Results – Mutagenic potential – STABILGREEN® K

Confirmation of genotype is performed for each batch of lyophilized bacteria before stock culture preparation. The last performance showed no abnormalities.

First Experiment

 Confirmation of the Criteria and Validity

All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. Nearly all determined values for the spontaneous revertant of the negative controls were in the normal range of the test laboratory (historical data of the laboratory). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and nearly all were within the historical control data ranges.

Solubility and Toxicity

In the first experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.

Mutagenicity

No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the test conditions. To verify this result, a further experiment was performed.

Survey of the Findings

The mean revertant values of the 3 replicates are presented in the following table.

Table Mean Revertants First Experiment

   Strain 

TA97a 

TA98 

TA100 

TA102 

TA1535 

   Induction 

 -S9

 +S9

 -S9

 +S9

 -S9

 +S9

 -S9

 +S9

 -S9

 +S9

   H2O 

Mean 

124

63

11

12

87

93

389

361

19

14

sd 

6.9 

7.5 

2.0 

2.6 

7.4 

12.4 

59.0 

36.3 

1.2 

1.2 

   DMSO 

Mean 

70

77

11

16

83

81

368

365

14

13

sd 

12.1 

5.5 

2.9 

6.4 

9.0 

0.6 

24.0 

101.3 

3.2 

4.6 

   Positive
   Controls* 

Mean 

513

487

305

55

756

1371

1183

1101

252

121

sd 

83.3 

26.0 

8.3 

7.1 

38.6 

524.9 

16.7 

150.1 

87.7 

21.2 

f(l) 

7.33 

6.32 

27.73 

3.44 

8.69 

16.93 

3.21 

3.02 

13.26 

9.31 

   5000
   μg/plate 

Mean 

82

88

10

21

86

96

311

340

21

15

sd 

6.7 

4.6 

4.2 

6.4 

9.2 

3.5 

64.7 

62.1 

8.1 

0.6 

f(l) 

1.17 

1.14 

0.91 

1.31 

1.04 

1.19 

0.85 

0.93 

1.50 

1.15 

   1500 
   μg/plate 

Mean 

87

85

17

19

92

76

292

337

18

13

sd 

3.5 

1.0 

4.5 

4.6 

18.0 

17.0 

48.7 

66.0 

4.6 

3.0 

f(l) 

1.24 

1.10 

1.55 

1.19 

1.11 

0.94 

0.79 

0.92 

1.29 

1.00 

   500 μg/plate 

Mean 

78

78

9

17

87

73

267

331

14

14

sd 

20.5 

9.7 

1.5 

5.7 

7.5 

8.2 

35.9 

9.2 

0.6 

2.6 

f(l) 

1.11 

1.01 

0.82 

1.06 

1.05 

0.90 

0.73 

0.91 

1.00 

1.08 

   150 μg/plate 

Mean 

89

96

13

12

86

86

320

331

16

17

sd 

21.7 

10.0 

2.1 

2.1 

3.8 

17.0 

17.1 

31.1 

10.7 

2.6 

f(l) 

1.27 

1.25 

1.18 

0.75 

1.04 

1.06 

0.87 

0.91 

1.14 

1.31 

   50 μg/plate 

Mean 

65

112

9

12

89

89

279

344

11

12

sd 

8.6 

7.4 

29

3.0 

6.4 

10.1 

34.0 

76.2 

1.5 

2.3 

f(l) 

0.93 

1.45 

0.82 

0.75 

1.07 

1.10 

0.76 

0.94 

0.79 

0.92 

f(l) = increase factor
* Different positive controls were used

Second Experiment

Confirmation of The Criteria and Validity

All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertant of the negative controls were in the normal range of the test laboratory (historical data of the laboratory). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and nearly all were within the historical control data ranges.

 Solubility and Toxicity

In the second experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.

Mutagenicity

No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the test conditions.

Survey of the Findings

The mean revertant values of the 3 replicates are presented in the following table.

Table Mean Revertants Second Experiment

   Strain 

TA97a 

TA98 

TA100 

   Induction 

 -S9

 +S9

 -S9

 +S9

 -S9

 +S9

   H2O 

Mean 

94

112

24

27

91

135

sd 

17.9 

16.4 

5.2 

3.5 

9.0 

28.1 

   DMSO 

Mean 

129

115

19

25

116

121

sd 

24.4 

27.0 

2.0 

6.1 

16.0 

24.4 

   Positive
   Controls* 

Mean 

568

584

717

82

693

644

sd 

112.6 

104.2 

164.4 

7.8 

12.2 

31.2 

f(l) 

4.40 

5.08 

37.74 

3.28 

7.62 

5.32 

   5000
   μg/plate 

Mean 

92

117

27

26

89

113

sd 

33.0 

9.9 

3.5 

4.9 

9.6 

14.6 

f(l) 

0.71 

1.02 

1.42 

1.04 

0.77 

0.93 

   2500
   μg/plate 

Mean 

123

143

19

24

102

111

sd 

20.4 

6.1 

2.3 

2.6 

16.8 

25.1 

f(l) 

0.95 

1.24 

1.00 

0.96 

0.88 

0.92 

   1250
   μg/plate 

Mean 

131

121

22

28

118

124

sd 

8.1 

2.3 

9.2 

7.6 

10.5 

11.8 

f(l) 

1.02 

1.05 

1.16 

1.12 

1.02 

1.02 

   625
   μg/plate 

Mean 

76

133

20

30

100

123

sd 

12.2 

14.0 

2.0 

0.6 

11.5 

18.1 

f(l) 

0.59 

1.16 

1.05 

1.20 

0.86 

1.02 

   313
   μg/plate 

Mean 

101

147

23

31

85

125

sd 

6.1 

21.4 

6.7 

5.1 

9.2 

32.5 

f(l) 

0.78 

1.28 

1.21 

1.24 

0.73 

1.03 

f(l) = increase factor
* Different positive controls were used.

Mutagenicity of Test Item

The test item STABILGREEN®K (Polyglyceryl-4 Oleyl Ether, Dioleyl Phosphate) showed no increase in the number of revertant in all bacteria strains in both experiments.

Nearly all negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that STABILGREEN®K (Polyglyceryl-4 Oleyl Ether, Dioleyl Phosphate) is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.

Acceptability of Study

In all experiments, no precipitation of the test item was observed at any of the tested concentrations up to 5000 μg/plate.
In the first and the second experiment, the test item caused no cytotoxicity towards all bacteria strains

The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 109 bacteria/mL.

Nearly all of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. Nearly all numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory (historical data of the laboratory) but all were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens.

Since all criteria for acceptability have been met, the study is considered valid.

DEVIATIONS
Deviations from the Study Plan

No deviations from the study plan were observed.

Deviations from the Guideline

None known.

RECORDING

One original of study plan and final report, respectively, all raw data of the study and all documents mentioned or referred to in study plan or final report will be kept in the GLP Document Archive of the test facility for 15 years. After that, the sponsor’s instructions will be applied (shipment of documentation to sponsor). A retain sample of the test item will be kept in the GLP Substance Archive for 15 years; then, the retain sample will be discarded. Number of originals which will be sent to the sponsor: 1