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Genetic toxicity: in vitro

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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:
15 June 1992 to 17 September 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guidelines and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Physical state: liquid

Method

Target gene:
S. typhimurium: Histidine synthesis

E. coli: Tryptophan synthesis
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):
- Type and identity of media: The broth used to grow overnight cultures of tester strains was Vogel-Bonner salt solution supplemented with 2.5% (w/v) Oxoid Nutrient Broth No. 2. Bottom agar (25 mL per 15 x 100 mm petri dish) was added to the Vogel-Bonner minimal medium E and supplemented with 1.5% (w/v) agar and 0.2% (w/v) glucose. Top agar consisting of 0.7 % w/v agar and 0.5 % w/v sodium chloride was supplemented with 10 mL of 0.5 mM histidine/ biotin solution per 100 mL agar. When S9 mix was required, 2.0 mL of the supplemented top agar was used in the overlay. However, when S9 was not required, water was added to the supplemented top agar (0.5 mL of water per 2 mL of supplemented top agar) and the resulting 2.5 mL of diluted supplemented top agar was used for the overlay.
- Periodically checked for genotype stability: Yes. The presence of the pKM101 plasmid was confirmed for tester strains TA98 and TA100 by demonstration of resistance to ampicillin (10 µg). The presence of the rfa wall mutation was confirmed by testing the sensitivity of each culture to crystal violet (10 µg).
- Periodically "cleansed" against high spontaneous background: Yes. The mean numbers of spontaneous revertants per plate in the vehicle controls that are characteristic of the respective strains were demonstrated by plating 100 µg aliquots of the culture along with the appropriate vehicle on selective media.
Additional strain / cell type characteristics:
other: rfa and uvrB mutations present in all strains together with R factor in TA98 and TA100
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: The broth used to grow overnight cultures of tester strains was Vogel-Bonner salt solution supplemented with 2.5% (w/v) Oxoid Nutrient Broth No. 2. Bottom agar (25 mL per 15 x 100 mm petri dish) was added to the Vogel-Bonner minimal medium E and supplemented with 1.5% (w/v) agar and 0.2% (w/v) glucose. Top agar consisting of 0.7 % w/v agar and 0.5 % w/v sodium chloride was supplemented with 10 mL of 0.11 mg/mL tryptophan solution per 100 mL agar. When S9 mix was required, 2.0 mL of the supplemented top agar was used in the overlay. However, when S9 was not required, water was added to the supplemented top agar (0.5 mL of water per 2 mL of supplemented top agar) and the resulting 2.5 mL of diluted supplemented top agar was used for the overlay.
- Periodically checked for genotype stability: Yes. The presence of the pKM101 plasmid was confirmed for tester strains TA98 and TA100 by demonstration of resistance to ampicillin (10 µg). The presence of the rfa wall mutation was confirmed by testing the sensitivity of each culture to crystal violet (10 µg).
- Periodically "cleansed" against high spontaneous background: Yes. The mean numbers of spontaneous revertants per plate in the vehicle controls that are characteristic of the respective strains were demonstrated by plating 100 µg aliquots of the culture along with the appropriate vehicle on selective media.
Additional strain / cell type characteristics:
other: contains uvrA
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
MUTAGENICITY ASSAY
Salmonella typhimurium
3330, 1000, 333, 100, 33.3, 10.0 µg/plate (with S9)
100, 33.3, 10.0, 3.33, 1.00, 0.33 µg/plate (without S9)

Escherichia coli
10000, 3330, 1000, 333, 100, 33.3 µg/plate (with and without S9)

CONFIRMATORY ASSAY
Salmonella typhimurium
3330, 1000, 333, 100, 33.3, 10.0 µg/plate (with S9)
333, 100, 33.3, 10.0, 3.33, 1.00 µg/plate (without S9)

Escherichia coli
3330, 1000, 333, 100, 33.3, 10.0 µg/plate (with S9)
10000, 3330, 1000, 333, 100, 33.3 µg/plate (without S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
plated for all tester strains both in the presence and absence of S9 with no test material
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
other: 2-aminoanthracene, ICR-191
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Exposure duration: plates were inverted and incubated for 48 ± 2 hours at 37 ± 2 ºC.

