Bronopol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 April - 7 Dec 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bronopol

EC Number:

200-143-0

EC Name:

Bronopol

Cas Number:

52-51-7

Molecular formula:

C3H6BrNO4

IUPAC Name:

2-bromo-2-nitropropane-1,3-diol

Method

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: histidine deficient

Metabolic activation:

with and without

Metabolic activation system:

Prepared from livers of male Sprague-Dawley rats induced with a single i.p. injection of 500 mg/kg bw Arochlor 1254 in corn oil five days prior t sacrifice; co-factors were added.

Test concentrations with justification for top dose:

Cytotoxicity tests: 50, 158, 500, 1581, 5000 µg/plate in absence and presence of S9
Mutagenicity tests: 1-64 µg/plate in absence and presence of S9

Vehicle / solvent:

deionized water

Controls

Untreated negative controls:

other: not applicable

Negative solvent / vehicle controls:

yes

True negative controls:

other: not applicable

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-1,2,-phenylene diamine, nitrofurantoin, cumene hydroperoxide, 2-aminoantracene

Details on test system and experimental conditions:

Plate incorporation test: The bacterial suspensions (0.1 mL) were mixed with soft agar (2.0 mL), 0.1 mL of Bronopol stock solutions or vehicle control, 0.5 mL S9 mix (in presence of metabolic activation) or buffer (in absence of S9) before being poured onto minimal agar plates.
Preincubation test: The bacterial suspensions (0.1 mL) were mixed with 0.1 mL of Bronopol stock solutions or vehicle control, 0.5 mL S9 mix (in presence of metabolic activation) or buffer (in absence of S9) and incubated for 20 min at 37°C. Soft agar (2.0 mL) was then added to the mixture before being poured onto minimal agar plates
The plates (three per concentration, negative and positive controls in each test) were then incubated for 48 hours at 37°C.
Total bacterial counts were also determined (soft agar with 5x higher histidine concentration) using two plates each in absence and presence of S9.
Examination for toxicity was (reduction of) background growth and number of mutant colonies and examination for mutagenicity was frequency of revertant colonies

Evaluation criteria:

The test was considered to be positive if a reproducible and dose-related increase in mutant counts of at least one strain was seen. The increase should be at least:
• twice that of negative controls for strains TA 1535, TA 100, TA 98
• threefold that of negative controls for strain TA 1537
• about 100 mutant colonies higher than negative controls for strain TA 102

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the cytotoxicity test marked toxicity was seen at >/=50 µg/plate for TA 98, TA 1537, TA 100 in presence or absence of S9 and for TA 1535 ( S9), at >/=158 µg/plate for TA 1535 (+S9) and TA 102 (+/- S9).
In the plate incorporation assay, cytotoxicity was seen in absence of S9 at 64 µg/plate for TA 1535 and TA 100. The same applied to TA 100, TA 1537, TA 98 and TA 102 in absence of S9 in the preincubation test. Also in the preincubation assay cytotoxicity was seen at >/=32 µg/plate in absence of S9 for TA 1535.

In the plate incorporation test mutant counts about twice the concurrent control level were seen in several concentrations in strain TA 1535 without metabolic activation. This was not considered to be relevant as this was in absence of a dose relationship, as there were no increases in the plate incorporation test and as the mutant counts observed with Bronopol were within the range of the normal fluctuation of negative controls.
In comparison to historical controls (ranging from median 8, semi-Q range 2 to median 10 semi-Q range 2), the concurrent control of TA 1535 in the plate incorporation test was relatively low (6).
Positive control compounds gave a clear positive result.

Any other information on results incl. tables

Strain/concen-tration [µg/plate]	Number of mutant cells
	Plate incorporation test		Preincubation test
	— S9	+ S9	— S9	+ S9
TA 1535:  0                    1                    2                    4                    8                   16                   32                   64 Positive control	6 10 13 15 11 13 13 0 580	10 10 9 11 9 11 10 9 183	11 10 12 11 10 5 0 0 542	12 10 11 12 17 11 9 13 123
TA 100:     0                    1                    2                    4                    8                   16                   32                   64 Positive control	127 121 116 118 113 97 58 0 277	151 137 146 152 154 160 160 133 1719	114 112 104 114 118 137 66 0 272	108 104 86 104 107 111 118 120 1311
TA 1537:  0                    1                    2                    4                    8                   16                   32                   64 Positive control	10 7 8 10 5 9 11 2 114	8 7 10 8 9 9 9 9 298	12 13 12 9 10 11 8 0 115	13 17 14 13 18 15 12 19 248
TA 98:       0                    1                    2                    4                    8                   16                   32                   64 Positive control	36 35 30 42 40 31 36 13 221	37 38 46 45 46 38 45 54 1767	32 37 31 26 28 25 23 0 192	31 31 34 25 25 29 36 32 1458
TA 102:     0                    1                    2                    4                    8                   16                   32                   64 Positive control	251 255 285 272 276 268 247 229 413	240 245 245 253 269 251 262 264 704	213 208 228 220 211 209 189 43 401	199 207 162 190 204 244 214 216 456

Applicant's summary and conclusion

Conclusions:

No mutagenic potential of Bronopol was detected in S. typhimurium strains in presence and absence of metabolic activation.