N-bromosuccinimide

Genetic toxicity: in vitro

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:

8 April 2004 to 17 January 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline Study according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

N-bromosuccinimide

EC Number:

204-877-2

EC Name:

N-bromosuccinimide

Cas Number:

128-08-5

Molecular formula:

C4H4BrNO2

IUPAC Name:

1-bromopyrrolidine-2,5-dione

Details on test material:

- Name of test material (as cited in study report): "N-Bromo Succinimide"
- Physical state: Light yellow powder
- Lot/batch No.: SSIN 044
- Solubility in water: 14.8 g/L at 20 °C
- Melting range: 174 - 179 °C
- Stability under conditions of storage: Stable.
- Expiration date of the lot/batch: 30 June 2005
- Storage condition of test material: In the dark, may be used under light.

Method

Target gene:

his- (s. typhimurium)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 5

Species / strain / cell type:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

first experiment: 1667, 556, 185, 62, 21, 7 µg/plate
second experiment: 5000, 1667, 556, 185, 62, 21 µg/plate
third experiment: 5000, 3333, 2222 µg/plate

Vehicle / solvent:

DMSO was used as solvent for the test substance and for the negative control group.

Details on test system and experimental conditions:

The exposure was performed according to the "Plate Incorporation Assay", in which bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in liquid state. The number of viable cells in the overnight-culture is in the range of 200 000 000 cells per mL.
For each sample the following solutions were combined:
• 0.1 mL of the overnight culture of the bacteria,
• 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
• 0.1 mL of the appropriate test- or reference substance solution and
• 2 mL of top agar.
The combined solutions were mixed and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).

Bacterial strains of S. typhimurium (TA97a, TA98, TA100, TA102 and TA1535) were obtained from Prof. Bruce N. Ames, Berkely, California. The bacteria were stored in small portions in a solutions of 6 % DMSO in PBS in liquid nitrogen.

Counting of colonies: The plates of the strains with a low spontaneous revertant rate, i.e. TA98 and TA1535 were counted visually by marking the colonies with a felt tipped pen. The plates of the other strains were photographed with a video camera and the picture files were scanned for colonies by a computer program.

The results of the first experiment were verified by additional experiments.

Triplicate repetitions were run for each dose group in each of the separate experiments that were conducted, for the control groups six-fold repetitions were run.

Groups and concentrations:
Test substance (first experiment): 1667, 556, 185, 62, 21, 7 µg/plate (3 samples)
Test substance (second experiment): 5000, 1667, 556, 185, 62, 21 µg/plate (3 samples)
Test substance (third experiment): 5000, 3333, 2222 µg/plate (3 samples)
control (DMSO): 100 µl (6 samples)
Positive controls:
Strain TA97a: 4-NOPD 10 µg (without S9), DMBA 10 µg (with S9)
Strain TA98: 2-NF 2 µg (without S9), 2-AA 1 µg (with S9)
Strain TA100: Sodium-azide 2 µg (without S9), 2-AA 2µg (with S9)
Strain TA102: t-BHPO 50 µg (without S9), DHA 50 µg (with S9)
Strain TA1535: Sodium-azide 1 µg (without S9), 2-AA 2 µg (with S9)

Evaluation criteria:

Means and standard deviations were calculated for the number of mutants in every concentration group.
The criteria for a positive result were:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
• For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: 250 % of the amount of the spontaneous revertants.
• For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: 167 % of the amount of the spontaneous revertants.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Some Toxicity observed, see attachment

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Without metabolisation: no genotoxicity. With metabolisation: first experiment no genotoxicity, second experiment genotoxicity at 5000 µg/plate. Additional experiment: no genotoxicity.

Cytotoxicity / choice of top concentrations:

other: Some Toxicity observed, see attachment

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Without metabolisation: No genotoxicity. With metabolisation: First experiment no genotoxicity, second experiment genotoxicity at 5000 µg/plate. Additional experiment: Verification at genotoxicity at 5000 µg/plate

Cytotoxicity / choice of top concentrations:

other: Some Toxicity observed, see attachment

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Without metabolisation: First experiment no genotoxicity; Second experiment genotoxicity at 556 µg/plate. With metabolisation: First experiment no genotoxicity, Second experiment genotoxicity at 5000 and 1667 µg/plate. Additional experiment: no genotox.

Cytotoxicity / choice of top concentrations:

other: Some Toxicity observed, see attachment.

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRECIPITATION:
No precipitation of the test substance was seen in any of the concentration groups.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary test a missing bacterial background lawn was noted at concentration of 1667 µg/plate and above.

COMPARISON WITH HISTORICAL CONTROL DATA:
The numbers of spontaneous revertants were comparable with the historic control data for the negative controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main test the test substance was toxic to the bacteria at higher concentrations. Without metabolic activation, toxicity (no bacterial background or only microcolonies instead of a bacterial background) was observed in some plates from 1667 µg/plate on. Metabolic activation decreased the toxicity; with metabolic activation only a reduced bacterial background was observed in strain TA98 at 5000 µg/plate.

Any other information on results incl. tables

Positive control substances:

All positive control substances increased the mutation frequency to more than the threshold values.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive in strain TA 102

According to the results, "N-Bromo Succinimide" is mutagenic in the Ames test with the strain TA102 with metabolic activation because a reproducible increase of the revertant numbers was obtained.

Executive summary:

Method

"N-BROMO SUCCINIMIDE" was tested for mutagenic activity with the "Salmonella typhimurium Reverse Mutation Test" (Ames Test). The study was conducted in accordance with the OECD-guideline 471 and directive 2000/32/EC, part B.13/14.

The test substance, dissolved in DMSO, was tested at concentrations ranging from 7 µg to 5000 µg per plate according to the "direct plate incorporation method" without external metabolisation as well as with an external metabolising system (S9-mix). As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used. Negative and positive controls were included. An independent repetition of the experiment was performed.

 

Results

Positive controls:

All positive control groups showed significantly increased mutation frequencies which demonstrate the sensitivity of the test system.

 

Test substance:

Toxicity:

The test substance was toxic to the bacteria at higher concentrations. Without a metabolic activation, toxicity (no bacterial background or only microcolonies instead of a bacterial background) was observed in some plates from 1667 µg/plate on. Metabolic activation decreased the toxicity; with metabolic activation only a reduced bacterial background was observed in strain TA98 at 5000 µg/plate.

 

Solubility:

No precipitation of the test substance was seen in any of the concentration groups.

 

Mutagenicity:

In presence of a metabolising system the test substance increased the number of revertant colonies in the strains TA97a, TA100 and TA102 to more than the threshold values (167 % of the controls for strains TA97a, TA100 and TA102). The increase was near the threshold values, was observed only in the highest concentrations, and was reproducible only in strain TA102.

Without a metabolising system one single, non reproducible increase of the revertant numbers was obtained with strain TA97a at 556 µg/plate. Also this increase was near the threshold value.

Altogether there are some evidences for a mutagenic action of "N-BROMO SUCCINIMIDE", the mutagenicity is, however, a borderline one.

  

Conclusion

 According to the results obtained in this study, "N-BROMO SUCCINIMIDE" is mutagenic in the Ames test with the strain TA102 with metabolic activation because a reproducible increase of the revertant numbers was obtained.