Registration Dossier

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:
From January 16, 2006 to February 2, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was performed according to OECD Guideline 471 and EU method B.13/B14, with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Erythromycin A-9 Oxime (E) Hydrochloride.
- Physical state: white powder.
- Lot/batch No.: B5310059
- Storage condition of test material: at ambient temperature, in the dark.
- Other:
pH: 6.16 (1% solution in deionised water, w/v, determined with a pH-Meter WTW pH340)

Method

Species / strain
Species / strain / cell type:
other: TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from Aroclor 1254
Test concentrations with justification for top dose:
Whole first experiment and second experiment with strains TA 97a, TA100 and TA1535: 0.09, 0.26, 0.8, 2.3, 7 and 21 µg/plate (with and without S9).
Second experiment with strains TA98 and TA102: 0.8, 2.3, 7, 21, 62 and 185 µg/plate (with and without S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was not enough soluble in water; DMSO is a common vehicle for the Ames test.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
2-nitrofluorene
sodium azide
other: 2-Aminoanthracene, 1, 8-Dihydroxy-anthraquinone, 4-Nitro-o-phenylenediamine, t-Butyl-hydroperoxide
Remarks:
Without S9: 4NOPD, 2 NF, Sodium-azide, tBHPO. With S9: DMBA, 2AA,DHA.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 12h (overnight)
- Exposure duration: 2 days.

NUMBER OF REPLICATIONS: triplicate repetitions were run for each dose group in each of the two separeate experiments. Control groups: six-fold repetitions.

NUMBER OF CELLS EVALUATED: 2 to 3 x 10E9 cells/mL

DETERMINATION OF CYTOTOXICITY
- Method: growth of bacterial background.

Evaluation criteria:
Means and standard deviations were calculated for the number of mutants in every concentration group.
The criteria for positive results are:
A reproducible increase of the number of revertants to more than the following thresholds values for at least one of the concentrations:
- For the strains with a low spontaneous revertant rate i.e. TA98 and TA 1535: the 2.5 fold of the amount of the spontaneous revertants.
- For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: the 1 2/3 fold of the amount of spontaneous revertants.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA97a, TA98, TA100, TA102 and TA1535.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Strains TA97a, TA100 and TA1535: toxic at 21µg/plate; strains TA102 and TA98: toxic at 185 µg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test substance was seen in any of the concentration groups.

RANGE-FINDING/SCREENING STUDIES:
The concentrations for the first experiment were set according to a preliminary toxicity test. The test substance was toxic at 21 µg/plate and above. At 7 µg/plate and below the bacterial background lawn was normal. Therefore, 21 µg/plate was chosen as the highest concentration.

The concentrations for the second experiment were changed according to the first one only for strains TA98 and TA102. No toxicity was found in these strains at 21 µg/plate. Therefore, the concentrations were increased two steps.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Mean number or revertants per plate for strain TA97a

Without metabolic activation

First experiment

Second experiment

Conc.

µg/plate

 

Revertants / plate

Conc.

µg/plate

 

Revertants / plate

mean

SD

N

mean

SD

N

21

Toxic

0.0

3

21

Toxic

0.0

3

7

103.7

6.7

3

7

110.3

20.5

3

2.3

95.3

16.9

3

2.3

124.7

12.5

3

0.8

112.0

13.5

3

0.8

117.0

32.4

3

0.26

119.0

3.5

3

0.26

118.7

16.2

3

0.09

118.0

19.3

3

0.09

129.0

23.3

3

solvent

103.3

8.4

6

solvent

125.2

15.5

6

positive

349.0

10.8

3

positive

368.0

19.1

3

With metabolic activation

First experiment

Second experiment

Conc.

µg/plate

 

Revertants / plate

Conc.

