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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

2 Ames tests are available on two different qualities of the registered substance (Legal entity compositions A and B). Both showed negative results with and without metabolic activation.


The in vitro micronucleus performed on the registered substance showed negative results with and without metabolic activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17/07/2019 to 12/08/2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)
Version / remarks:
June 2012
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella strains: histidine locus
E. coli strain: Tryptophan locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: rfa- (increase permeability of bacterial cells to larger molecules); uvrB- (inactivation of the excision repair system) for all salmonella strains, and plasmid pKM101 R factor for TA98 and TA100 (to enhance chemical and UV induced mutagenesis)
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: uvrA- : DNA repair deficiency which enhances its sensitivity to some mutagenic compounds
Cytokinesis block (if used):
Not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
Rat liver S9
- source of S9
: purchased from Moltox, Lot No. 4061 (Exp 1) and 4123 (Exp 2)
- method of preparation of S9 mix :
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9: 5.0 mL; 1.65 M KCl/0.4 M MgCl2: 1.0 mL; 0.1 M Glucose-6-phosphate: 2.5 mL; 0.1 M NADP 2.0 mL; 0.2 M Sodium phosphate buffer (pH 7.4): 25.0 mL; Sterile distilled water: 14.5 mL.
A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
- concentration: 10% of S9 in the S9-mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): S9 Quality Control and Production Certificates are available in the study report.
Test concentrations with justification for top dose:
Experiment 1: The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were originally assayed in triplicate against each tester strain.

However, the test item induced excessive toxicity (resulting in an insufficient number of non-toxic dose concentrations) to all of the tester strains dosed in the absence of metabolic activation and, therefore, part of the experiment was repeated using an amended dose range of 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate.

Experiment 2:
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
All tester strains (absence of S9-mix): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50 μg/plate.
All tester strains (presence of S9 mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl Sulfoxide (DMSO)
- Justification for choice of solvent/vehicle:
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house.
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
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene (2AA)
Remarks:
with metabolic activation
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments
: 2

METHOD OF TREATMENT/ EXPOSURE:
The test item was suspected to be volatile, therefore all testing was performed using the pre-incubation method (20 minutes at 37 ± 3 °C) except for the untreated controls. For volatile substances, the pre-incubation method may increase exposure of the bacteria to the test item in comparison to the standard plate incorporation method.

TREATMENT SCHEDULE:
- Preincubation period: 20 minutes
- Exposure duration/duration of treatment:
the plates were incubated in sealed anaerobic jars or bags at 37 ± 3 °C for between 48 and 72 hours

SELECTIVE AGENT: histidine, Tryptophan deficient agar

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction of the growth of the bacterial background lawn by microscopically assessment of the plate's thinning

METHODS FOR MEASUREMENTS OF GENOTOXICIY
: presence of revertant colonies using an automated colony counting system.
Rationale for test conditions:
In accordance with the OECD TG 471 guidelines.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see tables 3 to 6
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium, other: TA98 and TA100 rfa, uvrB, pKM101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see tables 3 to 6
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium, other: TA1535 and TA1537, rfa, uvrB
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see tables 3 to 6
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: No test item precipitate was
observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Results for the negative controls (spontaneous mutation rates) were considered to be acceptable (see tables 1 and 2 below). The vehicle (dimethyl sulphoxide) 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 or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.


Ames test:
- Signs of toxicity: in the experiment 1, the maximum dose level of the test item was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. However, the test item induced excessive toxicity to all of the tester strains dosed in the absence of metabolic activation (S9-mix) and, therefore, part of the experiment was repeated using an amended dose range of 0.015 to 50 μg/plate.
The test item induced a toxic response with weakened bacterial background lawns noted to all of the tester strains dosed in the absence of metabolic activation (S9-mix) at and above 15 μg/plate. In the presence of metabolic activation (S9-mix), weakened bacterial background lawns were initially noted from 150 μg/plate (all of the Salmonella strains) and 500 μg/plate (WP2uvrA).

In the experiment 2, the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of metabolic activation (S9-mix) from 15 μg/plate (all of the Salmonella strains) and 50 μg/plate (WP2uvrA). In the presence of metabolic activation (S9-mix), weakened bacterial background lawns were noted from 150 μg/plate (TA100 and TA1537) and 500 μg/plate (TA1535,TA98 and WP2uvrA).

- Mean number of revertant colonies per plate and standard deviation: There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in both experiments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, as well as the number of data)
- Positive historical control data: the current positive HCD dataset is presented in the full study report
- Negative (solvent/vehicle) historical control data: The combined Vehicle and untreated control HCD dataset is presented in the full study report

Table 1: Spontaneous Mutation Rates (Concurrent Negative Controls) in Experiment 1



































































Number of revertants (mean number of colonies per plate)
Base-pair substitution typeFrameshift type
TA100TA1535WP2uvrATA98TA1537
96293426 29
103          (104)23            (23)34            (35)39(34)25            (25)
112163836 21
147213844 15
120         (135)†15           (20)†36           (38)†31(35)†13           (13)†
138244030 10

Table 2: Spontaneous Mutation Rates (Concurrent Negative Controls) in Experiment 2

















































Number of revertants (mean number of colonies per plate)
Base-pair substitution typeFrameshift type
TA100TA1535WP2uvrATA98TA1537
1319 2327 15 
112          (120)14(14)28            (26)24(23)9(11)
11620 2718 9 

 


Table 3: Results of Experiment 1 - without metabolic activation (pre-incubation method)


































































































