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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:
9 November 2009 - 3 December 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD Guideline 471. GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Benzenamine, reaction products with aniline hydrochloride and nitrobenzene, hydrochlorides
EC Number:
309-913-1
EC Name:
Benzenamine, reaction products with aniline hydrochloride and nitrobenzene, hydrochlorides
Cas Number:
101357-16-8
Molecular formula:
Not applicable. Multiconstituent substance.
IUPAC Name:
Benzenamine, reaction products with aniline hydrochloride and nitrobenzene, hydrochlorides
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): C.I. Solvent Black 5 (analogue CAS 11099-03-9).
- Lot/batch No.: 10137087
- Physical state: Blackish brown powder
- Storage condition of test material: Room temperature.
- Stability and uniformity were property has been confirmed.

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Without metabolic activation (all strains): 0.610, 1.22, 2.44, 4.88, 9.77 and 19.5 μg/plate.
With metabolic activation (TA100, TA1535, TA98 and TA1537): 19.5, 39.1, 78.1, 156, 313 and 625 μg/plate.
With metabolic activation (WP2uvrA): 156, 313, 625, 1250, 2500 and 5000 μg/plate.
Vehicle / solvent:
DMSO
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamida (AF2)
Remarks:
TA100 (0.1 µg/mL), WP2uvrA (0.2 µg/mL), TA98 (1 µg/mL)
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
sodium azide
Remarks:
TA1535 (5 µg/mL)
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: 9-aminoacridine hydrochloride hydrate (9AA)
Remarks:
TA1537 (800 µg/mL)
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
TA100 (10 µg/mL), TA1535 (20 µg/mL), WP2uvrA (100 µg/mL), TA98 (5 µg/mL), TA1537 (20 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min at 37 ºC
- Exposure duration: 48 hours at 47 ºC

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY: Yes
Evaluation criteria:
Results are found to be positive when revertant frequency increase 2 times or more compared with vehicle control, with a dose-response relationship and the results are reproducible.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation was observed in the doses with metabolic activation

RANGE-FINDING/SCREENING STUDIES:
First dose-finding test: With and without metabolic activation, for all test strains. Dosages: 5, 15, 50, 150, 1500 and 5000 μg /plate.
Second dose-finding test: Without metabolic activation, for all test strains. Dosages: 0.384, 0.960, 0.240, 6 and 15 μg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
Negative and positive controls according with historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytototixicy was observed at highest dose both with and without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Results obtained in the main test:

Metabolic activation

µg/plate

Revertant colonies / plate (triplicate, average)

TA100

TA1535

WP2uvrA

TA98

TA1537

Without

DMSO

89 ± 10

7 ± 1

25 ± 2

19 ± 4

7 ± 3

0.610

89 ± 6

4 ± 1

21 ± 2

23 ± 6

4 ± 1

1.22

93 ± 11

6 ± 2

24 ± 4

18 ± 4

5 ± 1

2.44

95 ± 3

8 ±2

21 ± 7

17 ± 2

5 ± 3

4.88

72 ± 3

6 ± 1

21 ± 5

16 ± 9

6 ± 2

9.77

86 ± 11*

4 ± 3 *

24 ± 4 *

21 ± 3 *

5 ± 3 *

19.5

82 ± 9 *

6 ± 1 *

25 ± 3 *

21 ± 2 *

2 ± 1 *

AF-2

409 ± 16

--

427 ±

551 ± 21

--

NaN3

--

251 ± 5

--

--

--

9AA

--

--

--

--

404 ± 65

With

DMSO

98 22

7 ± 2

28 ± 4

30 ± 13

9 ± 1

19.5

118 ± 2

9 ± 4

--

44 ± 6

8 ± 3

39.1

117 ± 13

9 ± 3

--

43 ± 5

9 ± 1

78.1+

97 ± 5

7 ± 4

--

33 ± 6

8 ± 2

156 +

103 ± 14

5 ± 1

29 ± 3

33 ± 3

6 ± 3

313 +

101 ± 2

7 ± 2

27 ± 3

24 ± 2

6 ± 1

625 +

115 ± 5 *

8 ± 3 *

23 ± 6

26 ± 9 *

7 ± 4 *

1250 +

--

--

21 ± 2

--

--

2500 +

--

--

18 ± 1

--

--

5000 +

--

--

17 ± 5

--

--

2AA

687 ± 41

147 ± 10

193 ± 18

548 ± 27

189 ± 10

(--) Not tested

(+) Precipitation

(*) Cytotoxicity

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative (with and without metabolic activation)

Test item did not induce gene mutation in bacteria in a reverse mutation assay with and without metabolic activation.
Executive summary:

A bacterial reverse mutation assay was performed on test item according to OECD Guideline 471 preincubation method. Based on preliminary works, Salmonella typhimurium TA100, TA1535, TA98, TA1537 strains and Escherichia coli WP2uvrA strain were exposed up to 19.5 µg/mL without metabolic activation and up to 625 µg/mL (S. typhimurium strains) and 5000 µg/mL (WP2 uvrA) with metabolic activation. Negative solvent and positive controls were performed satisfactorily. Precipitation of the test item and cytotoxicity was detected in some concentrations. Test item was determined to be nonmutagenic under test conditions since a 2 -fold or greater and dose-dependent increase in revertant colonies was not observed in any test strain with or without metabolic activation. Based on increase in revertant colonies compared to the negative control, test item was determined to be nonmutagenic under test conditions.

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