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Diss Factsheets

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:
2009 -07-28 till 2009-08-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well performed OECD and GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

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
Chemical structure
Reference substance name:
1,3-dihydro-5-nitro-2H-benzimidazol-2-one
EC Number:
202-282-2
EC Name:
1,3-dihydro-5-nitro-2H-benzimidazol-2-one
Cas Number:
93-84-5
Molecular formula:
C7H5N3O3
IUPAC Name:
5-nitro-1,3-dihydro-2H-benzimidazol-2-one
Details on test material:
- Name of test material (as cited in study report): Nitrolon trocken
- Physical state: solid
- Analytical purity: 99.66 % (w/w)
- Lot/batch No.: DALA 064142
- Expiration date of the lot/batch: May 08, 2011 (Statement of producer)
- Stability under test conditions: > 72h in DMSO at room temperature
- Storage condition of test material: At room temperature

Method

Species / strain
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
pre-experiment (= experiment I): 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo(a)pyrene.
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation test

DURATION
- Exposure duration: incubation for at least 48 hours


NUMBER OF REPLICATIONS: triplicate plating


DETERMINATION OF CYTOTOXICITY: By reduction in the number of spontaneous revertants or by clearing of the bacterial background lawn.


Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
other: positive in strains TA 1537 and TA 98 in the absence of metabolic activation
Cytotoxicity / choice of top concentrations:
other: no, only minor toxic effects only in strain WP2 uvrA
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Study Name: 1280600

Study Code: Harlan-CCR 1280600

Experiment: 1280600 VV Plate

Date Plated: 28/07/2009

Assay Conditions:

Date Counted: 04/08/2009

Metabolic

Activation

Test

Group

Dose Level

(per plate)

Revertant Colony Counts (Mean ±SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without Activation

DMSO

17 ± 3

12 ± 4

31 ± 5

141 ± 15

53 ± 3

Untreated

16 ± 3

11 ± 2

31 ± 2

159 ± 7

50 ± 10

Nitrolon trocken

3 µg

20 ± 1

9 ± 4

26 ± 3

148 ± 16

44 ± 3

10 µg

15 ± 5

8 ± 2

25 ± 4

157 ± 8

47 ± 4

33 µg

15 ± 2

11 ± 4

32 ± 8

152 ± 6

45 ± 7

100 µg

15 ± 1

15 ± 1

33 ± 2

137 ± 22

44 ± 9

333 µg

15 ± 2

19 ± 1

55 ± 10

136 ± 28

36 ± 2

1000 µg

13 ± 1

35 ± 4

87 ± 8

136 ± 4

31 ± 3

2500 µg

12 ± 0

55 ± 1

117 ± 16

130 ± 15

24 ± 6

5000 µg

10 ± 3P M

51 ± 8P M

68 ± 2P M

95 ± 12P M

8 ± 2P M

NaN3

10 µg

1884 ± 88

2079 ± 167

4-NOPD

10 µg

354 ± 12

4-NOPD

50 µg

84 ± 13

MMS

3.0 µL

933 ± 33

With Activation

DMSO

20 ± 2

19 ± 4

33 ± 1

171 ± 10

66 ± 4

Untreated

14 ± 4

16 ± 2

35 ± 7

175 ± 20

62 ± 7

Nitrolon trocken

3 µg

19 ± 3

16 ± 1

36 ± 5

171 ± 25

66 ± 10

10 µg

20 ± 2

16 ± 5

32 ± 3

177 ± 14

67 ± 11

33 µg

19 ± 4

18 ± 3

35 ± 1

168 ± 16

59 ± 20

100 µg

20 ± 4

16 ± 2

37 ± 2

158 ± 13

57 ± 1

333 µg

18 ± 2

21 ± 2

33 ± 6

168 ± 5

43 ± 2

1000 µg

17 ± 1

28 ± 3

55 ± 4

145 ± 2

33 ± 2

2500 µg

18 ± 1

39 ± 14

56 ± 3

144 ± 12

32 ± 3

5000 µg

14 ± 1P M

34 ± 1P M

29 ± 1P M

95 ± 7P M

11 ± 3P M

2-AA

2.5 µg

402 ± 49

328 ± 24

2039 ± 141

2958 ± 153

2-AA

10.0 µg

351 ± 15

Key to Positive Controls

Key to Plate Postfix Codes

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

P

M

Precipitate

Manual count

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
other: positive without metabolic activation in TA 1537 and TA 98

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome strainsTA 1537 and TA 98 in the absence of metabolic activation.
Therefore, Nitrolon trocken is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

The test item Nitrolon trocken was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escheri­chia coli strain WP2 uvrA.

The assay was performed with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:           3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal back­ground growth in all strains with and without metabolic activation.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in almost all strains. Only in strain WP2 uvrA a reduction in the number of revertants (below the indication factor of 0.5) was observed at 5000 µg/plate in the absence and presence of metabol­ic activation.

Precipitation of the test item occurred in the test tubes from 1000 µg/plate up to 5000 µg/plate and on the incubated agar plates at 5000 µg/plate. The undissolved particles had no influence on the data recording.

Substantial and dose dependent increases in revertant colony numbers were observed following treatment with Nitrolon trocken in strains TA 1537 and TA 98 in the absence of metabolic activation. The number of colonies exceeded the threshold of twice in strain TA 98 at 1000 µg/plate and above, and the number of colonies exceeded the threshold of trice in strain TA 1537 at 1000 µg/plate and above.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.