Test PeriodFrom: 01 August 2019To: 04 August 2019
S9-Mix (-)Dose Level Per PlateNumber of revertants (mean) +/- SD
Base-pair substitution strainsFrameshift strains
TA100TA1535WP2uvrATA98TA1537
Solvent Control (DMSO)130          (133)
131          4.9#
139		17             (16)
15              1.0
16		44             (33)
27              9.3
29		24           (27)
30            3.1
26		13            (14)
14             0.6
14
0.015 µg141
134          (141)
148           7.0		13
16             (15)
15              1.5		29
40             (33)
29              6.4		25
30           (28)
29            2.6		13
13            (16)
23             5.8
0.05 µg146
127          (134)
130          10.2		14
17             (16)
18              2.1		25
17             (21)
22              4.0		29
24           (25)
22            3.6		9
10            (11)
15             3.2
0.15 µg126
164          (153)
168          23.2		15
18             (16)
16              1.5		31
34             (32)
32              1.5		27
33           (30)
31            3.1		19
13            (15)
13             3.5
0.5 µg147
154          (146)
138           8.0		8
22             (13)
10              7.6		37
33             (33)
30              3.5		29
28           (28)
26            1.5		16
22            (17)
12             5.0
1.5 µg162          (148)
135          13.5
148		12             (14)
17              2.5
14		48             (41)
28             11.5
48		35           (32)
27            4.6
35		18            (14)
11             3.6
13
5 µg160          (153)
143           8.9
156		14             (15)
22              6.1
10		34             (34)
31              3.0
37		36           (32)
35            6.7
24		12            (15)
10             6.4
22
15 µg143 S
158 S        (158)
172 S         14.5		11 S
17 S           (14)
15 S            3.1		31 S
37 S            (33)
31 S             3.5		38 S
39 S          (35)
29 S           5.5		16 S
14 S          (15)
14 S           1.2
50 µg0 V
0 V            (0)
0 V           0.0		17 S
11 S           (14)
13 S            3.1		10 V
12 V           (13)
16 V            3.1		26 S
33 S          (30)
32 S           3.8		0 T
0 T            (0)
0 T            0.0

 


ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
S Sparse bacterial background lawn
T Toxic, no bacterial background lawn
V Very weak bacterial background lawn
# Standard deviation


 


Table 4: Results of Experiment 1 - with metabolic activation (pre-incubation method)

























































































































Test PeriodFrom: 25 July 2019To: 28 July 2019
S9-Mix (+)Dose Level Per PlateNumber of revertants (mean) +/- SD
Base-pair substitution strainsFrameshift strains
TA100TA1535WP2uvrATA98TA1537
Solvent Control (DMSO)95            (91)
86           4.5#
91		15             (13)
9               3.8
16		40             (41)
44              2.3
40		26           (31)
30            6.1
38		21            (22)
21             1.2
23
1.5 µg92
83            (87)
85            4.7		17
16             (14)
8               4.9		46
31             (36)
30              9.0		36
40           (35)
28            6.1		24
16            (19)
16             4.6
5 µg96
98            (95)
90            4.2		29
17             (20)
13              8.3		55
37             (48)
52              9.6		32
37           (32)
27            5.0		26
21            (24)
25             2.6
15 µg90
107          (101)
106           9.5		21
24             (20)
16              4.0		56
45             (48)
42              7.4		36
29           (32)
31            3.6		25
24            (25)
25             0.6
50 µg91
103           (94)
88            7.9		16
27             (19)
14              7.0		44
42             (43)
44              1.2		33
34           (31)
25            4.9		22
32            (26)
24             5.3
150 µg0 V            (0)
0 V           0.0
0 V		18 S           (18)
23 S            4.5
14 S		41             (37)
37              3.5
34		32 S          (29)
28 S           2.6
27 S		15 S          (19)
27 S           6.9
15 S
500 µg0 T            (0)
0 T            0.0
0 T		0 T             (0)
0 T             0.0
0 T		37 S            (40)
47 S             5.8
37 S		0 V           (0)
0 V           0.0
0 V		0 V            (0)
0 V            0.0
0 V
1500 µg0 T
0 T            (0)
0 T            0.0		0 T
0 T             (0)
0 T             0.0		35 S
45 S            (37)
32 S             6.8		0 V
0 V           (0)
0 V           0.0		0 T
0 T            (0)
0 T            0.0
5000 µg0 T
0 T            (0)
0 T            0.0		0 T
0 T             (0)
0 T             0.0		56 S
57 S            (55)
52 S             2.6		0 T
0 T            (0)
0 T            0.0		0 T
0 T            (0)
0 T            0.0
Positive controls S9-Mix (+)Name Dose Level
No. of Revertants		2AA2AA2AABP2AA
1 µg2 µg10 µg5 µg2 µg
1571        (1494)
1522         93.6
1390		241           (273)
260            40.1
318		138            (148)
155             9.1
152		121         (116)
101          13.6
127		165          (172)
170            7.6
180

BP Benzo(a)pyrene
2AA 2-Aminoanthracene
S Sparse bacterial background lawn
T Toxic, no bacterial background lawn
V Very weak bacterial background lawn
# Standard deviation


 


Table 5: Results of Experiment 2 - without metabolic activation (pre-incubation method)

























































































































Test PeriodFrom: 08 August 2019To: 11 August 2019
S9-Mix (-)Dose Level Per PlateNumber of revertants (mean) +/- SD
Base-pair substitution strainsFrameshift strains
TA100TA1535WP2uvrATA98TA1537
Solvent Control (DMSO)113          (113)
122          9.5#
103		11             (11)
9               2.0
13		23             (25)
23              4.0
30		26           (25)
17            8.0
33		16            (12)
10             3.5
10
0.015 µg113
110          (110)
106           3.5		13
8              (12)
16              4.0		35
17             (29)
35             10.4		19
17           (20)
25            4.2		12
6              (9)
9              3.0
0.05 µg129
119          (121)
115           7.2		12
11             (11)
10              1.0		29
23             (31)
41              9.2		27
31           (25)
17            7.2		4
7              (7)
11             3.5
0.15 µg100
123          (112)
113          11.5		11
6               (9)
11              2.9		38
24             (31)
30              7.0		25
29           (25)
20            4.5		11
6              (9)
11             2.9
0.5 µg108
98           (103)
102           5.0		5
15              (8)
5               5.8		23
23             (24)
26              1.7		13
13           (19)
32           11.0		18
20            (16)
11             4.7
1.5 µg114          (111)
109           2.5
111		10             (10)
13              2.5
8		30             (29)
31              3.2
25		25           (27)
16           12.1
40		12            (12)
9              3.5
16
5 µg111          (111)
101           9.5
120		14             (10)
8               3.5
8		24             (20)
21              4.0
16		20           (20)
21            1.0
19		14            (12)
11             1.7
11
15 µg86 S
86 S          (85)
83 S           1.7		11 S
9 S            (11)
13 S            2.0		31
25             (27)
26              3.2		15 S
33 S          (23)
21 S           9.2		7 S
9 S            (8)
8 S            1.0
50 µg89 S
70 S          (84)
93 S          12.3		10 S
6 S             (8)
9 S             2.1		15 S
29 S            (26)
35 S            10.3		0 V
0 V           (0)
0 V           0.0		0 V
0 V            (0)
0 V            0.0
Positive controls S9-Mix (-)Name Dose Level
No. of Revertants		ENNGENNGENNG4NQO9AA
3 µg5 µg2 µg0.2 µg80 µg
376          (344)
258          75.3
398		769          (1341)
2145          716.6
1110		517            (498)
499            19.5
478		156         (181)
186          23.4
202		211          (187)
201           33.3
149

