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Toxicological information

Genetic toxicity: in vitro

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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:
04 Jan - 22 Feb 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study. Due to cytotoxicity more marked with pre-incubation, of the six tested doses, only four instead of 5 doses were analysable in the second assay in tester strain TA100 (+S9) as recommended by OECD guideline 471.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Groupe interministeriel des produits chimiques, Paris, France
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: TA 1535, TA 1537, TA 98 and TA 100 carry a mutation of the uvr B gene and the deep rough mutation (rfa), TA100 and TA98 contain the R-factor plasmid (pkM101). TA 102 carries the deep rough mutation (rfa) and contains the R-factor plasmid (pAQ1)
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
Preliminary toxicity test:
- 50, 150, 500, 1500, 5000 µg/plate (with and without metabolic activation)
Experiment I:
- 5, 15, 50, 150, 500, 1000 µg/plate (without metabolic activation; TA 1535, TA 1537, TA 100 and TA 102)
- 5, 15, 50, 150, 500, 1500 µg/plate (without metabolic activation, TA 98)
- 15, 50, 150, 500, 1500, 3000 µg/plate (with metabolic activation, all tester strains)
Experiment II:
- 5, 15, 50, 150, 500, 1000 µg/plate (without metabolic activation, TA 1535)
- 1.5, 5, 15, 50, 150, 500 µg/plate (without metabolic activation, TA 1537)
- 5, 15, 50, 150, 500, 750 µg/plate (without metabolic activation, TA 98, TA 100 and TA 102)
- 5, 15, 50, 150, 500, 1500 µg/plate (with metabolic activation, TA 98, TA 102 and TA 1535, pre-incubation method)
- 5, 15, 50, 150, 500, 1000 µg/plate (with metabolic activation, TA 100 and TA 1537, pre-incubation method)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: The vehicle was chosen due to the solubility properties of the test item.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9-mix: sodium azide (TA 1535, TA 100), 9-amino-acridine (TA 1537), 2-nitro fluorene (TA 98), mitomycin c (TA 102); +S9-mix: 2-anthramine (TA 1535, TA 1537, TA 98, TA 100), benzo[a]pyrene (TA 102)
Remarks:
sodium azide (1 µg/plate), 9-amino-acridine (50 µg/plate), 2-nitro fluorene (2 µg/plate), mitomycin c (0.125 µg/plate), 2-anthramine (2/1 µg/plate without/with pre-incubation), benzo[a]pyrene (2 µg/plate without/with pre-incubation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation; pre-incubation (experiment II with metabolic activation)

DURATION
- Pre-incubation period: 60 minutes

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY
- Method: In order to choose the range of doses for the test, the toxic activity of the test item was determined. The toxicity assay was carried out in all the strains to be tested under the same conditions as the mutagenicity test with and without metabolic activation but using only one plate per dose instead of 3. The plates were incubated for approximately 44 hours at ca. 37°C, and the revertants were counted. The maximum dose in the preliminary toxicity assay was the maximum dose according to OECD Guideline 471 e.g. 5000 μg/plate. Toxicity was checked by microscopic examination of the background growth.
Evaluation criteria:
A test system is considered as mutagenic if
- a test item causing a positive response proportional to the dose for at least 3 doses with, for the highest increase, a value greater than or equal to 3 times the value for the solvent control, is considered positive in the assay (TA 1535 and TA 1537)
- a test item causing a positive response proportional to the dose for at least 3 doses with, for the highest increase, a value greater than or equal to 2 times the value for the solvent control, is considered positive in the assay (TA 98, TA 100 and TA 102)
In some borderline cases, an additional criterion to be considered is the comparison between the number of revertants induced by the test item and the laboratory historical control data. Indeed, an increase in each individual value that is above the highest value of corresponding historical control data can help supporting a conclusion such as “equivocal” or “weak” mutagen. If a test item causes a positive response during a single assay and that result cannot be reproduced in at least 1 independent assay, the initial positive result may be considered as not significant. If, in all experimental conditions examined, none of the above criteria are fulfilled, a test item is considered clearly negative and unable to induce mutations in this test system.
Statistics:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated. In addition, data was analysed by means of Dunnett's method allowing the comparison of the mean value for each dose to the mean value for the corresponding solvent control.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: 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:
Exp I: -S9: 1000 µg/plate: TA1535, TA1537, TA100; 1500 µg/plate: TA98; +S9: 1500 µg/plate: TA100; 3000 µg/plate: TA1535, TA1537, TA98; Exp II: -S9: 750 µg/plate: TA100; 1000 µg/plate: TA1535; +S9: 500 µg/plate: TA1535, TA1537, TA100; 1500 µg/plate: TA98
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp I: -S9: 1000 µg/plate: TA102; +S9: 3000 µg/plate: TA102; Exp II: -S9: 750 µg/plate: TA102; +S9: 150 µg/plate: TA102
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was fully soluble in the vehicle (sterile water).
- Precipitation: No precipitation was observed in the main mutagenicity assay (experiment I and II) neither in the presence nor in the absence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES
Cytotoxicity of the test item was assessed in a preliminary experiment resulting in:
- an important toxicity with the presence of microcolonies at the two highest tested doses of 5000 and 1500μg/plate without metabolic activation in strains TA1535, TA1537, TA100 and TA102
- an important toxicity with the presence of microcolonies at the highest tested dose of 5000μg/plate in all strains with metabolic activation and in strain TA98 without metabolic activation
- a moderate toxicity at the tested dose of 1500μg/plate in strain TA98 without metabolic activation and in strain TA100 with metabolic activation
- a slight toxicity at the tested dose of 500μg/plate in strains TA1535 ad TA1537 without metabolic activation and at the tested dose of 1500μg/plate in strains TA1535, TA1537, TA98 and TA102 with metabolic activation.
Therefore, the maximum doses retained for the first mutagenicity assay in the absence of metabolic activation were 1000 µg/plate (TA1535, TA1537, TA100 and TA102) and 1500μg/plate (TA 98) and 3000 µg/plate in all strains in the presence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA
The results were within the range of the historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY
Cytotoxicity was recorded at 1000 µg/plate (TA 1535, TA 1537, TA 100, TA 102) and 1500 µg/plate (TA 98) in the absence of metabolic activation and at 1500 µg/plate (TA 100) and 3000 µg/plate (TA 1535, TA 1537, TA 98, TA 102) in the presence of metabolic activation (experiment I). In the second experiment cytotoxicity was present at 750 µg/plate (TA 100, TA 102) and at 1000 µg/plate /TA 1535) in the absence of metabolic activation and at 150 µg/plate (TA 102), 500 µg/plate (TA 1535, TA 1537, TA 100) and 1500 µg/plate (TA 98) in the presence of metabolic activation.

