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Toxicological information

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:
1993-01-15 to 1993-02-02
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study does not include a test using E. coli or TA 102 and was only performed as plate incubation test and lack of data on the purity / quality of the sample tested.

Data source

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

Materials and methods

Test guideline
Qualifier:
no guideline followed
Guideline:
other: Study carried out in "the Salmonella / microsome test as described by Ames et al (1973a 1975) and Maron and Ames (1983)
Deviations:
yes
Remarks:
The study does not include a test using E. coli or TA 102 and was only performed as plat incubation test.
Principles of method if other than guideline:
The priciples of method correspond to the guideline OECD 471.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Hydroxyambran

Method

Target gene:
Strain Amino acid marker Other relevant mutations Plasmid
Salmonella
thyphimurium Histidine mutation Type of mutation Main DNA target Cell wall DNA-repair
TA 1535 hisG46 base pair substitution GC rfa uvrB- no
TA 100 hisG46 base pair substitution GC rfa uvrB- pKM101
TA 1537 hisG3076 frameshift GC rfa uvrB- no
TA 98 hisG3052 frameshift GC rfa uvrB- pKM101
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: see above
Metabolic activation:
with and without
Metabolic activation system:
S 9 mix from Aroclor 1254 induced Wistar rats livers
Test concentrations with justification for top dose:
8, 40, 200, 1000, 5000, 7500 µg/plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-Nitro-1,2-phenylene diamine; 2-Aminoanthracene
Details on test system and experimental conditions:
Test procedure:
In order to select the doses for the main test, a preliminary range finding was conducted with seven concentrations of the test article (10, 32, 100, 320, 1000, 3200 and 10000 µg/plate). In each case, 0.1 ml of the test article solution, o.1 ml of TA 100 bacterial suspension (overnight culture, about 109 cells/ml), 0.5 ml of buffer and 2 ml top agar were mixed and emptied onto petri dishes. In a parallel experiment, 0.1 ml of a bacterial suspension diluted to 10-6 and 0.1 ml of test article solution were pated onto nutrient broth agar plates.
The number of colonies, the number of revertants and the background growth were determined as a measure of the toxicity of HYDROXYAMBRAN. Bacteriotoxic effects were caused in the range from 32 to 10000 µg/plate. However, these effects were not extremely expressed, because the degree of reduction of count of revertants, background growth and of the number of viable cells persisted at the higher concentrations.
The following concentrations were therefore chosen for the main tests (4% and 10% S9): 8, 40, 200, 1000 and 5000 µg/plate. The HYDROXYAMBRAN sample was dissolved in DMSO. In the repetition test (4% S9) the highest test concentration was increased to 7500 µg/plate.

The main tests and the repetition test were performed as follows:
0.1 ml of each test article solution, 0.1 ml of bacterial suspension, 0,5 ml of S9 mix or buffer and 2.0 ml of top agar were mixed and poured onto petri dishes containing solid agar. The petri dishes were incubated at 37 °C for 48 hours and the revertant colonies were counted. Three plates were used for each strain and concentration level. Furthermore, the test was carried out with and without S9 mix. For the negative (solvent without test article) and positive control three plates each were used.
The bacterial suspension used were obtained from 16-houir cultures in nutrient broth which had been incubated at 37 °C and shaken at 90 rpm. These suspensions were used for the determination of mutant counts. The number of viable cells was established concomitantly when determining the titer.
After incubation for 48 hours at 37 °C, the colonies were counted by an ARTEK COUNTER MODEL 982B. Automatic counts were validated by manual counting in regular intervals.

The test was repeated in an independent experiment (with 10% S9).

The following criteria were used for the acceptance of an assay:
- The negative controls had to be within the expected range as defined by published data (Maron and Ames 1983)
- The positive controls had to show sufficient effects as defined by the laboratory’s experience
- The titer determination must have revealed a sufficient bacterial density in the suspension.
Evaluation criteria:
Assessment of mutagenicity and bacteriotoxicity:
A reproducible and dose-related increase of mutant counts for at least one strain is considered positive. For TA 98 and TA 1535 a twofold increase of revertants compared to the negative controls should be reached, whereas for TA 1537 a threefold increase should be attained. For TA 100 a 1.5-fold increase is regarded as an indication of potential mutagenicity. Otherwise the results are considered to be negative.