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
S Sparse bacterial background lawn
V Very weak bacterial background lawn
# Standard deviation


 


Table 6: Results of Experiment 2 - with metabolic activation (pre-incubation method)

























































































































Test PeriodFrom: 08 August 2019To: 11 August 2019
S9-Mix (+)Dose Level Per PlateNumber of revertants (mean) +/- SD
Base-pair substitution strainsFrameshift strains
TA100TA1535WP2uvrATA98TA1537
Solvent Control (DMSO)100          (104)
101          6.7#
112		14             (13)
14              2.3
10		31             (41)
48              8.7
43		26           (27)
29            1.5
27		18            (14)
11             3.6
13
0.15 µg115
106          (104)
92           11.6		6
14             (10)
11              4.0		36
46             (42)
45              5.5		30
25           (25)
21            4.5		12
10            (11)
10             1.2
0.5 µg94
112          (101)
98            9.5		14
15             (13)
11              2.1		29
40             (39)
49             10.0		27
31           (26)
19            6.1		14
7             (10)
8              3.8
1.5 µg80
102          (105)
133          26.6		18
12             (16)
17              3.2		41
47             (48)
57              8.1		21
23           (22)
21            1.2		15
12            (13)
12             1.7
5 µg108
130          (117)
113          11.5		6
8               (9)
13              3.6		47
42             (46)
48              3.2		17
28           (27)
35            9.1		17
18            (18)
19             1.0
15 µg116          (118)
119           1.5
118		9               (7)
7               2.5
4		50             (45)
39              5.6
46		33           (29)
27            3.5
27		9             (12)
14             2.6
13
50 µg98           (110)
122          12.0
111		21             (13)
11              7.6
6		32             (39)
43              5.9
41		29           (25)
19            5.1
26		17            (12)
13             5.6
6
150 µg64 S
67 S          (66)
66 S           1.5		11
9              (13)
18              4.7		34
42             (37)
35              4.4		35
34           (31)
24            6.1		6 S
7 S            (7)
9 S            1.5
500 µg0 T
0 T            (0)
0 T            0.0		11 S
6 S             (7)
5 S             3.2		40 S
32 S            (35)
33 S             4.4		0 V
0 V           (0)
0 V           0.0		0 V
0 V            (0)
0 V            0.0
Positive controls S9-Mix (+)Name Dose Level
No. of Revertants		2AA2AA2AABP2AA
1 µg2 µg10 µg5 µg2 µg
1306        (1366)
1397         52.3
1396		202           (217)
226            13.3
224		110            (110)
110             0.0
110		154         (159)
151          11.4
172		131          (147)
145           17.6
166

BP Benzo(a)pyrene
2AA 2-Aminoanthracene
S Sparse bacterial background lawn
T Toxic, no bacterial background lawn
V Very weak bacterial background lawn
# Standard deviation

Conclusions:
Under the conditions of this study, the test item was considered to be non-mutagenic in the presence and absence of S9 activation.
Executive summary:

ST 08 C 19 was tested to evaluate its potential mutagenicity in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA-. The study was performed according to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing and under GLP conditions. 


Bacteria were exposed to the test item diluted in DMSO vehicle using the pre-incubation method in two independent experiments at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).
Due to excessive toxicity observed without metabolic activation in the experiment 1, the concentration selected for the first experiment were in the range of 0.015 to 50 µg/plate without metabolic activation and 1.5 to 5000 µg/plate with metabolic activation. The concentration range for Experiment 2 was amended following the results of Experiment 1 and was 0.015 to 50 µg/plate without metabolic activation and 0.15 to 500 μg/plate with metabolic activation. Eight test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve four non toxic dose levels and to achieve the toxic limit of the test item.


The vehicle (dimethyl sulphoxide) 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 or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. Cytotoxicity was observed in all tester strains with and without metbaolic activation in both experiments (visible reduction in the growth of the bacterial background lawns).


There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in both experiments.


It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31-03-2021 to 11-05-2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: ICH S2(R1)
Version / remarks:
Guideline adopted June 2012 (ICH S2(R1) Federal Register
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine or tryptophan locus
Species / strain / cell type:
E. coli WP2 uvr A
Remarks:
bacteria
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
bacteria
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: Rat liver S9 (10% liver S9 in standard co-factors)
- source of S9: Purchased from recognised supplier (dates within full study report); S9 Microsomal fractions: Lot No.’s 4370 (Experiment 1) and 4359 (Experiment 2)
- method of preparation of S9 mix: Documented in the full study report. The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
- concentration or volume of S9 mix and S9 in the final culture medium: 10% S9.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): A Certificate of S9 QC and Production certificate including Activity is presented in the full study report.
Test concentrations with justification for top dose:
Experiment 1 (pre-incubation method): 0; 1.5; 5; 15; 50; 150; 500; 1500 and 5000 µg/plate.
Experiment 1-repeat (pre-incubation method): 0; 0.005; 0.015; 0.05; 0.15; 0.5; 1.5; 5; 15; 50 and 150 µg/plate.
Experiment 2 (pre-incubation method): 0; 0.005; 0.015; 0.05; 0.15; 0.5; 1.5; 5; 15; 50 and 150 µg/plate.
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 g/plate) were originally assayed in triplicate against each tester strain. However, the test item induced excessive toxicity (resulting in an insufficient number of non-toxic dose concentrations) to four of the tester strains dosed in the absence of S9-mix and, therefore, part of the experiment was repeated using an amended dose range as follows:
TA100: 0.005, 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 µg/plate.
WP2uvrA and TA98: 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50 µg/plate.
TA1537: 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.


Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (dimethylsulphoxide)

- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in DMSO at the same concentration in solubility checks performed in house. DMSO was therefore selected as the vehicle.

- Other: Formulated concentrations were adjusted/increased to allow for the stated water/impurity content. See 'Test Material Information' for further details.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene (2AA)
Remarks:
With metabolic activation S9
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
Remarks:
Without metabolic activation S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1. in medium; in agar (pre-incubation) ; Experiment 2. in medium; in agar (pre-incubation).
The test item was suspected to be volatile, therefore all testing was performed using the pre-incubation method (20 minutes at 37 ± 3 °C) except for the untreated controls. For volatile substances, the pre-incubation method may increase exposure of the bacteria to the test item in comparison to the standard plate incorporation method.

