Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-12-19 - 2002-01-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
other: USA, EPA (TSCA) OPPTS harmonised guidelines
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: black powder
- Storage conditions: room temperature in the dark

Method

Target gene:
Salmonella typhimurium LT2
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
10% liver S9-mix in standard co-factors
Test concentrations with justification for top dose:
Preliminary toxicity study: 0.15, 0.5, 1.5, 5, 15, 5, 150, 500, 1500 and 5000 µg/plate
Mutation study - Experiment 1: 15, 5, 150, 500, 1500 and 5000 µg/plate
Mutation study - Experiment 1: 1.5, 5, 15, 5, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl formamide
- Justification for choice of solvent/vehicle: Dimethyl formamide is an acceptable vehicle for use in this test system
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION: Exposure duration: 48 hours
NUMBER OF REPLICATIONS: in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

TESTER STRAINS:
The strains used in this assay were all mutants derived from Salmonella typhimurium LT2 and were those recommended for general screening.
TA100
TA 1535 sensitive to agents inducing base-pair substitution
TA102 sensitive to agents inducing frame-shift mutations
TA98
The strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 and were stored at -196°C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to determine the amino-acid requirement, presence of rfa, R factors, uvrB mutation and the spontaneous reversion rate.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 250427 06/06) and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.


PROCEDURE
Preliminary Toxicity Study
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100), 2 ml of molten, trace histidine supplemented, top agar, 0.1 ml of test material formulation, 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel- Bonner Minimal agar (30 ml/plate). Ten doses of the test material and a vehicle control (dimethyl formamide) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation Study - Experiment 1
Six concentrations of the test material (15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Mutation Study - Experiment 2
The second experiment was performed using methodology as described for Experiment 1, using fresh bacterial cultures, test material and control solutions. The test material dose range was amended following observations made from Experiment 1 and was as follows:
TA100 and TA1535 (with and without S9), TA98 (with S9 only): 1.5, 5, 15, 50, 150, 500, 1500 ug/plate
TA102 (with and without S9): 5, 15, 50, 150, 500, 1500, 5000 ug/plate
TA98 (without S9 only), TA1537 (with and without S9): 15, 50,150, 500,1500 ug/plate
Additional dose levels were included to allow for the toxicity of the test material, ensuring there were a minimum of four non-toxic doses and, in the case of TA100 and TA1535, to determine the level at which the test material first induced mutations.

ACCEPTANCE CRITERIA
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pkMlOl plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels. There should be no evidence of excessive contamination.

















Evaluation criteria:
The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression(5)) significant increase in the revertant count in at least one strain of bacteria.
Statistics:
not specified

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
15 - 500 µg/plate
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
15 - 150 µg/plate
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
10 - 500 µg/plate
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The test material was toxic initially at and above 1500 ug/plate to the strain of Salmonella used (TA100). The test material formulation and the S9-mix used in this experiment were both shown to be effectively sterile

Any other information on results incl. tables

Preliminary study

The number of revertant colonies for the toxicity assay were:

With (+) or without (-) Metabolic Activation Strain Dose (µg/plate)
0 0,15 0,5 1,5 5 15 50 150 500 1500 5000
- TA 100 94 73 72 93 112 160 231 257 310 0V 0V
+ TA100 148 104 93 126 147 283 322 300 255 249 0T
T = toxic, no bacterial background lawn
V = very weak bacterial background lawn

Mutation Study

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Study.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 to Table 5.

The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant decrease in the frequency of revertant colonies in all of the tester strains, both with and without S9-mix, at and above 1500 µg/plate. The test material was, therefore, tested up to either the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial tester strain type and presence or absence of S9-mix. A black, fine, powdery precipitate was observed at and above 5000 µg/plate, this did not prevent the scoring of revertant colonies.

Dose-related, reproducible and statistically significant increases were observed at sub-toxic dose levels of the test material. TA100 and TA1535 exhibited statistically significant increases from 15 to 500 µg/plate both with and without S9-mix. TA102 exhibited reproducible increases in the presence of S9 only from 15 to 150 µg/plate.