The criterion for a biologically significant bacteriotoxic effect is a reduction in the number of colonies/plate or revertants/plate or in background growth by more than 50% relative to the respective negative control.
Statistics:
Calculation of the mean value and the standard deviation from the counts of three plates

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
Remarks:
Bacteriotoxic effects were caused in the range from 32 to 10000 µg/plate. However, these effects were not extremely expressed.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Main test I (4% S9):
An increase of count of revertants as an indicator for mutagenic activity was observed in TA 1535 at the highest test concentration of 5000 µg/plate. This effect was irrespective of the presence or absence of metabolic activity.
Bacteriotoxic effects were induced in two of the for test organisms. With TA 1537 a reduction of the count of revertants was caused in the range from 40 to 5000 µg/plate in the absence of metabolic activation and, beginning at a concentration of 200 µg/plate, in the presence of metabolic activation.
With TA 100 this effect was observed in the range from 200 to 5000 µg/plate without metabolic activation and, beginning at a concentration of 1000 µg/plate, with metabolic activation. With test organisms TA 1535 and TA 98 no significant bacteriotoxic effects were observed.

Main test II (10% S9):
The second main test was performed with the same concentrations as main test I. The S9 concentration was increased to 10%.
No evidence of mutagenic activity of HYDROXYAMBRAN was found. Compared to negative controls (solvent), neither a biological relevant nor dose-related increase of the reversion rates were found. This observation was irrespective of the presence of S9 mix.
The results with regard to bacteriotoxicity with test organisms TA 1537 and TA 100 in the absence of metabolic activation was confirmed. With test organism TA 1537 the bacteriotoxic concentration was increased to 1000 µg/plate with metabolic activation (10% S9), but with test organism TA 100 no significant bacteriotoxic effect occurred in the presence of metabolic activation, although a slight reduction in the number of revertants was seen at 1000 and 5000 mg/plate. As in the first main test, TA 1535 and TA 98 revealed no bacteriotoxic effects irrespective of the absence or presence of metabolic activation.

Repetition test:
Because of the inconsistent results with regard to mutagenic activity, a test repetition was performed with the test organism TA 1535 in order to prove the results under the same test conditions as observed in the first main test (4% S9). And, with regard to a possible dose-related reaction, the highest test concentration was increased to 7500 mg/plate. Additionally, a repetition test (10% S9) was performed with test organism TA 1537.
Under these conditions no mutagenic activity was observed. Bacteriotoxic effects were caused with test organism TA 1537 in the range from 5000 to 7500 µg/plate in the absence of metabolic activation. In the presence of metabolic activation this effect was only observed at a concentration of 5000 µg/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Strain

Dose/Plate

First Main Test (4% S9)

Second Main Test (10% S9)

Repetition

(TA 1535 4% S9, TA 1537 10% S9)

 

 

Revertants per plate

Revertants per plate

Revertants per plate

 

 

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

 

 

M

Q

M

Q

M

Q

M

Q

M

Q

M

Q

TA 1535

Spont. Mut. Rate

22

0.6

-

-

19

1,3

-

-

24

1,3

-

-

 

DMSO

35

1

16

1

14

1

15

1

19

1

19

1

 

8

23

0.6

17

1.1

16

1,1

15

1,0

-

-

-

-

 

40

30

0.9

13

0.8

17

1,2

16

1,1

23

1,3

20

1.1

 

200

29

0.8

17

1,0

18

1,2

13

0,9

25

1,4

17

0.9

 

1000

50

1.4

25

1,6

15

1,0

17

1,1

17

0,9

17

0.9

 

5000

167

4.7

45

2.8

17

1.2

18

1.2

15

0.8

21

1.1

 

7500

-

-

-

-

-

-

-

-

17

0.9

23

1.2

 

Sodium azide

462

13.1

-

-

695

48.5

-

-

295

15.8

-

-

 

2-Aminoanthracene

-

-

219

13.4

-

-

223

14.5

-

-

135

7.3

 

Titer/ml

8.4 Exp+8

15.6 Exp+8

11.4 Exp+8

TA1537

Spont. Mut. Rate

13

0.8

-

-

14

1.5

-

-

18

1.0

-

-

 

DMSO

16

1

18

1

9

1

7

1

17

1

12

1

 

8

11

0.7

14

0.8

4

0.5

8

1.1

-

-

-

-

 

40

7

0.4

13

0.7

2

0.3

4

0.5

10

0.6

14

1.1

 

200

4

0.2

8

0.4

0

0.0

6

0.8

9

0.5

8

0.7

 

1000

4

0.3

4

0.2

0

0.0

2

0.3

11

0.7

7

0.6

 