The test item was accurately weighed and, on the day of each experiment, approximate
half-log dilutions prepared in high purity DMSO by mixing on a vortex mixer. Formulated concentrations were adjusted to allow for the stated water/impurity content (2.7%) of the test item. All test item preparation and dosing was performed under yellow safety lighting.

All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.


A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine (for S. typhimurium strains) or tryptophan (for E.coli strain) supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

DURATION:
Exposure duration:
Experiment 1 - All of the plates were pre-incubated in sealed, small volume containers, by application of 0.1 mL of the appropriate bacterial strain culture, 0.5 ml of phosphate buffer OR S9-mix (as appropriate) and 0.1 ml of the appropriate concentration of test item formulation, vehicle or 0.1 ml of appropriate positive control were incubated at 37 ± 3 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten trace amino-acid supplemented media. All of the plates were sealed in anaerobic jars or bags (one jar/bag for each concentration of test item/vehicle) during the incubation procedure (37 ± 3 ºC for between 48 and 72 hours) to minimize potential losses of the test item from the plates. After incubation, the plates were scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). A single manual count was performed due to revertant colonies spreading slightly which prevented an accurate automated count. Manual counts are considered to be equivalent to the machine counts.

Experiment 2 - As the result of Experiment 1 was considered negative, Experiment 2 was again performed using the pre-incubation method in the presence and absence of metabolic activation (S9 mix).

SELECTION AGENT (mutation assays): histidine-deficient agar

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:
In accordance with the OECD TG 471 guidelines.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537

A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments give clear positive or negative results, in some instances the data generated prohibit making a definite judgment about test item activity. Results of this type are reported as equivocal.
Statistics:
Mean & Standard deviation.
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See Tables 1 and 2
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:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See Tables 1 and 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See Tables 1 and 2
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:
See Tables 1 and 2
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:
See Tables 1 and 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The current Positive HCD dataset is presented in the full study report.
- Negative (solvent/vehicle) historical control data: The current background spontaneous revertant counts in concurrent untreated controls and/or or vehicle controls ; historic negative controls are presented in the full study report.

Table 1 : Test Results: Experiment 1 with and without metabolic activation and results of concurrent positive controls





















































































































































































































































































































































































































































































































































































































































































































































































































































S9-Mix
(-)		Dose LevelNumber of revertants (rounded mean) +/- SD
Per PlateBase-pair substitution strainsFrameshift strains
 TA100TA1535WP2uvrATA98TA1537
Solvent Control89-849-1117-1519-1918-15
(DMSO)909.0#102.1122.5234.5123.0
 74 13 15 14 15 
1.5 µg98-8113-1316-1720-1812-13
7614.7184.5191.7182.086.1
70 9 16 16 20 
5 µg58 S-6318-1622-2222-227-8
68 S5.0182.9202.5220.081.5
63 S 13 25 22 10 
15 µg65 S-6612-1212 S-1817 S-166-8
67 S1.0164.524 S6.014 S2.1123.2
66 S 7 19 S 18 S 7 
50 µg41 S-4317-109 S-1210 S-145 S-7
43 S1.586.210 S4.914 S4.012 S4.0
44 S 5 18 S 18 S 5 S 
150 µg0 T00 V00 V00 V00 V0
0 T0.00 V0.00 V0.00 V0.00 V0.0
0 T 0 V 0 V 0 V 0 V 
500 µg0 T00 T00 V00 T00 T0
0 T0.00 T0.00 V0.00 T0.00 T0.0
0 T 0 T 0 V 0 T 0 T 
1500 µg0 T00 T00 V00 T00 T0
0 T0.00 T0.00 V0.00 T0.00 T0.0
0 T 0 T 0 V 0 T 0 T 
5000 µg0 T00 T00 V00 T00 T0
0 T0.00 T0.00 V0.00 T0.00 T0.0
0 T 0 T 0 V 0 T 0 T 
Positive controlsNameENNGENNGENNG4NQO9AA
S9-Mix
(-)		Dose Level3 µg5 µg2 µg0.2 µg80 µg
No. of Revertants547-527592-1184548-483238-219131-145
53928.61575521.237792.420019.015012.7
494 1384 523 218 155 
S9-Mix
(+)		Dose LevelNumber of revertants (rounded mean) +/- SD
Per PlateBase-pair substitution strainsFrameshift strains
 TA100TA1535WP2uvrATA98TA1537
Solvent Control88-8711-1120-2030-2518-12
(DMSO)871.5#110.6172.5245.094.9
 85 12 22 20 10 
1.5 µg125-10610-922-2327-2215-16
9616.281.2317.1204.7192.6
98 10 17 18 14 
5 µg106-11110-821-1919-1811-14
1175.792.1192.0224.0153.1
109 6 17 14 17 
15 µg115-10016-1231-2326-2314-13
9914.093.6227.5276.7141.7
87 11 16 15 11 
50 µg96-1079-928-2721-2316-13
12012.1111.53710.5284.0103.0
106 8 16 21 13 
150 µg0 T07-718-2021 S-1917-12
0 T0.051.5254.023 S5.3114.2
0 T 8 18 13 S 9 
500 µg0 T09-915 S-2114 S-170 T0
0 T0.080.628 S6.717 S3.50 T0.0
0 T 9 19 S 21 S 0 T 
1500 µg0 T012 S-80 V00 V00 T0
0 T0.06 S3.50 V0.00 V0.00 T0.0
0 T 6 S 0 V 0 V 0 T 
5000 µg0 T00 T00 V00 T00 T0
0 T0.00 T0.00 V0.00 T0.00 T0.0
0 T 0 T 0 V 0 T 0 T 
Positive controlsName2AA2AA2AABP2AA
S9-Mix
(+)		Dose Level1 µg2 µg10 µg5 µg2 µg
No. of Revertants1932-1530259-244204-208113-128301-296
1194373.523513.32134.714818.031622.4
1463 237 206 123 272 

Table 1' - Test Results: Experiment 1 (repeat) without metabolic activation and results of concurrent positive controls




















































































































































































































































































































































