No reproducible increases in the frequency of revertant colonies were recorded for any of the remaining bacterial strains, with any dose of the test material, either with or without metabolic activation.

Judging by the type of Salmonella strains exhibiting dose-related increases (TA100, TA1535 and TA102), the test material was concluded to be a base-pair mutagen.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 1: Spontaneous Mutation Rates (Concurrent Negative Controls)
Experiment 1
Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 TA102 TA98 TA1537
94 (97) 27 (23) 318 (322) 26 (23) 6 (13)
82 21 325 23 18
116 22 322 20 16
                
Experiment 2
Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 TA102 TA98 TA1537
112 (101) 26 (22) 302 (308) 26 (21) 13 (12)
94 16 303 24 9
96 24 318 13 15

Table 2: Test Results: Experiment 1 - Without Metabolic Activation
Test Period From: 04 January 2002 To: 07 January 2002
With or without S9-Mix Test substance concentration (µg/plate) Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 TA102 TA98 TA1537
- 0 87
81
76
(81)
5.5#
32
28
26
(29)
3.1
313
341
328
(327)
14.0
24
26
19
(23)
3.6
14
14
8
(12)
3.5
- 15 222
210
270
$$$
(234)
31.7
34
46
50
$$$
(43)
8.3
309
329
335
(324)
13.6
36
24
25
(28)
6.7
11
9
12
(11)
1.5
- 50 380
352
360
$$$
(364)
14.4
57
65
75
$$$
(66)
9,0
329
330
365
(341)
20.5
28
27
32
(29)
2.6
14
12
16
(14)
2,0
- 150 280
336
291
$$$
(302)
29.7
78
73
66
$$$
(72)
6,0
305
344
368
(339)
31.8
23
31
44
(33)
10.6
11
19
22
(17)
5.7
- 500 252
244
248
$$$
(248)
4.0
64
54
61
$$$
(60)
5.1
281
250
274
(268)
16.3
28
27
31
(29)
2.1
16
15
13
(15)
1.5
- 1500 0V
0V
0V
(0)
0.0
0V
0V
0V
(0)
0.0
94
104
92
(97)
6.4
0V
0V
0V
(0)
0.0
0T
0T
0T
(0)
0.0
- 5000 0TP
0TP
0TP
(0)
0.0
0TP
0TP
0TP
(0)
0.0
0TP
0TP
0TP
(0)
0.0
0TP
0TP
0TP
(0)
0.0
0TP
0TP
0TP
(0)
0.0
Positive controls

S9-Mix

-
Name ENNG ENNG MMC 4NQO 9AA
Concentration (µg/plate) 3 5 0.5 0.2 80
No. Colonies per plate 548
513
438
(500)
56.2
453
311
289
(351)
89.0
809
919
924
(884)
65.0
110
117
112
(113)
3.6
1766
1924
2215
(1968)
227.8
ENNG
4NQO
9AA
MMC
P
T
V
$$$
#
N-ethyl-N'-nitro-N-nitrosoguanidine
4-Nitroquinoline-1-oxide
9-Aminoacridine
Mitomycin C
Precipiate
Toxic, no bacterial background lawn
Very weak bacterial background lawn
p < = 0.005
Standard deviation

Table 3: Test results: Experiment 1 - With metabolic activation
Test Period From: 04 January 2002 To: 07 January 2002

With or

without S9-Mix

Test substance concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 TA102 TA98 TA1537
+ 0 84
75
95
(85)
10.0#
18
15
8
(14)
5.1
324
381
361
(327)
14.0
29
40
34
(34)
5.5
18
19
17
(18)
1.0
+ 15 196
188
200
$$$
(195)
6.1
52
37
50
$$$
(46)
8.1
491
474
479
(324)
13.6
53
44
59
$$$
(52)
7.5
26
15
21
(21)
5.5
+ 50 312
220
236
$$$
(256)
49.2
53
41
54
$$$
(49)
7.2
483
454
478
(341)
20.5
50
50
48
**
(49)
1.2
16
28
22
(22)
6.0
+ 150 320
316
272
$$$
(303)
26.6
66
55
35
$$$
(52)
15.7
483
457
453
(339)
31.8
50
46
50
**
(49)
2.3
19
27
22
(23)
4.0
+ 500 276
263
274
$$$
(271)
7.0
43
46
46
$$$
(45)
1.7
346
319
326
(268)
16.3
23
34
40
(32)
8.6
13
15
25
(18)
6.4
+ 1500 0T
0T
0T
(0)
0.0
0T
0T
0T
(0)
0.0
155
136
142
(97)
6.4
0V
0V
0V
(0)
0.0
0T
0T
0T
(0)
0.0
+ 5000 0TP
0TP
0TP
(0)
0.0
0TP
0TP
0TP
(0)
0.0
24P
28P
24P
(0)
0.0
0TP
0TP
0TP
(0)
0.0
0TP
0TP
0TP
(0)
0.0

