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Genetic toxicity in vitro

Description of key information

Ames Test

Under the conditions of this study, the test material was considered to be non-mutagenic.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 November 2003 to 9 December 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
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 information:
Composition 1
Target gene:
- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
- Toxicity Test
A toxicity test, using strain TA 100 only, was performed to establish suitable dose levels for the mutation tests. One plate of each of the following concentrations of test material was prepared:
17, 50, 167, 500, 1667 and 5000 μg per plate (with and without S9 mix)

- Mutation Tests
The dose levels for the mutation assays, based on the results of the toxicity test, were:
17, 50, 167, 500, 1667 and 5000 μg per plate (with and without S9 mix)
Vehicle:
- Vehicle(s)/solvent(s) used: Sterile ultra-pure water
Negative controls:
yes
Solvent controls:
yes
Remarks:
Sterile ultra-pure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-aminoanthracene
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and pre-incubation

EXPERIMENT 1- PLATE INCORPORATION METHOD
Volumes of soft agar (2 mL) were dispensed into small sterile tubes. To this, 0.5 mL of S9 mix or 0.05 M phosphate buffer, pH 7.4 were added, followed by 0.1 mL of bacteria. The solvent or test solution (0.1 mL) was added last. The tube contents, which were continually cooling, were mixed and poured onto minimal medium plates, prepared in-house. These plates contained 20 mL of 1.5% purified agar, in Vogel-Bonner Medium E (Vogel et al (1956)) with 2% glucose. When the soft agar had set, the plates were inverted and incubated at 37°C for 2 or 3 days. All testing for this experiment was performed in triplicate.

EXPERIMENT 2- PRE-INCUBATION METHOD
Volumes of S9 mix or 0.05 M phosphate buffer, pH 7.4 (0.5 mL) were dispensed into small sterile tubes. This was followed by 0.1 ml of bacteria per tube and, finally, the solvent or test solution (0.1 mL per tube). The tube tops were then screwed on tightly and the tubes placed in a shaking incubator at 37°C for 20 min. After this, the tube tops were removed and 2 mL of soft agar added to each tube. The tube contents, which were continually cooling, were mixed and then poured onto agar plates, as above. When the soft agar had set, the plates were inverted and incubated at 37°C for 2 or 3 days. All testing for this experiment was performed in triplicate.

INCUBATION AND SCORING
After incubation, the colonies were counted using a Cardinal Colony Counter set at maximum sensitivity, i.e. colonies of 0.1 mm or more in diameter were counted. The data was captured electronically using a validated software system. The plates were also examined for precipitates and microscopically, for microcolony growth.

NUMBER OF REPLICATIONS: 3

ACCEPTABILITY CRITERIA
A test was considered acceptable if, for each strain:
i) the bacteria demonstrated their typical responses to crystal violet, ampicillin and u.v. light.
ii) at least 2 of the vehicle control plates were within the following ranges: TA 1535, 4-30; TA 1537, 1-20; TA 98, 10-60; TA 100, 60-200 and E. coli WP2uvrA 1-60.
iii) on at least 2 of the positive control plates there were at least x 2 the mean vehicle control mutant numbers per plate, or in the case of TA 100, at least x 1.5 the mean vehicle control mutant numbers per plate.
iv) no toxicity or contamination was observed in at least 4 dose levels.
v) in cases where a mutagenic response was observed, no more than one dose level was discarded before the dose that gave the highest significant mean colony number.

Evaluation criteria:
Where the acceptibility criteria were met, a significant mutagenic response was recorded if there was:
i) for S. typhimurium strains TA 1535, TA 1537, and TA 98 and for E. coli, at least a doubling of the mean concurrent vehicle control values at some concentration of the test material. For S. typhimurium strain TA 100, a 1.5-fold increase over the control value was considered significant. If the mean colony count on the vehicle control plates was less than 10, then a value of 10 was assumed for assessment purposes. In such cases, a minimum count of 20 was required before a significant mutagenic response was registered.
ii) a dose related response, although at high dose levels this relationship could be inverted because of, for example, (1) toxicity to the bacteria generally, (2) specific toxicity to the mutants and (3) inhibition of foreign compound metabolising enzymes where mutagens require metabolic activation by the liver.
iii) a reproducible effect in independent tests.
Key result
Species / strain:
other: S. typhimurium TA1535, TA1537, TA98, TA100; E. coli WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
> Toxicity Test
No toxicity to the bacteria was observed and no precipitation of the test material occurred in either the presence or the absence of S9 mix.

> Mutation Tests
- Vehicle Control
The vehicle control values were generally within the normal ranges experienced at the Test Facility and reported in the literature with these strains of S. typhimurium and E. coli, (Ames et al, 1975; Gatehouse et al, 1994). The exception was TA 98 in the first mutation assay, in the absence of S9 mix. On this occasion the mean vehicle control counts for TA 98 were lower than the acceptance criterion (see test rejection)
- Positive Control
The results obtained in the positive control groups were generally within the normal ranges expected for each bacterial strain and activation condition. In the first mutation assay retest (see test rejection), a poor response was obtained for TA 1537 with 2-aminoanthracene. Poor responses were also obtained in the second mutation assay for all strains with 2-aminoanthracene. These parts of both assays were repeated successfully.
- Test material
The test material did not induce mutagenic activity in any of the 5 bacterial strains used, in either activation condition.
There was no toxicity to the bacteria and no precipitation of the test item was observed in either mutation assay, in either the presence or the absence of S9 mix.