NUMBER OF REPLICATIONS: test concentrations were performed in triplicate

EVALUATION PROCEDURE
The condition of the bacterial background lawn was evaluated for evidence of cytotoxicity and test material precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate. The numbers of revertant colonies per plate for the vehicle and negative controls and all plates containing test material were counted manually. The numbers of revertant colonies per plate for positive controls were counted by automated colony counter.
Evaluation criteria:
Test data from individual experiments was considered valid if:
a) tester strain cultures exhibit sensitivity to crystal violet
b) cultures of tester strains TA98 and TA100 exhibit resistance to ampicillin
c) tester strain cultures exhibit a characteristic number of spontaneous revertants per plate when plated along with the vehicle under selective conditions
d) positive controls must exhibit a 3-fold increase over the value of the vehicle control for that strain
e) a minimum of three non-toxic doses are required to evaluate assay data.

Once the criteria for a valid assay have been met, responses observed in the assay are evaluated as follows:

Tester strains TA98, TA100 and WPuvrA-
For a test material to be considered positive, it must produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test material.

Tester strains TA1535 and TA1537
For a test material to be considered positive, it must produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test material.

Results and discussion

Test results
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Test material handling
In acetone the test material formed a cloudy yellow homogenous suspension at 200 mg/mL which was the most concentrated stock dilution of the test material prepared. The test material formed a solution at 20 mg/mL and remained a solution at all subsequent dilutions prepared for the mutagenicity assay.

- Dose Range Finding Study
Doses to be tested in the mutagenicity assay were selected based on the results of the dose rangefinding study conducted on the test material using tester strains TA100 and WP2uvrA- in both the presence and absence of a 10% S9 mix (one plate per dose). Ten doses of test material, from 10,000 to 10.0 µg/plate, were tested. Under the conditions of the study, cytotoxicity was observed with tester strain TA100 at 667 µg/plate and above in the presence of S9 and at 33.3 µg/plate and above in the absence of S9 as evidenced by a thinning and/or disappearance of the bacterial background lawn and a reduction in the number of revertants per plate. With tester strain WP2uvrA-, cytotoxicity was observed at 10,000 µg/plate in the presence of S9 and at 667 µg/plate in the presence of S9 and at 667 µg/plate in the absence of S9 as evidenced by a slight thinning of the bacterial background lawn and/or a reduction in the number of revertants per plate.

- S9 Optimisation Study
An S9 optimisation study was performed using tester strains TA98 and TA100 with the highest non-cytotoxic dose of test material, 333 µg/plate, and four concentrations of S9 mix (5%, 10%, 20% and 80% S9 homogenate per mL of S9 mix). Under the conditions of the study, no effect was observed due to varying the S9 homogenate concentration in this experiment. In the absence of an effect, a 10% S9 mix was used in the mutagenicity assay.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Mutagenicity Assay
In the initial mutagenicity assay, all data were acceptable and no positive increases in the number of revertants per plate were observed with any of the tester strains either in the presence or absence of S9. After a review of the data generated in the initial mutagenicity assay, it was decided to increase the doses treated with the Salmonella tester strains in the absence of S9 and to decrease the doses tested with the E. coli tester strain in the presence of S9 due to the observed levels of cytotoxicity.

 

Confirmatory Mutagenicity Assay
In the confirmatory mutagenicity assay, all data were acceptable and no positive increases in the number of revertants per plate were observed with any of the tester strains either in the presence or absence of S9. In the absence of S9, greater cytotoxicity was observed than demonstrated in the initial mutagenicity assay. For this reason, it was decided to retest all strains in the absence of S9 in a further confirmatory assay.

 

Further Confirmatory Assay (in the absence of S9)
All data with the Salmonella tester strains were acceptable and no positive increases in the number of revertants per plate were observed. The cytotoxicity observed in this experiment was similar to that observed in the initial mutagenicity assay. No data were generated with tester strain WP2uvrA- due to an unacceptable mean vehicle control value for this strain. Tester strain WP2uvrA- was retested in a further experiment in which all data were acceptable and no positive increase in the number of revertants per plate was observed in the absence of S9.

