Registration Dossier

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Follows a recognised guideline and performed to GLP standards.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity test concentrations: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Definitive test concentrations: 50, 150, 500, 1500 and 5000 μg/plate.
Vehicle / solvent:
trisodium 6,6',6"-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoate was fully soluble in sterile distilled water at 50 mg/ml but was insoluble in DMSO at the same concentration in solubility checks performed in-house. Sterile distilled water was selected as the vehicle.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Migrated to IUCLID6: , 9-Aminoacridine, 4-Nitroquinoline-1-oxide, 2-Aminoanthracene, Benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

- Exposure duration: 48 hours
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statisitical methods as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive results, in some instances the data generated will prohibit a definitive judgement about the test material. Results of this type will be reported as equivocal.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test: trisodium 6,6',6"-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoate was found to be non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). See table 1 for results of this test.
Results for the definitive test can be seen in table 2-5.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 results of the preliminary toxicity test:

With (+) or without (-) S9-mix Strain Dose
0 0.15 0.5 1.5 5 15 50 150 500 1500 5000
- TA100 124 99 125 C 115 142 111 89 113 120 126
+ TA100 109 95 123 117 124 143 132 117 109 104 102
- WP2uvrA- 24 19 38 24 26 30 32 20 31 29 26
+ WP2uvrA- 32 33 31 33 32 36 36 36 34 38 37

C= contaminated

Table 2. Definitive test 1 - without metabolic activation

Test Period From 03 August 08   To 06 August 08
Number of revertants (mean number of colonies per plate)
Base-pair substitution type   Frameshift type
With or without S9-Mix Test substance concentration (μg/plate) TA100 TA1535 WP2uvrA- TA 98 TA1537
- 0 123 (113)
8.7#
16 18
(1.5)
30 (30)
0.6
19 (14)
4.7
7 (8)
2.3
109 19 29 10 7
107 18 30 12 11
- 50 122 (123)
7.1
13 (12)
1.2
34 (32)
6.7
19 (15)
3.5
9 (9)
2.5
117 11 38 15 11
131 13 25 12 6
- 150 135 (126)
9.0
16 (20)
3.6
21 (23)
3.2
20 (18)
1.7
9 (11)
2.6
117 23 27 17 10
126 21 22 17 14
- 500 126 (123)
4.2
22 (19)
2.6
33 (30)
3.0
25 (17)
7.2
6 (10)
3.2
124 17 30 12 11
118 18 27 13 12
- 1500 106 (108)
3.5
19 (15)
3.5
27 (29)
5.9
17 (20)
3.1
10 (11)
1.2
106 12 25 19 10
112 15 36 23 12
- 5000 121 (121)
4.5
21 (20)
2.1
25 (24)
3.1
19 (17)
2.1
14 (12)
1.5
116 22 27 15 11
125 18 21 16 12
Positive controls
S9 Mix
-
Name  ENNG ENNG ENNG 4NQO 9AA
Concentration 3 5 2 0.2 80
No. colonies per plate 390 (352)
34.6
264 (291)
26.1
503 (525)
19.3
107 (119)
21.1
844 (823)
42.3
345 316 540 106 774
322 293 531 143 850

Table 3 definitive test 1 - with metabolic activiation

Test Period From 03 August 08   To 06 August 08
Number of revertants (mean number of colonies per plate)
Base-pair substitution type   Frameshift type
With or without S9-Mix Test substance concentration (μg/plate) TA100 TA1535 WP2uvrA- TA 98 TA1537
+ 0 113 (114)
16.9#
13 (14)
1.0
32 (33)
1.7
19 (23)
5.5
6 (9)
5.2
110 15 35 29 15
100 14 32 20 6
+ 50 106 (121)
13.2
10 (11)
1.7
39 (36)
2.9
12 (23)
10.3
12 (12)
1.5
126 10 34 26 10
131 13 34 32 13
+ 150 110 (104)
5.7
12 (11)
1.2
31 (33)
2.6
31 (27)
5.1
14 (11)
3.5
102 10 36 28 7
99 10 32 21 11
+ 500 125 (113)
17.4
14 (13)
1.7
32 (29)
5.8
32 (27)
5.0
3 (6)
3.1
121 14 22 26 5
93 11 32 22 9
+ 1500 99 (117)
16.5
10 (13)
2.5
37 (32)
5.0
20 (21)
6.1
9 (6)
3.0
122 13 31 16 6
131 15 27 28 3
+ 5000 133 (119)
11.9
14 (13)
0.6
38 (35)
2.9
19 (23)
3.8
4 (9)
4.4
111 13 33 25 11
114 13 33 26 12
Positive controls
S9 Mix
+
Name  2AA 2AA 2AA BP 2AA
Concentration 1 2 10 5 2
No. colonies per plate 1000 (896)
99.9
221 (239)
42.9
380 (357)
30.6
235 (242)
28.2
440 (390)
48.2
886 288 322 273 385
801 208 368 218 344