Any other information on results incl. tables

Table 1: Results of the preliminary cytotoxicity test

Toxicity Assay

Dose in µg/plate

TA 1535

TA 1537

TA 98

TA 100

TA 102

T

P

T

P

T

P

T

P

T

P

Test item without S9-mix

0

50

150

500

1500

5000

-

-

-

+

+++

+++

-

-

-

-

-

-

-

-

-

+

+++

+++

-

-

-

-

-

-

-

-

-

-

++

+++

-

-

-

-

-

-

-

-

-

-

+++

+++

-

-

-

-

-

-

-

-

-

-

+++

+++

-

-

-

-

-

-

Top Dose in first mutagenicity assay

 

1000

1000

1500

1000

1000

Test item with S9-mix

0

50

150

500

1500

5000

-

-

-

-

+

+++

-

-

-

-

-

-

-

-

-

-

+

+++

-

-

-

-

-

-

-

-

-

-

+

+++

-

-

-

-

-

-

-

-

-

-

++

+++

-

-

-

-

-

-

-

-

-

-

+

+++

-

-

-

-

-

-

Top dose in first mutagenicity assay

 

3000

3000

3000

3000

3000

T: toxicity (- non toxic: + slightly toxic: ++ moderately toxic: +++ strongly toxic)

P: precipitation (- absence: + slight precipitate: ++ moderate precipitate: +++ important precipitate)

Table 2: Results of mutagenicity assay (experiment I)

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

TA 102

TA98

TA1537

0

93.8 ± 6.1

14.0 ± 2.8

107.7 ± 23.9

16.2 ± 1.9

6.8 ± 3.4

5

85.7 ± 7.1

14.3 ± 3.5

105.3 ± 15.5

10.3 ± 1.2

6.0 ± 3.0

15

97.3 ± 3.5

17.3 ± 5.9

112.7 ± 9.2

14.3 ± 2.1

4.7 ± 0.6

50

95.0 ± 11.3

16. 7 ± 1.5

127.3 ± 8.1

15.7 ± 4.2

4.0 ± 1.0

150

85.3 ± 6.5

13.0 ± 2.6

114.7 ± 1.2

10.7 ± 6.4

3.7 ± 3.8

500

82.7 ± 29.1

15.3 ± 4.5

100.7 ± 32.9

12.0 ± 4.0

4.0 ± 2.6

1000

18.7 ± 1.5

9.3 ± 2.1

50.0 ± 2.0

-

**

1500

-

-

-

4.3 ± 2.1

-

Positive controls, –S9

Name

NaN

NaN

MM-c

2-NF

9-AA

Concentrations

(μg/plate)

1

1

0.125

2

50

Mean No. of colonies/plate

(average of 3 ± SD)

