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Genetic toxicity: in vitro

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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:
21 December 2000 - 30 March 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study under GLP conditions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
: OECD GLP (1997)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Physical state: Orthorhombic crystal
- Purity: 98.2 %
- Impurities: No data
- Lot/batch No.: GH01
- Manufacuture: Tokyo Chemical Industry Co., Ltd.
- Stability under test conditions: Stable during the test period (confirmed by other related test)
- Storage condition of test material: in cool, light-blocking and air-tight conditions

Method

Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Rat liver, induced with phenobarbital and 5,6-benzoflavone
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver, induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
- Dose determination test: 5, 10, 50, 100, 500, 1000, and 5000 μg/plate
- Mutagenicity test: 312.5, 625, 1250, 2500, and 5000 μg/plate
- Confirmation test: 312.5, 625, 1250, 2500, and 5000 μg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: Distilled water for injection (Lot No. 00603D, Japanese Pharmacopeia, Fuso Pharmaceutical Industries, Ltd.)
- Justification for choice of solvent/vehicle: The test substance was soluble in water at 55.5 g/dL, and stable. Therefore distilled water for injection was selected as a vehicle.
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
Remarks:
: Distilled water for injection (Lot No. 00603D, Japanese Pharmacopeia, Fuso Pharmaceutical Industries, Ltd.)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) (Lot No. CKQ1402, Purity: 99.0%, Wako Pure Chemical Industries, Ltd.)
Remarks:
without S9 (S. typhimurium TA98, TA100; E. coli WP2 uvr A)
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 (S. typhimurium TA1535)

Migrated to IUCLID6: (SA) (Lot No. ELJ6565, Purity: 99.7%, Wako Pure Chemical Industries, Ltd.)
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 (S. typhimurium TA1537)

Migrated to IUCLID6: (9-AA) (Lot No. 106F06681, Purity: 97%, SIGMA CHEMICAL Co., Ltd.)
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2-AA) (Lot No. ELJ6826, Purity: 97.4%, Wako Pure Chemical Industries, Ltd.)
Remarks:
with S9 (all strains)
Details on test system and conditions:
METHOD OF APPLICATION
- Dose determination test and mutagenicity test: plate incorporation method
- Confirmation test: preincubation method


DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours


NUMBER OF PLATES
- Dose determination test: 2 plates/dose
- Mutagenicity test and confirmation test: 3 plates/dose


DETERMINATION OF CYTOTOXICITY
- Method: Growth inhibition of the bacterial background lawn was microscopically observed in each plate comparing to the solvent control.


OTHER EXAMINATIONS
- Precipitation of the test substance was microscopically observed.
- Colony counting for the solvent controls and the test substance groups was conducted with naked eyes, and for the positive control groups was conducted with a colony counter (Olympus, OL-502A, Yoshikawa Kogyo Co., Ltd.). In use of colony counter, the number of the bacterial colony was counted three times per plate and calculated the average for each plate.

Evaluation criteria:
A test substance was judged to be mutagenic (+) when a dose-related increase in the number of revertant colony was observed, the number of revertant colonies per plate in the test substance group was more than twice that of the negative control (solvent control) and when a reproducibility of test result was observed, or when a evident increase in the number of revertant colony was observed at one dose and a reproducibility of the test result was observed. A test substance for which the results do not meet the above criteria is judged to be non-mutagenic (-).
Growth in hibition was detected by increasing in colonies diameter or clearing of the background lawn of tester strains as compared with that of negative control.
Statistics:
A statistical analysis was not performed.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: E. coli WP2 uvr A
Remarks:
Migrated from field 'Test system'.
Additional information on results:
DOSE DETERMINATION TEST (PLATE INCORPORATION METHOD)
Results are shown in Table 1.
Neither precipitation of the test substance nor bacterial growth inhibition by the test substance was observed in any dose groups of the test substance. Therefore five concentration of the test substance was set at 312.5 to 5000 μg/plate with a common ration of 2 in mutagenicity test.


MUTAGENICITY TEST (PLATE INCORPORATION METHOD)
Results are shown in Table 2.
The test substance did not show any significant dose-related increase in the number of revertant colonies to at least twice as many as that of the solvent control in any test strains with or without metabolic activation.
Neither precipitation of the test substance nor bacterial growth inhibition by the test substance was observed in any dose groups of the test substance.


CONFIRMATION TEST (PREINCUBATION METHOD)
Results are shown in Table 3.
The test substance showed similar findings to the mutagenicity test. Therefore, the reproducibility of test results were confirmed.
Neither precipitation of the test substance nor bacterial growth inhibition by the test substance was observed in any dose groups of the test substance.