Table 1: Mutagenicity Assay Results (means)

With S9

Dose (µg)/plate

TA98

TA100

TA1535

TA1537

Background lawn

With S9

Dose (µg)/plate

WP2uvrA-

Background lawn

Vehicle control

25

138

22

4

1

Vehicle control

24

1

Negative control

32

120

20

7

1

Negative control

23

1

Test material

10.0

31

129

21

12

1

Test material

33.3

22

1

33.3

38

130

21

6

1

100

14

1

100

26

135

16

7

1

333

21

1

333

27

114

16

6

1

1000

9

1

1000

20

41

9

4

1

3330

6

1sp

3330

10

8

2

1

2sp

10000

0

6mp

Positive control

1118

1152

303

208

1

Positive control

302

1

Without S9

Without S9

Vehicle control

17

95

17

5

1

Vehicle control

16

1

Negative control

18

99

16

8

1

Negative control

21

1

Test material

0.33

21

93

17

7

1

Test material

33.3

17

1

1.00

15

102

13

4

1

100

11

1

3.33

16

94

9

8

1

333

19

1

10.0

15

108

13

6

1

1000

21

1

33.3

12

81

12

6

2

3330

18

2

100

9

70

16

5

2

10000

17

2

Positive control

178

608

516

362

1

Positive control

484

1

Table 2: Confirmatory Assay Results (means)

With S9 Dose (µg)/plate TA98 TA100 TA1535 TA1537 Background lawn With S9 Dose (µg)/plate WP2uvrA- Background lawn
Vehicle control 36 132 14 8 1 Vehicle control 23 1
Negative control 35 137 11 10 1 Negative control 25 1
Test material 10.0 31 138 11 6 1 Test material 10.0 18 1
33.3 32 149 13 11 1 33.3 13 1
100 39 124 10 12 1 100 14 1
333 27 133 9 9 1 333 13 1
1000 23 47 7 5 2sp 1000 13 1
3330 11 2 1 1 2sp 3330 4 1sp
Positive control 1434 1594 419 213 1 Positive control 516 1
Without S9 Without S9
Vehicle control 18 97 11 6 1 Vehicle control 11 1
Negative control 18 107 11 5 1 Negative control 18 1
Test material 1.00 16 107 12 5 1 Test material 33.3 15 1
3.33 13 111 15 4 1 100 18 1
10.0 11 76 15 3 3 333 15 2
33.3 16 100 9 4 2 1000 8 2
100 10 66 9 0 3 3330 1 2sp
333 4 28 5 2 3 10000 0 3sp
Positive control 174 570 542 362 1 Positive control 1041 1

Table 3: Further Confirmatory Assay Results (means)

Without S9 Dose (µg)/plate TA98 TA100 TA1535 TA1537 Background lawn Without S9 (1) Dose (µg)/plate WP2uvrA- (1) Background lawn (1) WP2uvrA- (2) Background lawn (2)
Vehicle control 12 111 12 6 1 Vehicle control 176 1 18 1
Negative control 12 109 11 5 1 Negative control NC 1 19 1
Test material 1.00 19 112 11 8 1 Test material 33.3 NC 1 20 1
3.33 16 111 11 3 1 100 NC 1 20 1
10.0 11 96 11 6 1 333 NC 1 15 1
33.3 16 98 12 5 1 1000 NC 1 16 2
100 10 69 13 3 2 3330 NC 2 1 2sp
333 0 35 0 0 3 10000 NC 3 0 3sp
Positive control 211 861 692 353 1 Positive control NC 1 721 1

NC = not counted due to unacceptable mean vehicle control value

Background Lawn Evaluation Codes

1 = normal

2 = slightly reduced

3 = moderately reduced

sp = slight precipitate

mp = moderate precipitate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of the study, in both an initial and confirmatory assay, the test material did not cause a positive increase in the number of revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9).
Executive summary:

The potential of the test material to cause gene mutation in bacterial strains was determined following a methodology similar to that outlined in the standardised guideline OECD 471. During the study, four strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and one strain of Escherichia coli (WP2uvrA-) were treated in the presence and absence of a rat liver derived metabolic activation system (S9 mix). In two separate assays, the test material did not induce any significant increases in the number of revertant colonies, in any of the tester strains, either in the presence or absence of S9 mix.