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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

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 resultsopen allclose all
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
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
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
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
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.