Positive

controls

S9-Mix

+

Name 2AA 2AA DAN BP 2AA

Concentration

(µg/plate)

1 2 10 5 2

No. Colonies

per plate

1469
1309
1638
(1472)
164.5
177
185
183
(182)
4.2
967
588
600
(718)
215.4
257
264
165
(229)
55.2
446
469
418
(444)
25.5
BP
2AA
DAN
P
T
V
**
$$$
#
Benzo(a)pyrene
2-Aminoanthracene
1,8-Dihydroxyanthraquinone
Precipiate
Toxic, no bacterial bachground lawn
Very weak bacterial background lawn
p < = 0.01
p < = 0.005
Standard deviation

Table 4: Test results: Experiment 2 - Without metabolic activation
Test Period From: 11 January 2002 To: 14 January 2002

With or

without S9-Mix

Test substance

concentration (µg/plate)

Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 TA102 TA98 TA1537
- 0 86
76
83
(82)
5.1#
15
21
17
(18)
3.1
300
372
348
(340)
36.7
14
17
20
(17)
3.0
6
6
15
(9)
5.2
- 1.5 76
70
68
(71)
4.2
13
18
16
(16)
2.5
N/T N/T N/T
- 5 92
79
93
(88)
7.8
14
18
9
(14)
4.5
343
320
341
(335)
12.7
N/T N/T
- 15 123
129
123
$$$
(125)
3.5
29
22
24
(25)
3.6
365
360
369
(365)
4.5
28
22
25
(25)
3.0
11
9
12
(11)
1.5
- 50 188
196
197
$$$
(194)
4.9
36
28
32
$$$
(32)
4.0
374
377
386
*
(379)
6.2
41
29
33
**
(34)
6.1
12
11
17
(13)
3.2
- 150 268
282
264
$$$
(271)
9.5
37
57
53
$$$
(49)
10.6
400
357
363
(373)
23.3
24
23
39
(29)
9.0
13
14
7
(11)
3.8
- 500 304
267
261
$$$
(277)
23.3
53
42
47
$$$
(47)
5.5
285
298
298
(294)
7.5
22
33
14
(23)
9.5
11
13
12
(12)
1.0
- 1500 0V
0V
0V
(0)
0.0
0V
0V
0V
(0)
0.0
129
C
137
(133)
5.7
0V
0V
0V
(0)
0.0
0V
0V
0V
(0)
0.0
- 5000 N/T N/T 0TP
0TP
0TP
(0)
0.0
N/T N/T

Positive

controls

S9-Mix

-

Name ENNG ENNG MMC 4NQO 9AA

Concentration

(µg/plate)

3 5 0.5 0.2 80

No. Colonies

per plate

473
411
438
(441)
31.1
255
255
244
(251)
6.4
852
801
912
(855)
55.6
114
104
106
(108)
173.5
2429
2132
2125
(2229)
173.5
ENNG
4NQO
9AA
MMC
C
N/T
P
T
V
*
**
$$$
#
N-ethyl-N'-nitro-N-nitrosoguanidine
4-Nitroquinoline-1-oxide
9-Aminoacridine
Mitomycin C
Contaminated
Not tested at this dose level
Precipiate
Toxic, no bacterial bachground lawn
Very weak bacterial background lawn
p < = 0.05
p < = 0.01
p < = 0.005
Standard deviation