5000

3

0.2

5

0.3

1

0.1

2

0.3

2

0.1

3

0.3

 

7500

-

-

-

-

-

-

-

-

3

0.2

7

0.6

 

9 Aminoacridine

206

12.9

-

-

43

4.6

-

-

213

12.5

-

-

 

2-Aminoanthracene

-

-

354

19.6

-

-

269

36.7

-

-

107

8.9

 

Titer/ml

6.4 Exp+8

9.4 Exp+8

9.7 Exp+8

TA 98

Spont. Mut. Rate

27

0.9

-

-

34

1.3

-

-

-

-

-

-

 

DMSO

32

1

33

1

26

1

25

1

 

 

 

 

 

8

28

0.9

38

1.2

22

0.8

22

0.9

 

 

 

 

 

40

21

0.7

26

0.8

17

0.6

24

1.0

 

 

 

 

 

200

18

0.6

25

0.7

15

0.6

23

0.9

 

 

 

 

 

1000

16

0.5

23

0.7

13

0.5

19

0.8

 

 

 

 

 

5000

17

0.5

22

0.7

16

0.6

19

0.8

 

 

 

 

 

7500

-

-

-

-

-

-

-

-

 

 

 

 

 

4-Nitro-1.2-phenylene diamine

730

23.1

-

-

843

32.0

-

-

 

 

 

 

 

2-Aminoanthracene

-

-

904

27.1

-

-

1087

44.1

 

 

 

 

 

Titer/ml

4.3 Exp+8

14.1 Exp+8

 

TA 100

Spont. Mut. Rate

188

1.0

-

-

148

0.9

-

-

-

-

-

-

 

DMSO

184

1

175

1

161

1

169

1

 

 

 

 

 

8

148

0.8

174

1.0

135

0.8

180

1.1

 

 

 

 

 

40

141

0.8

179

0.8

101

0.6

180

1.1

 

 

 

 

 

200

46

0.3

90

0.5

65

0.4

172

1.0

 

 

 

 

 

1000

39

0.2

75

0.4

45

0.3

82

0.3

 

 

 

 

 

5000

51

0.3

69

0.4

46

0.3

79

0.3

 

 

 

 

 

7500

-

-

-

-

-

-

-

-

 

 

 

 

 

Sodium azide

731

4.0

-

-

772

4.8

-

-

 

 

 

 

 

2-Aminoanthracene

-

-

932

5.3

-

-

1358

8.0

 

 

 

 

 

Titer/ml

3.5 Exp+8

6.0 Exp+8

 

 

M=mean value from 3 plates; Q= Quotient MTest: MDMSO; - S9 = without S9 mix; + S9 = with S9 mix

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In summary, the results indicate that HYDROXYAMBRAN caused no mutagenic effects at concentrations ranging from 8 to 7500 µg/plate in the four tester strains (Salmonella thyphimurium TA 1535, TA 100, TA 1537 and TA 98) in the plate incubation test with and without metabolic activation.

An indication for mutagenicity in the first main test with and without metabolic activation with TA 1535 at 5000 µg/plate was not confirmed in the second main test and in the repetion test too.
These effects observed in the first main test with TA 1535 (increase of revertants relative to the control) could possibly be explained by a disturbance in the test due to further differing bacteriotoxic effects of the test article HYDROXYANBRAN.

In summary, bacteriotoxic effects were observed in the range from 32 to 7500 µg/plate
Executive summary:

The mutagenicity of HYDROXYAMBRAN was determined in the four bacterial tester strains (Salmonella thyphimurium TA 1535, TA 100, TA 1537 and TA 98) in the plate incubation test with and without metabolic activation at concentrations ranging from 8 to 7500 µg/plate in accordance with OECD 471 (May 26, 1983).

 

An indication for mutagenicity in the first main test with and without metabolic activation with TA 1535 at 5000 µg/plate was not confirmed in the second main test and in the repetion test too.

These effects observed in the first main test with TA 1535 (increase of revertants relative to the control) could possibly be explained by a disturbance in the test due to further differing bacteriotoxic effects of the test article HYDROXYANBRAN.

 

In summary, the results indicate that HYDROXYAMBRAN caused no mutagenic effects at concentrations ranging from 8 to 7500 µg/plate in the four tester strains (Salmonella thyphimurium TA 1535, TA 100, TA 1537 and TA 98) with and without metabolic activation.

Bacteriotoxic effects were observed in the range from 32 to 7500 µg/plate