µg/plate

 

Revertants / plate

mean

SD

N

mean

SD

N

21

Toxic

0.0

3

21

Toxic

0.0

3

7

112.3

12.4

3

7

133.0

17.6

3

2.3

121.3

21.5

3

2.3

133.7

11.0

3

0.8

140.7

8.0

3

0.8

151.7

11.7

3

0.26

150.3

20.0

3

0.26

134.3

29.5

3

0.09

127.3

4.0

3

0.09

142.3

3.1

3

solvent

128.0

14.2

6

solvent

141.8

11.5

6

positive

609.0

162.5

3

positive

991.3

33.7

3

Table 2: Mean number of revertants per plate for strain TA98

Without metabolic activation

First experiment

Second experiment

Conc.

µg/plate

 

Revertants / plate

Conc.

µg/plate

 

Revertants / plate

mean

SD

N

mean

SD

N

21

6.3

1.5

3

185

Toxic

0.0

3

7

11.7

2.5

3

62

0.0

0.0

3

2.3

11.0

3.0

3

21

8.0

1.0

3

0.8

11.0

2.6

3

7

9.7

5.1

3

0.26

11.7

1.5

3

2.3

10.7

3.8

3

0.09

12.3

2.1

3

0.8

11.0

5.0

3

solvent

10.5

2.6

6

solvent

12.7

3.2

6

positive

147.7

6.4

3

positive

130.0

29.3

3

With metabolic activation

First experiment

Second experiment

Conc.

µg/plate

 

Revertants / plate

Conc.

µg/plate

 

Revertants / plate

mean

SD

N

mean

SD

N

21

8.7

3.1

3

185

Toxic

0.0

3

7

13.3

0.6

3

62

0.0

0.0

3

2.3

15.7

1.5

3

21

7.0

3.6

3

0.8

13.7

4.9

3

7

12.3

3.1

3

0.26

14.7

0.6

3

2.3

14.3

0.6

3

0.09

15.0

2.0

3

0.8

10.7

5.1

3

solvent

13.2

2.9

6

solvent

13.2

2.6

6

positive

311.0

38.3

3

positive

237.7

28.5

3

Table 3: Mean number of revertants per plate for strain TA100

Without metabolic activation

First experiment

Second experiment

Conc.

µg/plate

 

Revertants / plate

Conc.

µg/plate

 

Revertants / plate

mean

SD

N

mean

SD

N

21

Toxic

0.0

3

21

toxic

0.0

3

7

139.3

10.1

3

7

42.7

5.7

3

2.3

177.7

32.9

3

2.3

61.3

4.9

3

0.8

151.3

12.9

3

0.8

75.0

11.8

3

0.26

165.7

24.6

3

0.26

82.3

15.0

3

0.09

185.7

19.4

3

0.09

81.7

13.7

3

solvent

176.2

16.2

6

solvent

78.5

11.0

6

positive

416.3

22.7

3

positive

487.0

39.7

3

With metabolic activation

First experiment

Second experiment

Conc.

µg/plate

 

Revertants / plate

Conc.

µg/plate

 

Revertants / plate

mean

SD

N

mean

SD

N

21

Toxic

0.0

3

21

Toxic

0.0

3

7

182.0

7.8

3

7

60.7

8.5

3

2.3

181.7

10.1

3

2.3

77.3

9.2

3

0.8

181.0

8.7

3

0.8

84.0

17.8

3

0.26

177.0

12.3

3

0.26

90.7

25.0

3

0.09

157.7

14.0

3

0.09

109.0

35.3

3

solvent

177.3

8.3

6

solvent

94.0

15.3

6

positive

1397.7

29.5

3

positive

1396.3

60.7

3

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

According to the results obtained in this study, the test substance is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535, with and without an external metabolising system up to the limit of toxicity.
Executive summary:

The test substance was tested for mutagenic activity with the “Salmonella typhimurium Reverse Mutation Test” (Ames test). The study was conducted according to OECD Guideline 471 and B.13 EU Method, with GLP. The test substance was dissolved in DMSO. The test test was performed according to the “direct plate incorporation method”. As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used. Negative and positive controls were included. An independent repetition of the experiment was performed. According to the results obtained in this study, the test substance is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535, with and without an external metabolising system up to the limit of toxicity.