- Test Rejection
The first mutation assay in the presence of S9 mix was rejected due to an operator error in the preparation of the S9 mix. In the retest, TA 1537 was rejected due to the poor response obtained with 2-aminoanthracene. In the absence of S9 mix, TA 98 was repeated due to a dosing error at 17 μg per plate and also the vehicle control counts were outwith the acceptance criterion. WP2uvrA was also repeated due to a dosing error at 500 μg per plate.
In the second mutation assay, all strains were repeated in the presence of S9 mix due to poor responses obtained with 2-aminoanthracene. Also in this test TA 100 was rejected in the absence of S9 mix due to a dosing error at 500 μg per plate. These parts of the tests were repeated successfully.

Table 1: Summary of Results from Experiment 1

Mean Number of Revertant Colonies Per Plate in the Absence of S9 Mix

Test item

Dose level (µg per plate)

TA1535

TA1537

TA98*

TA100

WP2uvrA*

Water

100 µL

22

5

22

153

36

Test material

17

22

5

21

141

43

50

25

7

16

150

44

167

23

7

21

131

40

500

20

5

15

145

41

1667

26

8

18

141

36

5000

22

8

13

131

41

Positive controls

Compound

NaN3

9AA

2NF

NaN3

ENNG

Dose level (µg per plate)

1

80

1

1

2

Mean

412

4411

1310

1014

270

Mean Number of Revertant Colonies Per Plate in the Presence of S9 Mix

Test item

Dose level (µg per plate)

TA1535

TA1537*

TA98

TA100

WP2uvrA

Water

100 µL

21

17

39

114

7

Test material

17

29

15

32

124

8

50

17

18

38

125

14

167

26

15

36

114

11

500

27

22

31

124

10

1667

31

17

35

105

14

5000

31

15

36

128

12

Positive controls

Compound

2AAN

2AAN

2AAN

2AAN

2AAN

Dose level (µg per plate)

2

2

0.5

0.5

20

Mean

602

526

2721

4449

246

 * Results from re-tests

 

NaN3: sodium azide

9AA: 9-aminoacradene

2NF: 2-nitrofluorene

ENNG: N-ethyl-N-nitro-N-nitrosoguanidine

2AAN: 2-aminoanthracene

Table 2: Summary of results from Experiment 2

Mean Number of Revertant Colonies Per Plate in the Absence of S9 Mix

Test item

Dose level (µg per plate)

TA1535

TA1537

TA98

TA100*

WP2uvrA

Water

100 µL

11

10

22

86

8

Test material

17

14

9

17

97

13

50

17

12

21

89

6

167

10

8

18

89

8

500

11

7

19

86

9

1667

10

10

20

80

7

5000

12

9

20

85

7

Positive controls

Compound

NaN3

9AA

2NF

NaN3

ENNG

Dose level (µg per plate)

1

80

1

1

2

Mean

283

3038

1610

1014

387

Mean Number of Revertant Colonies Per Plate in the Presence of S9 Mix

Test item

Dose level (µg per plate)

TA1535

TA1537

TA98

TA100

WP2uvrA

Water

100 µL

15

14

21

76

7

Test material

17

13

12

26

79

6

50

11

13

22

75

9

167

15

7

24

72

13

500

14

11

22

76

8

1667

11

14

20

74

7

5000

9

5

18

74

7

Positive controls

Compound

2AAN

2AAN

2AAN

2AAN

2AAN

Dose level (µg per plate)

2

2

0.5

0.5

20

Mean

65

36

79

190

11

  * Results from re-test

 

NaN3: sodium azide

9AA: 9-aminoacradene

2NF: 2-nitrofluorene

ENNG: N-ethyl-N-nitro-N-nitrosoguanidine

2AAN: 2-aminoanthracene

Conclusions:
It was concluded that the test material was not mutagenic to Salmonella typhimurium or Escherichia coli when tested in sterile, ultra-pure water up to a pre-determined maximum concentration of 5000 μg per plate.
Executive summary:

The mutagenic potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 471 and EU Method B.13/14, under GLP conditions.

During the study the test material was tested for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli WP2uvrA at concentrations ranging from 17 to 5000 μg per plate. The solvent used was sterile, ultra-pure water.

Two independent tests (one direct plate and one pre-incubation) were conducted on agar plates in the presence and absence of an Aroclor-1254 induced rat liver preparation and the co-factors required for mixed-function oxidase activity (S9 mix). The first mutation assay was repeated in the presence of S9 mix only due to an operator error.

Concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix.

No mutagenic activity was observed in any of the 5 bacterial strains, in either activation condition.

There was no toxicity to the bacteria and no precipitation of the test material was observed in either mutation assay, in either the presence or the absence of S9 mix.

It was concluded that the test material was not mutagenic to Salmonella typhimurium or Escherichia coli when tested in sterile, ultra-pure water up to a pre-determined maximum concentration of 5000 μg per plate.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames Test

The mutagenic potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 471 and EU Method B.13/14, under GLP conditions.

During the study the test material was tested for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli WP2uvrA at concentrations ranging from 17 to 5000 μg per plate. The solvent used was sterile, ultra-pure water.

Two independent tests (one direct plate and one pre-incubation) were conducted on agar plates in the presence and absence of an Aroclor-1254 induced rat liver preparation and the co-factors required for mixed-function oxidase activity (S9 mix). The first mutation assay was repeated in the presence of S9 mix only due to an operator error.

Concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix.

No mutagenic activity was observed in any of the 5 bacterial strains, in either activation condition.

There was no toxicity to the bacteria and no precipitation of the test material was observed in either mutation assay, in either the presence or the absence of S9 mix.

It was concluded that the test material was not mutagenic to Salmonella typhimurium or Escherichia coli when tested in sterile, ultra-pure water up to a pre-determined maximum concentration of 5000 μg per plate.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.