24 August 2009†

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

0

90

103

111

(101)

10.6#

19

13

14

(15)

3.2

26

26

32

(28)

3.5

35

42

37

(38)

3.6

15

21

19

(18)

3.1

-

50

96

97

89

(94)

4.4

13

11

14

(13)

1.5

30

27

30

(29)

1.7

27

41

41

(36)

8.1

16

9

18

(14)

4.7

-

150

76

77

123

(92)

26.9

14

13

19

(15)

3.2

41

35

31

*

(36)

5.0

47

27

45

(40)

11.0

18

16

19

(18)

1.5

-

500

99

103

87

(96)

8.3

9

16

19

(15)

5.1

25

31

32

(29)

3.8

35

32

30

(32)

2.5

17

19

16

(17)

1.5

-

1500

84

74

81

(80)

5.1

13

25

12

(17)

7.2

24

23

21

(23)

1.5

27

35

38

(33)

5.7

18

17

17

(17)

0.6

-

5000

92 P

117 P

109 P

(106)

12.8

48 P

74 P

60 P

$$$

(61)

13.0

15 P

13 P

15 P

(14)

1.2

29 P

48 P

48 P

(42)

11.0

15 P

19 P

15 P

(16)

2.3

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

529

543

646

(573)

63.9

375

455

378

(403)

45.3

569

732

798

(700)

117.9

117

118

123

(119)

3.2

530

479

488

(499)

27.2

 


        Experimental procedure performed at later date due to high solvent control counts in original test

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

*         p£0.05

$$$     p£0.005

#        Standard deviation


Table 3 Test Results: Range-Finding Test– With Metabolic Activation

Test Period

From: 16 August 2009

To: 19 August 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

+

0

118

112

81

(104)

19.9#

11

16

15

(14)

2.6

22

37

22

(27)

8.7

37

38

20

(32)

10.1

4

12

14

(10)

5.3

+

50

76

89

68

(78)

10.6

11

12

10

(11)

1.0

29

27

29

(28)

1.2

30

29

36

(32)

3.8

14

5

11

(10)

4.6

+

150

91

97

80

(89)

8.6

8

10

5

(8)

2.5

32

30

31

(31)

1.0

21

15

31

(22)

8.1

5

7

9

(7)

2.0

+

500

110

124

110

(115)

8.1

9

20

14

(14)

5.5

25

32

35

(31)

5.1

31

19

22

(24)

6.2

12

11

12

(12)

0.6

+

1500

130

110

101

(114)

14.8

12

14

10

(12)

2.0

32

26

29

(29)

3.0

26

32

24

(27)

4.2

8

8

5

(7)

1.7

+

5000

140 P

117 P

111 P

(123)

15.3

35 P

21 P

35 P

$$$

(30)

8.1

33 P

31 P

29 P

(31)

2.0

20 P

19 P

18 P

(19)

1.0

9 P

14 P

9 P

(11)

2.9

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1911

2195

1800

(1969)

203.7

192

216

207

(205)

12.1

378

341

414

(378)

36.5

95

96

125

(105)

17.0

465

364

518

(449)

78.2

 


BP      Benzo(a)pyrene

2AA    2-Aminoanthracene

P        Precipitate

$$$     p£0.005

#        Standard deviation

See Overall remarks for rest of tables

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative EXAMPLE

The test material was, therefore, considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction.

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It alsoets the requirents of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The dose range for Experiment 2 ranged between 1.5 and 5000 µg/plate and had to be amended for several of the bacterial strains because of toxicity due to the exposure method (pre-incubation instead of plate incorporation).

Additional dose levels and an expanded dose range were selected (where applicable) in order to achieve both four non-toxic dose levels and the toxic limit of the test material.

In addition, a third experiment was performed to confirm whether a statistically significant increase in TA1535 revertant colony frequency, noted in the range-finding test, was real or spurious. The experiment was carried out using bacterial strain TA1535, in the presence and absence of S9 and employed a narrowed test material dose range of 1000, 2000, 3000, 4000 and 5000 µg/plate. This experiment was a repeat of the methodology used in the range-finding test (plate incorporation).

Results.

The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

In the range-finding test (plate incorporation method) the test material caused no visible reduction in the growth of the bacterial background lawns at any dose level either in the presence or absence of S9. In the main test (pre-incubation method) the test material induced toxicity to the bacterial background lawns of Salmonella strains TA100, TA1535 and TA1537 (absence of S9) and TA1535 (presence of S9) at 5000 µg/plate. No toxicity was noted to any of the remaining bacterial strains. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. A test material precipitate (greasy in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Results fromthe first experiment (range-finding test, plate incorporation method) showed small but statistically significant increases in the frequency of TA1535 revertant colonies at a test material dose level of 5000 µg/plate in the absence of metabolic activation (S9). Smaller increases were also observed at the same dose level in the presence of S9. However, no increases were noted in Experiment 2 (main test) which employed the pre-incubation method (toxicity was noted to the bacterial background lawns at 5000 µg/plate).A third, confirmatory experiment was, therefore, performed which was designed to confirm the reproducibility and relevance of the responses. The experiment employed a tightened test material dose range (1000 to 5000 µg/plate) and used the same experimental conditions as the range-finding test (plate incorporation method). In this experiment, small, statistically significant increases were noted (absence of S9 only) at 4000 and 5000 µg/plate.

Although a small but consistent response was observed for TA1535 at the highest dose levels (plate incorporation method only), it was considered doubtful that the test material was causing a true mutagenic effect for the following reasons:

There was no response noted for Salmonella strain TA100 (the sister strain to TA1535) which raised the first concern as to whether the TA1535 response should be classified as being positive as the only difference between the two strains is that TA100 contains the plasmid pKM101, which generally makes this strain the more sensitive of the two for mutagen detection (8). This concern was further strengthened by the evaluation of the dose-response curve, which was a plateau at dose levels above 4000 µg/plate.The inclusion of the intermediary dose levels (1000, 2000, 3000 and 4000 µg/plate)should have induced a much clearer response (if the response was truly positive there should have been a clear, dose-related increase).The main test proved that the test material exhibited toxic effects under certain circumstances or exposure concentrations (the pre-incubation modification exposes the bacteria to a much higher concentration for the incubation period). Therefore, the small increases in colony numbers were considered an artefact resulting from a modest level of toxicity to this strain of bacteria (TA1535). There are a number of factors that may affect the spontaneous or background frequency of colony numbers and the threshold nature of the response seen in this study may indicate that the response was caused by an indirect mechanism causing an increase in the spontaneous rate of mutation rather than mutagenicity (direct or indirect).A possible mechanism may be that low level toxicity has caused a selective effect on the number of bacterial cells plated out onto Vogel-Bonner agar plates, resulting in an increase in non-revertant histidine-dependent bacteria.

Conclusion.

The test material was, therefore, considered to be non-mutagenic under the conditions of this test.

(8) Prival M J and Zeiger E (1998)Mutation Research,412, 251-260.

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