S9-Mix
(-)		Dose LevelNumber of revertants (rounded mean) +/- SD
Per PlateBase-pair substitution strainsFrameshift strains
 TA100WP2uvrATA98TA1537
Solvent Control80-9020-2221-236-12
(DMSO)11319.7#221.5222.1155.5
 78 23 25 16 
0.005 µg79-82N/TN/TN/T
842.5
82 
0.015 µg79-8726-2313-13N/T
947.6203.1141.0
89 22 12 
0.05 µg80-8211-1912-138-8
9612.7267.5121.780.6
71 19 15 7 
0.15 µg85-8925-2118-1416-8
884.6155.585.346.9
94 24 16 4 
0.5 µg98-10012-1813-166-10
928.6215.2172.366.4
109 21 17 17 
1.5 µg97-9314-1915-168-8
10010.2195.0106.661.5
81 24 23 9 
5 µg93 S-9217-2513-1510-12
94 S2.6238.6248.6112.1
89 S 34 7 14 
15 µg85 S-8025 S-2316 S-133-13
77 S4.226 S3.812 S2.6158.7
79 S 19 S 11 S 20 
50 µgN/T19 S-1613 S-169 S-11
15 S3.116 S3.55 S7.2
13 S 20 S 19 S 
150 µgN/TN/TN/T0 V0
0 V0.0
0 V 
Positive controlsNameENNGENNG4NQO9AA
S9-Mix
(-)		Dose Level3 µg2 µg0.2 µg80 µg
No. of Revertants466-484518-450191-204104-105
50017.042059.019717.68817.0
485 412 224 122 

The original dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate.  However, the test item induced excessive toxicity to four of the tester strains dosed in the absence of metabolic activation (S9-mix) and, therefore, part of the experiment was repeated (Table 1') using an amended dose range between 0.005 and 150 µg/plate, depending on tester strain type.  The dose range was amended, following the results of Experiment 1, and ranged between 0.005 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix.  


Table 2.- Test Results: Experiment 2 with and without metabolic activation and results of concurrent positive controls





























































































































































































































































































































































































































































































































































































































































































































































































































































































































































S9-Mix
(-)		Dose LevelNumber of revertants (rounded mean) +/- SD
Per PlateBase-pair substitution strainsFrameshift strains
 TA100TA1535WP2uvrATA98TA1537
Solvent Control134-12017-1718-2126-198-11
(DMSO)10415.0#106.5232.9186.1175.5
 121 23 23 14 7 
0.005 µg149-133N/TN/TN/TN/T
13115.1
119 
0.015 µg126-122N/T19-1411-14N/T
1193.6124.0114.6
121 12 19 
0.05 µg108-11523-1813-1613-186-9
1185.8144.7182.9204.0133.8
118 16 18 20 7 
0.15 µg117-10015-1720-1915-198-11
8815.3171.5162.6244.7123.1
94 18 21 17 14 
0.5 µg120-13312-1426-2127-2111-10
11131.2226.8146.1247.482.1
169 9 22 13 12 
1.5 µg137-11014-1629-2316-129-7
8825.0124.7157.1123.571.5
104 21 24 9 6 
5 µg112-10713-1329-198-207-9
1128.7121.0139.02710.491.5
97 14 14 25 10 
15 µg86 S-9913 S-1229-1910 S-136-6
90 S19.712 S0.6119.221 S7.050.6
122 S 12 S 17 8 S 6 
50 µgN/T14 S-1225 S-230 V00 V0
11 S1.719 S3.50 V0.00 V0.0
11 S 25 S 0 V 0 V 
150 µgN/T0 V0N/TN/T0 V0
0 V0.00 V0.0
0 V 0 V 
Positive controlsNameENNGENNGENNG4NQO9AA
S9-Mix
(-)		Dose Level3 µg5 µg2 µg0.2 µg80 µg
No. of Revertants679-7001451-1287347-280168-19977-77
65955.21080189.3355123.620230.1780.6
763 1331 137 228 77 
S9-Mix
(+)		Dose LevelNumber of revertants (rounded mean) +/- SD 
Per PlateBase-pair substitution strainsFrameshift strains 
 TA100TA1535WP2uvrATA98TA1537 
Solvent Control125-12012-1319-2320-198-8 
(DMSO)1205.5#151.5295.1145.062.5 
 114 13 22 24 11  
0.05 µg130-118N/TN/TN/TN/T 
12113.2 
104  
0.15 µg158-139N/TN/T25-2310-11 
12517.2212.1111.0 
133 22 12  
0.5 µg149-136N/T21-2332-2711-11 
13011.3232.0254.4110.0 
129 25 24 11  
1.5 µg131-12411-1223-2419-199-11 
12810.2131.2231.7162.592.9 
112 13 26 21 14  
5 µg113-11314-1431-3032-2716-13 
1174.0104.0245.6225.066.1 
109 18 35 26 17  
15 µg120-11311-1122-2919-1910-11 
1158.7101.5377.5153.5121.2 
103 13 28 22 12  
50 µg106-11112-1030-2536-247-9 
13118.0102.0254.51810.792.5 
96 8 21 17 12  
150 µg162 S-1208 S-621-2422 S-239 S-12 
105 S36.55 S2.1253.126 S2.615 S3.1 
94 S 4 S 27 21 S 11 S  
500 µgN/T0 V017 S-160 V00 T0 
0 V0.014 S1.70 V0.00 T0.0 
0 V 17 S 0 V 0 T  
1500 µgN/T0 T00 V0N/TN/T 
0 T0.00 V0.0 
0 T 0 V  
5000 µgN/T0 T0N/TN/TN/T 
0 T0.0 
0 T  
Positive controlsName2AA2AA2AABP2AA 
S9-Mix
(+)		Dose Level1 µg2 µg10 µg5 µg2 µg 
No. of Revertants1086-1220179-185214-241143-127211-197 
1121202.51827.920357.112615.518115.1 
1453 194 307 112 199  

ENNG          N-ethyl-N'-nitro-N-nitrosoguanidine


4NQO          4-Nitroquinoline-1-oxide


9AA            9-Aminoacridine


N/T             Not tested at this dose level


S                Sparse bacterial background lawn


V               Very weak bacterial background lawn


#           Standard deviation

Conclusions:
In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test ST 04 C 21 was considered to be non-mutagenic.
Executive summary:

Introduction:


The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MOE, the OECD Guidelines for Testing of Chemicals No. 471 “Bacterial Reverse Mutation Test”, 21 July 1997 as updated in 2020, Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, the ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749) and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.