Table 5: Test results: Experiment 2 - With metabolic activation
Test Period From: 11 January 2002 To: 14 January 2002
With or without S9-Mix Test substance concentration (µg/plate) Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 TA102 TA98 TA1537
+ 0 80
113
92
(95)
16.7#
12
14
12
(13)
1.2
391
393
370
(385)
12.7
29
44
41
(38)
7.9
18
13
16
(16)
1.5
+ 1,5 83
90
98
(90)
7.5
13
9
12
(11)
2.1
N/T 34
32
27
(31)
3.6
N/T
+ 5 113
106
127
(115)
10.7
19
25
14
*
(19)
5.5
402
434
402
(413)
18.5
32
36
35
(34)
2.1
N/T
+ 15 194
183
202
$$$
(193)
9.5
23
24
33
$$$
(27)
5.5
491
437
468
*
(465)
27.1
41
35
40
(39)
3.2
17
17
20
(18)
1.7
+ 50 303
252
317
$$$
(291)
34.2
38
42
42
$$$
(41)
2.3
543
529
563
$$$
(545)
17.1
38
39
27
(35)
6.7
18
24
26
*
(23)
4.2
+ 150 321
370
310
$$$
(334)
31.9
48
62
47
$$$
(52)
8.4
472
524
515
$$$
(504)
27.8
35
35
40
(37)
2.9
24
17
17
(19)
4.0
+ 500 267
297
305
$$$
(290)
20.0
31
32
36
$$$
(33)
2.6
388
378
275
(347)
62.6
33
32
25
(30)
4.4
13
12
14
(13)
1.0
+ 1500 0V
0V
0V
(0)
0.0
0V
0V
0V
(0)
0.0
196
189
195
(193)
3.8
0V
0V
0V
(0)
0.0
0V
0V
0V
(0)
0.0
+ 5000 N/T N/T 34 SP
43 SP
23 SP
(33)
10.0
N/T N/T
Positive controls

S9-Mix

+
Name 2AA 2AA DAN BP 2AA
Concentration (µg/plate) 1 2 10 5 2
No. Colonies per plate 1728
1747
1746
(1740)
10.7
299
254
286
(280)
23.2
916
904
755
(858)
89.7
165
215
174
(185)
26.7
611
611
636
(619)
14.4
BP
2AA
DAN
P
T
V
*
$$$
#
Benzo(a)pyrene
2-Aminoanthracene
1,8-Dihydroxyanthraquinone
Precipitate
Toxic, no bacterial background lawn
Very weak bacterial background lawn
p < = 0.05
p < = 0.005
Standard deviation

Applicant's summary and conclusion

Conclusions:
The test material was considered to be mutagenic under the conditions of this test.
Executive summary:

The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines and was conducted in compliance with GLP.

Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with suspensions of the test material using the Ames plate incorporation method at up to seven dose levels (0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate), in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 15 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following observations made in Experiment 1 and ranged between 1.5 and 5000 µg/plate depending on bacterial strain type and presence or absence of S9-mix. Additional dose levels were used in both experiments (where applicable) to allow for the toxicity of the test material ensuring there were a minimum of four non-toxic doses.

The vehicle (dimethyl formamide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant decrease in the frequency of revertant colonies in all of the tester strains, both with and without S9-mix, at and above 1500 µg/plate. The test material was, therefore, tested up to either the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial tester strain type and presence or absence of S9-mix. A black, fine, powdery precipitate was observed at and above 5000 µg/plate, this did not prevent the scoring of revertant colonies.

Dose-related, reproducible and statistically significant increases were observed at sub-toxic dose levels of the test material. TA100 and TA1535 exhibited statistically significant increases from 15 to 500 µg/plate both with and without S9-mix. TA102 exhibited reproducible increases in the presence of S9 only from 15 to 150 µg/plate.

No reproducible increases in the frequency of revertant colonies were recorded for any of the remaining bacterial strains, with any dose of the test material, either with or without metabolic activation.

Judging by the type of Salmonella strains exhibiting dose-related increases (TA100, TA1535 and TA102), the test material was concluded to be a base-pair mutagen.

The test material was considered to be mutagenic under the conditions of this test.