Definitive test 2 - without metabolic activation

Test Period From 03 August 08   To 06 August 08
Number of revertants (mean number of colonies per plate)
Base-pair substitution type   Frameshift type
With or without S9-Mix Test substance concentration (μg/plate) TA100 TA1535 WP2uvrA- TA 98 TA1537
- 0 95 (96)
1.2#
21 (22)
1.2
32 (27)
4.6
14 (16)
2.5
10 (11)
1.0
97 21 26 16 11
95 23 23 19 12
- 50 112 (106)
8.7
19 (22)
5.8
22 (22)
2.5
19 (17)
3.5
11 (10)
5.0
96 19 20 13 15
110 29 25 19 5
- 150 92 (104)
12.6
20 (21)
1.5
25 (31)
6.5
15 (17)
2.1
11 (11)
0.6
102 23 31 19 10
117 21 38 18 11
- 500 104 (104)
6.5
24 (23)
1.5
16 (20)
3.5
13 (13)
1.5
15 (12)
2.6
97 23 20 11 11
110 21 23 14 10
- 1500 97 (106)
8.1
16 (21)
5.7
30 (31)
6.6
18 (18)
0.6
12 (11)
2.3
107 27 25 19 8
113 19 38 18 12
- 5000 106 (101)
5.0
25 (22)
3.5
29 (29)
2.5
21 (18)
4.4
12 (10)
4.4
102 22 27 20 13
96 18 32 13 5
Positive controls
S9 Mix
-
Name  ENNG ENNG ENNG 4NQ 9AA
Concentration 3 5 2 0.2 80
No. colonies per plate 388 (430)
38.2
260 (263)
10.3
496 (478)
60.6
194 (196)
6.7
1474 (1035)
381.7
438 254 410 203 845
463 274 527 190 785

Table 4 Definitive test 2 - with metabolic activation

Test Period From 03 August 08   To 06 August 08
Number of revertants (mean number of colonies per plate)
Base-pair substitution type   Frameshift type
With or without S9-Mix Test substance concentration (μg/plate) TA100 TA1535 WP2uvrA- TA 98 TA1537
+ 0 97 (98)
1.2#
13 (11)
1.5
36 (38)
2.1
25 (22)
2.6
14 (12)
3.8
99 10 40 20 8
99 11 37 21 15
+ 50 86 (89)
3.6
11 (13)
2.5
42 (32)
10.6
27 (27)
0.0
18 (13)
5.6
93 16 34 27 14
88 13 21 27 7
+ 150 76 (86)
10.0
8 (11)
3.5
34 (32)
2.0
31 (23)
7.2
9 (11)
2.6
96 11 30 18 14
85 15 32 19 10
+ 500 90 (89)
11.1
12 (12)
2.0
29 (33)
3.5
26 (24)
3.5
19 (14)
4.7
99 10 35 26 12
77 14 35 20 10
+ 1500 79 (74)
7.8
15 (12)
2.3
46 (38)
7.1
25 (28)
2.3
5 (8)
3.0
65 11 32 29 8
78 11 37 29 11
+ 5000 86 (93)
6.1
15 (14)
1.0
22 (33)
9.5
20 (22)
2.1
10 (10)
2.5
97 14 40 24 7
96 13 36 23 12
Positive controls
S9 Mix
+
Name  2AA 2AA 2AA BP 2AA
Concentration 1 2 10 5 2
No. colonies per plate 959 (950)
15.0
211 (251)
52.2
403 (299)
119
210 (222)
19.7
471 (422)
42.5
933 232 324 212 399
959 310 169 245 396

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

trisodium 6,6',6"-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoate is considered to be non-mutagenic under the conditions of this test.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium and E. coli WP2 uvr A were exposed to trisodium 6,6',6"-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoate at concentrations of 0, 50, 150, 500, 1500, 5000 µg/plate in the presence and absence of mammalian metabolic activation.

 

The positive controls induced the appropriate responses in the corresponding strains.  There was no evidence of induced mutant colonies over background.

 

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.