Methods:


Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames pre-incubation method at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).  The original dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate.  However, the test item induced excessive toxicity to four of the tester strains dosed in the absence of metabolic activation (S9-mix) and, therefore, part of the experiment was repeated using an amended dose range between 0.005 and 150 µg/plate, depending on tester strain type.  The dose range was amended, following the results of Experiment 1, and ranged between 0.005 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix.  Eight test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non‑toxic dose levels as required by the test guideline and were selected based on the cytotoxicity noted in Experiment 1.  Both experiments were performed using the pre-incubation methodology because the volatility of the test item was considered to be incompatible with the plate incorporation method.  Each test was performed using fresh cultures of the bacterial strains and fresh test item formulations.


Results: 


The number of revertant counts for the solvent (dimethyl sulphoxide (DMSO)) control plates were 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 metabolic activation (S9-mix) were validated.


Depending on bacterial strain type and presence or absence of S9-mix, the maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate or the toxic limit.  In the first mutation test, the test item induced a visible reduction in the growth of the bacterial background lawn of all of the tester strains, initially from 5 and 150 µg/plate in the absence and presence of S9‑mix, respectively.


Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) or the toxic limit was employed in the second mutation test.  The test item once again induced a toxic response with a visible reduction in the growth of the bacterial background lawns noted to all of the tester strains, initially from 15 and 150 µg/plate in the absence and presence of S9‑mix, respectively.


No test item precipitate was observed on the plates at any of the doses tested either in the presence or in the absence of S9-mix in Experiments 1 and 2.


There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in Experiment 1. 


Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in Experiment 2. 


Under the conditions of this test, the test item was considered to be non-mutagenic.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22-07-2019 to 31-10-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: Whole blood cultures from Human male volunteer (non-smoking, 18-35y old). The volunteer has been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly
Cytokinesis block (if used):
Cytochalasin B at a final concentration of 4.5 %g/mL
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: MOLTOX from Trinova Biochem - from liver tissue of males Sprague-Dawley rats (5-6 weeks old), lot No 4061 expiry date 14 feb 2021
- method of preparation of S9 mix: The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM).
- concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The S9 was pre-tested for acceptability by the supplier prior to purchase and was supplied with a relevant “Quality Control & Production Certificate” which is presented in the annex of the study report.
Test concentrations with justification for top dose:
The selection of the maximum dose level for the Main Experiment (Mutation Test) was based on toxicity for all of the exposure groups in the presence and absence of S9.

test concentrations chosen for the main test:
- 4h without S9 mix: 0 to 64 µg/mL (0, 8, 16, 32, 40, 48, 56 and 64 μg/mL)
- 4h with S9 mix: 0 to 128 µg/mL (0, 16, 32, 64, 72, 80, 88, 96 and 128 μg/mL)
- 24h continuous exposure without S9 mix: 0 to 64 µg/mL (0, 8, 16, 32, 40, 48, 56 and 64 μg/mL)

The 24-hour continuous exposure group was repeated due to not meeting the maximum permissible dose level stated in the OECD 487 Test Guideline.
The dose range of test item used was 0, 16, 32, 48, 56, 64, 72, 96, 112 and 128 μg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The test item was insoluble in aqueous media at 20 mg/mL but was fully miscible in DMSO at 200 mg/mL in solubility checks performed in-house.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcine (DEME-C)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate for cultures treated with the test item, quadruplicate for the vehicle,
- Number of independent experiments: the results obtained in the 4h exposure without metabolic action were repeated with a second experiment in increasing the exposure time to 24h. Part of the main experiment was repeated on a separate day due not meeting the OECD Guideline requirements for determining the maximum dose level analyzed for micronuclei. Therefore, the 24-hour continuous exposure group was repeated where the maximum concentration should be limited by toxicity.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not applicable
- Test substance added in medium: 0.1 mL of the appropriate solution of vehicle control or test item was added to each culture.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: not applicable
- Exposure duration/duration of treatment: 4 or 24h
- Harvest time after the end of treatment (sampling/recovery times): after exposure with test item (or vehicle or positive control) cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:

- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure: cytochalasine B, used at 4.5 µg/mL, cells are treated at the end of the exposure period with the test item and during 24h

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry with gentle warming. Each slide was permanently labeled with the appropriate identification data. When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
The micronucleus frequency in 1000 binucleated cells was analyzed per culture (2000 binucleated cells per concentration for the test item and positive control and 4000 binucleated cells for the vehicle controls). Cells with 1, 2 or more micronuclei were recorded and included in the total.

- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not applicable

- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- cytokinesis-block proliferation index. A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI (Cytokinesis Block Proliferation Index) value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B.
Rationale for test conditions:
In accordance with OECD 487 Test guideline.
Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase when evaluated with an appropriate trend test.
3. The results in all evaluated dose groups are within the range of the laboratory historical control data.
The test system is then considered to be unable to induce chromosome breaks and/or gain or loss.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. The increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
Statistics:
The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei.
The dose-relationship (trend-test) was assessed using a linear regression model. An arcsin square-root transformation was applied to the percentage of binucleated cells containing micronuclei (excluding positive controls). A linear regression model was then applied to these transformed values with dose values fitted as the explanatory variable. The F-value from the model was assessed at the 5% statistical significance level.
Species / strain:
lymphocytes: From Human blood donor
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: There was no significant change in pH when the test item was dosed into media
- Data on osmolality: the osmolality did not increase by more than 50 mOsm.
- Precipitation and time of the determination: A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at 128 μg/mL in the presence of S9 only.
- Definition of acceptable cells for analysis: The qualitative assessment of the slides determined that the toxicity and precipitate was similar to that observed in the Cell Growth Inhibition Test and that there were binucleate cells suitable for scoring at the maximum dose level of test item in the absence of S9 only (64 μg/mL). In the presence of S9, the maximum dose level of the test item with binucleate cells suitable for scoring was 96 μg/mL.
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES (if applicable):
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 125 μg/mL and 250 μg/mL in the 4-hour exposure groups in the absence and presence of metabolic activation (S9), respectively and at and above 62.5 μg/mL in the 24-hour continuous exposure group.
Hemolysis was observed following exposure to the test item at and above 125 μg/mL in the 4-hour exposure groups and at and above 250 μg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 31.25 μg/mL in the exposure groups in the absence of S9 and up to 62.5 μg/mL in the presence of S9. The test item induced evidence of toxicity in all of the exposure groups.
The selection of the maximum dose level for the Main Experiment (Mutation Test) was based on toxicity for all of the exposure groups in the presence and absence of S9.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See tables.
For all test methods and criteria for data analysis and interpretation: See tables. Also see above “Evaluation” and “Statistics” fields.
- Concentration-response relationship where possible: See tables.
- Statistical analysis: See tables.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: See tables.
o In the case of the cytokinesis-block method: CBPI and/or ; distribution of mono-, bi- and multi-nucleated cells : see tables.
o Other observations when applicable (complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells): see tables.