VALIDITY OF THE TEST
The number of the revertant colonies in the positive control group showed evident increase comparing to that of the solvent control at any tests.
The number of revertant colonies in both solvent control and positive control were within the expected range (mean ± 2S.D.) to the background data in this laboratory.

Any other information on results incl. tables

Table 1. Reverse mutation test of beta-alanine in S.typhimurium and E.coli (Dose determination test)

With (+) or without (-)
S9 mix
Test substance concentration
(μg/plate)
Number of revertants (number of colonies/plate)a)
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
S9 mix
(-)
Solvent control 129
126 (128)
11
18 (15)
41
27 (34)
25
31 (28)
11
10 (11)
5 133
134 (134)
15
21 (18)
24
33 (29)
26
29 (28)
16
13 (15)
10 137
137 (137)
 8
 9 (9)
25
47 (36)
37
37 (37)
15
12 (14)
50 167
150 (159)
14
12 (13)
35
34 (35)
22
29 (26)
15
15 (15)
100 124
122 (123)
11
11 (11)
47
32 (40)
34
38 (36)
 9
15 (12)
500 135
136 (136)
 9
16 (13)
31
43 (37)
32
29 (31)
 8
 7 (8)
1000 111
118 (115)
12
12 (12)
35
32 (34)
31
23 (27)
10
12 (11)
5000 111
127 (119)
 8
11 (10)
39
40 (40)
26
27 (27)
13
12 (13)
S9 mix
(+)
Solvent control 156
145 (151)
14
18 (16)
42
49 (46)
42
42 (42)
11
17 (14)
5 162
155 (159)
10
14 (12)
28
31 (30)
41
45 (43)
 8
 7 (8)
10 160
162 (161)
14
15 (15)
40
40 (40)
42
26 (34)
13
12 (13)
50 152
145 (149)
16
18 (17)
45
47 (46)
42
38 (40)
13
14 (14)
100 160
155 (158)
17
12 (15)
56
54 (55)
42
37 (40)
13
18 (16)
500 135
150 (143)
15
 7 (11)
47
37 (42)
44
44 (44)
21
17 (19)
1000 123
147 (135)
10
18 (14)
35
40 (38)
46
43 (45)
18
12 (15)
5000 114
119 (117)
13
25 (19)
39
41 (40)
35
37 (36)
13
11 (12)
Positive control not requiring
S9 mix
Name AF-2 SA AF-2 AF-2 9-AA
Concentration (μg/plate) 0.01 0.5 0.01 0.1 80
Number of colonies/plate 470
532 (501)
556
529 (543)
268
242 (255)
477
536 (507)
498
544 (521)
Positive control requiring
S9 mix
Name 2-AA 2-AA 2-AA 2-AA 2-AA
Concentration (μg/plate) 1 2 10 0.5 2
Number of colonies/plate 920
1000 (960)
230
251 (241)
852
913 (883)
603
616 (610)
230
269 (250)

a): The average number of colonies in each concentration.

Solvent: Distilled water for injection

Table 2. Reverse mutation test of beta-alanine in S.typhimurium and E.coli (Mutagenicity test)

With (+) or without (-)
S9 mix
Test substance concentration
(μg/plate)
Number of revertants (number of colonies/plate)a)
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
S9 mix
(-)
Solvent control 149
142 (149)
156 
15
 7 (12)
15 
31
24 (27)
25
30
30 (31)
32
 7
 9 (9)
12
312.5 127
131 (131)
134 
16
10 (12)
11
26
28 (30)
35
39
42 (39)
35
12
11 (11)
10
625 136
139 (140)
144
9
19 (14)
15
34
23 (27)
24
32
27 (29)
27
10
10 (10)
 9
1250 152
144 (148)
147
14
17 (13)
 8
27
26 (29)
33
35
43 (38)
36
13
11 (11)
10
2500 130
128 (130)
132
12
13 (13)
14
21
40 (30)
30
24
29 (28)
31
 8
12 (9)
 8
5000 148
143 (148)
152
15
17 (16)
16
29
30 (29)
28
38
33 (35)
33
10
 8 (12)
17
S9 mix
(+)
Solvent control 152
162 (154)
149
 7
19 (14)
16
35
31 (32)
29
37
34 (40)
49
12
 9 (13)
17
312.5 147
150 (148)
146
19
17 (20)
24
37
32 (33)
29
48
47 (45)
40
17
18 (17)
16
625 145
149 (148)
150
15
16 (16)
18
36
35 (34)
31
50
47 (47)
44
13
18 (14)
11
1250 132
148 (148)
164
27
14 (20)
18
29
41 (35)
35
46
50 (43)
33
13
12 (11)
 9
2500 161
158 (159)
158
16
18 (15)
12
30
22 (30)
37
50
46 (48)
47
10
14 (13)
16
5000 131
143 (140)
146
18
24 (20)
19
27
35 (31)
32
32
38 (35)
36
10
14 (12)
13
Positive control not requiring
S9 mix
Name AF-2 SA AF-2 AF-2 9-AA
Concentration (μg/plate) 0.01 0.5 0.01 0.1 80
Number of colonies/plate 478
509 (506)
532
476
561 (520)
524
170
225 (200)
206
526
462 (500)
513
570
578 (569)
560
Positive control requiring
S9 mix
Name 2-AA 2-AA 2-AA 2-AA 2-AA
Concentration (μg/plate) 1 2 10 0.5 2
Number of colonies/plate 853
949 (920)
959
239
211 (217)
202
886
993 (935)
927
636
654 (632)
607
207
266 (248)
272

