Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Sep - 21 Oct 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Guidelines for Toxicity Testings of New Chemical Substances
Version / remarks:
Notification No. 1121002 of Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, No. 2 of the Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry, & No. 031121002 of Environmental Policy Bureau, Ministry of the Environment, Japan, November 21, 2003
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon (for S. typhimurium strains) and trp operon (for E. coli strain)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Dose-range finding test: 1.2, 4.9, 20, 78, 313, 1250 and 5000 μg/plate
Main test: 39, 78, 156, 313, 625 and 1250 µg/plate

In the dose-range finding test, growth inhibition by the test substance was observed at 1250 μg/plate and above in all strains with and without metabolic activation. Therefore, 1250 μg/plate was selected as top dose for all strains with and without metabolic activation in the main test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was insoluble in water. A solubility test showed that the test substance was soluble at 50 mg/mL in DMSO.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2); 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine 2HCl (ICR-191); 2-Aminoanthracene (2-AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: duplicates each in the dose-range finding study and in the main test


DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition
Evaluation criteria:
The test substance was judged to be positive with regard to mutagenicity, if the number of revertant colonies on the test plates increased significantly in comparison with the control plates (twice as that of the solvent control), and in addition dose-response and reproducibility were observed.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: TA1537, TA1535, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1250 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1250 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed either with or without metabolic activation
- Contamination: In a sterility test on the test solution and the S9 mix, no growth of bacteria was observed.

RANGE-FINDING/SCREENING STUDIES: In a preliminary dose-range finding test concentrations of 1.2, 4.9, 20, 78, 313, 1250 and 5000 μg/plate were tested. Growth inhibition by the test substance was observed at 1250 μg/plate and above in all strains with and without metabolic activation. And no precipitation of the test substance on the plates was observed either with or without metabolic activation. Therefore, 1250 μg/plate dose was selected as top dose for all strains both with and without metabolic activation in the main test.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: please refer to table 3
- Negative (solvent/vehicle) historical control data: please refer to table 3

Any other information on results incl. tables

Table 1. Results of dose-range finding test

With or without S9mix Dose (μg/plate) Number of revertant colonies/plate
Base-pair substitution type Frame-shift type
TA100 TA1535 WP2uvrA TA98 TA1537
-S9mix Solvent Control (DMSO) 136 24 33 23 10
134 (135) 24 (24) 21 (27) 27 (25) 9 (10)
1.2 121 19 10 17 12
116 (119) 19 (19) 21 (16) 18 (18) 13 (13)
4.9 102 17 20 19 7
142 (122) 24 (21) 21 (21) 29 (24) 9 (8)
20 124 14 16 23 5
103 (114) 17 (16) 24 (20) 21 (22) 9 (7)
78 135 15 28 13 8
110 (123) 20 (18) 20 (24) 20 (17) 8 (8)
313 133 22 23 19 9
149 (141) 20 (21) 22 (23) 12 (16) 13 (11)
1250 0* 0* 17* 0* 0*
0*(0)* 0*(0)* 20*(19)* 0*(0)* 0*(0)*
5000 0* 0* 11* 0* 0*
0*(0)* 0*(0)* 23*(17)* 0*(0)* 0*(0)*
+S9mix Solvent Control (DMSO) 132 15 28 34 20
139 (136) 18 (17) 33 (31) 35 (35) 25 (23)
1.2 146 17 27 32 22
148 (147) 13 (15) 19 (23) 27 (30) 30 (26)
4.9 139 14 27 34 19
121 (130) 12 (13) 28 (28) 38 (36) 23 (21)
20 121 11 25 29 19
139 (130) 8 (10) 34 (30) 39 (34) 17 (18)
78 133 10 29 30 20
131 (132) 8 (9) 28 (29) 26 (28) 17 (19)
313 159 13 24 33 20
130 (145) 17 (15) 20 (22) 22 (28) 20 (20)
1250 124* 11* 31* 21* 10*
114*(119)* 14*(13)* 28*(30)* 19*(20)* 16*(13)*
5000 89* 0* 13* 18* 0*
101*(95)* 0*(0)* 18*(16)* 17*(18)* 0*(0)*
Positive control not requiring S9mix Name AF-2 NaN3 AF-2 AF-2 ICR-191
Dose (μg/plate)          
0.01 0.5 0.01 0.1 1
Number of colonies/plate 550 401 125 520 2209
510 (530) 436 (419) 137 (131) 541 (531) 2186 (2198)
Positive control requiring S9mix Name B[a]P 2AA 2AA B[a]P B[a]P
Dose (μg/plate)          
5 2 10 5 5
Number of colonies/plate 867 369 524 213 83
859 (863) 331 (350) 484 (504) 219 (216) 90 (87)

AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3: Sodium azide

ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine2HCl

2AA: 2-Aminoanthracene

B[a]P: Benzo[a]pyrene

*: The growth inhibition of the tested bacterium by the test substance was observed.