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate: see tables


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- All the positive control items produced a statistically significant increase in the number of binucleated cells with micronuclei (statistically significant p ≤ 0.01 or were within the 95% Historic Control Data limit range presented in the full study report).
- Negative (solvent/vehicle) historical control data: All vehicle (DMSO) controls had frequencies of mono- and binucleated cells with micronuclei within the range expected for normal human lymphocytes. (Within the 95% Historic Control Data limit range presented in the full study report).

Table 1. CBPI Data - Preliminary Toxicity Test - 4-hour exposure without Metabolic Activation (S9)


































































































Treatment/ Concentration (µg/mL)Mononucleate CellsBinucleate CellsMultinucleate CellsCBPIcCytostasis (% )
Vehicle (DMSO)264219171.480
29319017
7.81263226111.500‡
15.63245240151.540‡
31.2531418061.3821
62.5NBNBNB--
125 P HNBNBNB--
250 P HNBNBNB--
500 P HNBNBNB--
1000 P HNBNBNB--
2000 P HNBNBNB--

c Mean value for vehicle
- Not selected for scoring
NB No binucleate cells or insufficient binucleate cells for scoring
P Precipitate observed at end of exposure period in blood-free cultures
H Hemolysis observed at the end of exposure in blood cultures
‡ Cytostasis reported as 0 as the CBPI value is equal to or higher than the solvent control
DMSO Dimethyl sulphoxide


 


Table 2: CBPI Data - Preliminary Toxicity Test - 4-hour exposure with Metabolic Activation (S9)


































































































Treatment / Concentration (µg/mL)Mononucleate CellsBinucleate CellsMultinucleate CellsCBPIcCytostasis (% )
Vehicle (DM SO)225227481.630
23523134
7.81211244451.670‡
15.63247219341.579
31.25205266291.650‡
62.525923831.4922
125 HNBNBNB--
250 P HNBNBNB--
500 P HNBNBNB--
1000 P HNBNBNB--
2000 P HNBNBNB--

c Mean value for vehicle
- Not selected for scoring
NB No binucleate cells or insufficient binucleate cells for scoring
P Precipitate observed at end of exposure period in blood-free cultures
H Hemolysis observed at the end of exposure in blood cultures
‡ Cytostasis reported as 0 as the CBPI value is equal to or higher than the solvent control
DMSO Dimethyl sulphoxide


Table 3: CBPI Data - Preliminary Toxicity Test - 24-hour exposure without Metabolic Activation (S9)


































































































Treatment / Concentration (µg/mL)Mononucleate CellsBinucleate CellsMultinucleate CellsCBPIcCytostasis (% )
Vehicle (DM SO)144274821.790
19426145
7.81175259661.781
15.63150270801.860‡
31.25162296421.764
62.5 PNBNBNB--
125 PNBNBNB--
250 P HNBNBNB--
500 P HNBNBNB--
1000 P HNBNBNB--
2000 P HNBNBNB--

c Mean value for vehicle
- Not selected for scoring
NB No binucleate cells or insufficient binucleate cells for scoring
P Precipitate observed at end of exposure period in blood-free cultures
H Hemolysis observed at the end of exposure in blood cultures
‡ Cytostasis reported as 0 as the CBPI value is equal to or higher than the solvent control
DMSO Dimethyl sulphoxide


Table 4: CBPI Data – Main Experiment - 4-Hour Exposure without Metabolic Activation (S9)






















































































































































































Treatment/ Concentration (µg/mL)ReplicateMononucleate CellsBinucleate CellsMultinucleate CellsCBPIMean CBPIMean Cytostasis (% )
Vehicle (DMSO)A1214249371.651.590
A2240234261.57
B1274187391.53
B2240224361.59
8A------
B----
16A254212341.561.557
B266204301.53
32A273206211.501.4818
B288195171.46
40A29120181.431.4425
B289196151.45
48A34615131.311.2951
B37112631.26
56A36413331.281.2557
B39011001.22
64AFBFBFB---
BFBFBFB-
MMC 0.2A296194101.431.4720
B25024821.50

- Not selected for scoring
FB Too few binucleate cells available for scoring
MMC Mitomycin C


DMSO Dimethyl sulphoxide


Table 5: CBPI Data – Main Experiment - 4-Hour Exposure with Metabolic Activation (S9)







































































































































































































Treatment/ Concentration (µg/mL)ReplicateMononucleate CellsBinucleate CellsMultinucleate CellsCBPIMean CBPIMean Cytostasis (% )
Vehicle (DMSO)A1243237241.571.570
A2205256411.67
B1257217261.54
B2288217221.50
16A------
B----
32A------
B----
64A------
B----
72A271219101.481.4325
B31618131.37
80A26623041.481.4423
B31118671.40
88A37312611.261.3244
B31318431.38
96A35814201.281.2655
B39011801.23
128 PANBNBNB---
BNBNBNB-
CP 5A34515701.311.3047
B35913921.29

- Not selected for scoring
P Precipitate observed at the end of exposure period in blood-free cultures
NB No binucleates cells available for scoring
CP Cyclophosphamide
DMSO Dimethyl sulphoxide


Table 6: CBPI Data – Main Experiment - 24-Hour Exposure without Metabolic Activation (S9)






















































































































































































Treatment/ Concentration (µg/mL)ReplicateMononucleate CellsBinucleate CellsMultinucleate CellsCBPIMean CBPIMean Cytostasis (% )
Vehicle (DMSO)A1196287171.641.720
A2142342161.75
B1171315141.69
B2123354231.80
8A------
B----
16A------
B----
32A------
B----
40A------
B----
48A201281181.631.686
B156328161.72
56A19530141.621.6510
B180310121.67
64A24425631.521.5228
B24725031.51
DC    0.075A33016731.351.3749
B31917291.38

- Not selected for scoring
DC Demecolcine
DMSO Dimethyl sulphoxide


Table 7: CBPI Data – Main Experiment Repeat - 24-Hour Exposure without Metabolic Activation (S9)
























































































































































































