624.0 ± 97.3

477.3 ± 258.8

480.0 ± 142.2

230.7 ± 31.9

450.7 ± 117.1

+

0

110.5 ± 16.1

12.0 ± 3.7

179.3 ± 27.1

21.7 ± 4.1

6.7 ± 1.9

+

15

102.7 ± 5.5

8.7 ± 2.9

210.0 ± 28.8

24.0 ± 4.0

6.0 ± 1.0

+

50

104.0 ± 5.0

12.3 ± 2.5

193.3 ± 17.5

19.3 ± 8.1

6.7 ± 3.2

+

150

84.3 ± 2.5

9.0 ± 2.0

155.3 ± 25.0

19.0 ± 5.2

8.0 ± 2.6

+

500

94.7 ± 5.7

7.7 ± 0.6

189.3 ± 12.2

27.0 ± 6.6

5.0 ± 4.6

+

1500

31.3 ± 4.9

11.0 ± 2.6

126.0 ± 30.0

24.0 ± 7.9

3.0 ± 2.6

+

3000

**

**

22.7 ± 6.7

**

**

Positive controls, +S9

Name

2-AA

2-AA

BaP

2-AA

2-AA

Concentrations

(μg/plate)

2

2

2

2

2

Mean No. of colonies/plate

(average of 3 ± SD)

1738.7 ± 268.8

418.7 ± 66.2

912.0 ± 224.0

1888.0 ± 279.0

154.7 ± 24.1

**: not analyzable due to toxicity

NaN: sodium azide

MM-c: mitomycin c

2 -NF: 2 -nitrofluorene

9 -AA: 9 -aminoacridine

2 -AA: 2 -anthramine

BaP: benz[a]pyrene

-: concentration not assessed

Table 3: Results of mutagenicity assay (experiment II)

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

TA 102

TA98

TA1537

0

114.3 ± 19.7

12.5 ± 3.2

165.3 ± 20.1

14.8 ± 2.0

5.7 ± 1.6

 

1.5

-

-

-

-

9.3 ± 2.1

5

109.7 ± 10.1

12.0 ± 3.5

196.0 ± 7.2

18.0 ± 5.2

9.0 ± 2.6

15

104.0 ± 6.0

11.0 ± 2.6

188.7 ± 26.6

14.0 ± 1.0

10.0 ± 5.0

50

91.0 ± 8.5

14.3 ± 6.4

173.3 ± 3.1

12.0 ± 1.0

7.7 ± 3.8

150

107.7 ± 12.2

15.3 ± 4.2

139.3 ± 8.1

14.0 ± 1.7

4.7 ± 0.6

500

93.3 ± 11.9

10.3 ± 4.0

141.3 ± 16.0

15.3 ± 4.2

6.7 ± 0.6

 

750

65.0 ± 2.8

-

95.3 ± 21.6

13.3 ± 0.6

-

1000

-

8.0 ± 2.6

-

-

-

Positive controls, –S9

Name

NaN

NaN

MM-c

2-NF

9-AA

Concentrations

(μg/plate)

1

1

0.125

2

50

Mean No. of colonies/plate

(average of 3 ± SD)

544.0 ± 48.0

421.3 ± 21.2

490.7 ± 33.3

244.7 ± 36.7

580.0 ± 102.1

+

0

71.7 ± 5.9

11.2 ± 3.3

202.0 ± 29.4

20.2 ± 6.5

5.7 ± 1.2

+

5

74.0 ± 12.1

9.7 ± 2.1

178.0 ± 32.7

23.7 ± 7.5

5.7 ± 0.6

+

15

78.3 ± 6.7

10.0 ± 2.0

164.7 ± 9.5

21.3 ± 5.0

9.7 ± 2.3

+

50

81.7 ± 7.2

9.7 ± 1.5

146.0 ± 9.2

22.3 ± 2.5

8.3 ± 8.4

+

150

94.0 ± 6.9

10.3 ± 1.5

116.0 ± 5.3

18.3 ± 4.7

6.7 ± 3.1

+

500

**

6.0 ± 2.0

117.3 ± 11.4

16.3 ± 6.7

2.7 ± 1.5

 

1000

**

-

-

-

**

+

1500

-

**

72.0 ± 22.6

**

-

Positive controls, +S9

Name

2-AA

2-AA

BaP

2-AA

2-AA

Concentrations

(μg/plate)

1

1

2

1

1

Mean No. of colonies/plate

(average of 3 ± SD)

1280.7 ± 287.0

232.7 ± 20.2

536.0 ± 60.4

 

1152.0 ± 180.3

83.3 ± 12.7

**: not analyzable due to toxicity

NaN: sodium azide

MM-c: mitomycin c

2 -NF: 2 -nitrofluorene

9 -AA: 9 -aminoacridine

2 -AA: 2 -anthramine

BaP: benz[a]pyrene

-: concentration not assessed

Applicant's summary and conclusion

Conclusions:
A bacterial gene mutation assay (Ames test) was performed with the test material according to OECD TG 471 and in compliance with GLP. The test material was considered not to be mutagenic in the presence and absence of metabolic activation in the tester strains used.