The average number of colonies of two plates at each dose is shown in the ( ).

Table 2. Results of main test

With or without S9mix Dose (μg/plate) Number of revertant colonies/plate
Base-pair substitution type Frame-shift type
TA100 TA1535 WP2uvrA TA98 TA1537
-S9mix Solvent Control (DMSO) 130 30 37 27 16
151 (141) 28 (29) 31 (34) 16 (22) 17 (17)
39 152 23 30 21 19
126 (139) 26 (25) 28 (29) 16 (19) 20 (20)
78 144 30 26 19 18
138 (141) 33 (32) 22 (24) 20 (20) 18 (18)
156 120 26 25 20 26
140 (130) 32 (29) 31 (28) 15 (18) 21 (24)
313 149 29 24 21 12
123 (136) 33 (31) 30 (27) 18 (20) 13 (13)
625 125 33 29 28 12
122 (124) 22 (28) 26 (28) 19 (24) 8 (10)
1250 123* 19* 26* 13* 4*
90*(107)* 12*(16)* 20*(23)* 9*(11)* 4*(4)*
+S9mix Solvent Control (DMSO) 145 19 28 33 23
151 (148) 25 (22) 29 (29) 31 (32) 25 (24)
39 142 22 26 23 27
127 (135) 32 (27) 27 (27) 24 (24) 19 (23)
78 149 25 27 22 31
136 (143) 22 (24) 23 (25) 28 (25) 23 (27)
156 147 38 20 30 27
118 (133) 24 (31) 30 (25) 27 (29) 31 (29)
313 131 19 26 22 23
129 (130) 24 (22) 24 (25) 31 (27) 23 (23)
625 123 30 33 24 21
162 (143) 29 (30) 29 (31) 32 (28) 23 (22)
1250 107* 16* 26* 24* 10*
110*(109)* 24*(20)* 23*(25)* 24*(24)* 10*(10)*
Positive control not requiring S9mix Name AF-2 NaN3 AF-2 AF-2 ICR-191
Dose (μg/plate)          
0.01 0.5 0.01 0.1 1
Number of colonies/plate 562 445 147 542 2268
637 (600) 505 (475) 168 (158) 501 (522) 2061 (2165)
Positive control requiring S9mix Name B[a]P 2AA 2AA B[a]P B[a]P
Dose (μg/plate)          
5 2 10 5 5
Number of colonies/plate 643 346 485 202 72
681 (662) 390 (368) 436 (461) 204 (203) 92 (82)

AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3: Sodium azide

ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine2HCl

2AA: 2-Aminoanthracene

B[a]P: Benzo[a]pyrene

*: The growth inhibition of the tested bacterium by the test substance was observed.

The average number of colonies of two plates at each dose is shown in the ( ).

Table 3. Historical background data from Jun to Aug 2004

    without S9mix with S9mix
strains   mean - 3SD mean - 2SD mean mean + 2SD mean + 3SD n mean - 3SD mean - 2SD mean mean + 2SD mean + 3SD n
TA100 solvent control 74 88 115 143 156 349 78 94 126 157 173 324
positive control 433 477 563 649 693 349 491 578 750 922 1008 324
TA1535 solvent control 9 13 21 29 33 267 4 8 16 23 27 245
positive control 365 394 451 508 537 267 280 305 354 403 428 245
WP2 uvrA solvent control 12 16 26 36 40 229 15 20 30 39 44 221
positive control 105 119 148 176 190 229 364 402 478 553 591 221
TA98 solvent control 7 11 20 29 34 328 13 19 30 42 48 304
positive control 468 504 577 649 686 328 142 164 207 251 272 304
TA1537 solvent control 1 5 11 18 22 255 6 11 21 30 35 233
positive control 1683 1820 2093 2367 2503 255 42 52 71 91 101 233
TA1538 solvent control 7 9 14 20 22 20 9 14 24 34 39 20
positive control 549 589 668 747 787 20 68 85 117 150 166 20

SD: Standard deviation

Applicant's summary and conclusion

Conclusions:
Under the conditions of the Ames test the substance was not mutagenic in any of the five bacterial strains (TA1535, TA1537, TA98, TA100 and WP2 uvrA) tested with and without metabolic activation up to 5000 µg/plate. Cytotoxicity was observed at and above 1250 µg/plate.