a): The average number of colonies in each concentration.

Solvent: Distilled water for injection

Table 3. Reverse mutation test of beta-alanine in S.typhimurium and E.coli (Confirmation test)

With (+) or without (-)
S9 mix
Test substance concentration
(μg/plate)
Number of revertants (number of colonies/plate)a)
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
S9 mix
(-)
Solvent control 143
153 (147)
146 
13
12 (15)
19
29
19 (25)
27
29
31 (32)
37
10
14 (13)
16
312.5 155
138 (148)
151
14
12 (12)
10
29
32 (30)
29
41
44 (41)
38
17
14 (16)
17
625 143
158 (151)
151
17
11 (13)
11
38
13 (27)
29
30
40 (37)
40
14
10 (13)
16
1250 155
154 (155)
156
12
16 (13)
11
28
28 (29)
31
29
35 (33)
34
14
13 (12)
 9
2500 143
139 (143)
147
13
15 (14)
13
23
20 (27)
37
34
32 (37)
45
15
14 (13)
11
5000 133
126 (132)
136
17
11 (14)
13
20
30 (29)
38
25
38 (34)
38
 9
11 (11)
14
S9 mix
(+)
Solvent control 149
155 (153)
154
20
13 (14)
10
26
37 (33)
37
39
41 (44)
51
17
14 (15)
15
312.5 153
154 (155)
158
23
16 (18)
15
35
36 (33)
29
42
46 (45)
47
18
15 (17)
18
625 148
141 (142)
136
14
13 (16)
20
36
32 (34)
33
43
46 (46)
50
18
18 (18)
17
1250 149
143 (146)
147
16
22 (18)
17
30
38 (35)
37
42
39 (40)
38
10
14 (12)
13
2500 148
152 (150)
149
10
19 (14)
13
38
30 (36)
39
41
39 (42)
45
19
19 (18)
15
5000 145
166 (157)
160
26
22 (21)
14
31
31 (35)
44
41
48 (46)
48
10
19 (14)
14
Positive control not requiring
S9 mix
Name AF-2 SA AF-2 AF-2 9-AA
Concentration (μg/plate) 0.01 0.5 0.01 0.1 80
Number of colonies/plate 555
528 (543)
546
519
487 (514)
536
222
173 (186)
163
466
495 (496)
526
568
650 (591)
555
Positive control requiring
S9 mix
Name 2-AA 2-AA 2-AA 2-AA 2-AA
Concentration (μg/plate) 1 2 10 0.5 2
Number of colonies/plate 1041
988 (997)
962
270
241 (261)
272
960
904 (945)
970
597
566 (585)
593
198
224 (219)
236

a): The average number of colonies in each concentration.

Solvent: Distilled water for injection

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

This bacterial mutation study revealed that beta-alanine was negative with or without metabolic activation system.
Executive summary:

The study was conducted according to OECD TG 471 and 472 under GLP conditions. The mutagenicity of beta-alanine was examined using plate incorporation method inS. typhimurium TA 100, TA 98, TA 1535, TA 1537 and E. coli WP2uvr A, at concentrations up to 5000 μg/plate with or without metabolic activation system. In this study, the number of revertant colonies per plate with beta-alanine was less than twice that of the solvent control. Therefore, beta-alanine is judged to be non-mutagenic.