Treatment/ Concentration (µg/mL)ReplicateMononucleate CellsBinucleate CellsMultinucleate CellsCBPIMean CBPIMean Cytostasis (% )
Vehicle (DMSO)A11652351001.871.800
A2220214661.69
B1160245951.87
B2193227801.77
16A------
B----
32A------
B----
48A201247521.701.6815
B223224531.66
56A234232341.601.6025
B235231341.60
64A277194291.501.4939
B278204181.48
72 HA33016821.341.3556
B32117631.36
96 HA4118811.181.1681
B4376301.13
112 HAFBFBFB---
BFBFBFB-
128 HANBNBNB---
BNBNBNB-
DC    0.075A322155231.401.4050
B334132341.40

- Not selected for scoring
H Haemolysis observed at the end of exposure period
FB Few binucleate cells available for scoring
NB No binucleate cells available for scoring
DC Demecolcine


DMSO Dimethyl sulphoxide


Table 8: Cytostasis and Micronucleus Data – Main Experiment - 4-Hour Exposure without Metabolic Activation (S9)






















































































Treatment/ Concentration (μg/mL)ReplicateMean Cytostasis (% )Binucleated cells containing micronuclei
%Meanp -valuebTrend test
p -valued
Vehicle (DMSO)A100.300.33-0.451
A20.40
B10.40
B20.20
40A250.000.35-
B0.70
48A510.200.25-
B0.30
56A570.000.25-
B0.50
MMC 0.2A203.003.107.49E-20 ***
B3.20

b p-values are for comparison with the control using Chi-square test
d Linear Regression value calculated from individual Arcsin values
MMC Mitomycin C
*** P<0.001
DMSO Dimethyl sulphoxide


Table 9: Cytostasis and Micronucleus Data – Main Experiment - 4-Hour Exposure with Metabolic Activation (S9)






















































































Treatment/ Concentration (μg/mL)Replicate%
Cytostasis		Binucleated cells containing micronuclei
%Meanp -valuebTrend test
p -valued
Vehicle (DMSO)A100.000.28-0.238
A20.60
B10.30
B20.20
80A230.700.650.0301*
B0.60
88A440.500.450.2676
B0.40
96A550.100.350.6165
B0.60
CP 5A472.902.653.12E-17 ***
B2.40

b p-values are for comparison with the control using Chi-square test
d Linear Regression value calculated from individual Arcsin values
CP Cyclophosphamide
* P<0.05
*** P<0.001


DMSO Dimethyl sulphoxide


 


Table 10: Cytostasis and Micronucleus Data – Main Experiment - 24-Hour Exposure without Metabolic Activation (S9)






















































































Treatment/ Concentration (μg/mL)ReplicateMean Cytostasis (% )Binucleated cells containing micronuclei
%Meanp -valuebTrend test
p -valued
Vehicle (DMSO)A100.400.43-0.563
A20.40
B10.40
B20.50
48A60.200.55-
B0.90
56A100.200.15-
B0.10
64 PA280.200.45-
B0.70
DC 0.075A493.904.401.77E-28 ***
B4.90

b p-values are for comparison with the control using Chi-square test
d Trend test p-values using Linear regression model applied to control and test item concentrations
P Precipitate
DC Demecolcine
DMSO Dimethyl sulphoxide
*** P<0.001


Table 11: Cytostasis and Micronucleus Data – Main Experiment Repeat- 24-Hour Exposure without Metabolic Activation (S9)























































































Treatment/ Concentration (μg/mL)ReplicateMean Cytostasis (% )Binucleated cells containing micronuclei
%Mean %p -valuebTrend test
p -valued
Vehicle (DMSO)A100.200.35-0.222
A20.20
B10.90
B20.10
56A250.200.650.1017
B1.10
64A390.600.600.1646
B0.60
72A560.200.35-
B0.50
DC 0.075A507.205.957.37E-44 ***-
B4.70

b p-values are for comparison with the control using Chi-square test
d Linear Regression value calculated from individual Arcsin values
DC Demecolcine
DMSO Dimethyl sulphoxide
*** P<0.001


Table 12: Summary of Results




















































































































































Exposure ConditionTreatment/ Concentration (μg/mL)Cytostasis (% )% Binucleated cells containing
micronucleia
Meanp -valuebTrend test
p -valued
4-hour -S9DMSO00.33-0.451
40250.35-
48510.25-
56570.25-
MMC 0.2203.107.49E-20***-
4-hour +S9DMSO00.28-0.238
80230.650.0301*
88440.450.2676
96550.350.6165
CP 5472.653.12E-17***-
24-hour -S9DMSO00.43-0.563
4860.46-
56100.15-
64280.45-
DC 0.075494.401.77E-28 ***-
24-hour -S9 RepeatDMSO00.35-0.222
56250.650.1017
64390.600.1646
72560.35-
DC 0.075505.957.37E-44***-

a The number of micronucleated cells determined in a sample of 2000 binucleate cells (4000 for vehicle)
b p-values are for comparison with the control using Chi-square test
d Linear Regression value calculated from individual Arcsin values
P Precipitate observed at end of exposure period in blood-free cultures
MMC Mitomycin C
CP Cyclophosphamide
DC Demecolcine
* P<0.05
*** P<0.001
DMSO Dimethyl sulphoxide

Conclusions:
Under the conditions of this study, the registered substance was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
Executive summary:

An in vitro micronucleus test was performed on test item ST 08 C 19 according to the requirements of OECD TG 487 under GLP conditions to assess the test item ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (1.8% S9-mix).


Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.
The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by toxicity for the short-term exposure groups and by precipitate in the case of the 24-hour continuous exposure group. The dose levels selected for the Main Experiment were as follows:


- 4-hour without S9: 0, 8, 16, 32, 40, 48, 56, 64 µg/mL


- 4-hour with S9 (2%): 0, 16, 32, 64, 72, 80, 88, 96, 128 µg/mL


- 24-hour without S9: 0, 8, 16, 32, 40, 48, 56, 64 µg/mL


Part of the main experiment was repeated on a separate day due not meeting the OECD Guideline requirements for determining the maximum dose level analyzed for micronuclei. Therefore, the 24-hour continuous exposure group was repeated where the maximum concentration should be limited by toxicity with the following dose range:


- 24-hour without S9: 0, 16, 32, 48, 56, 64, 72, 96, 112, 128 µg/mL


All vehicle (dimethyl sulphoxide (DMSO)) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.
The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.


The test item was toxic to human lymphocytes but did not induce any statistically significant and biologically relevant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that either induced approximately 50% reduction in CBPI or was the lowest precipitating dose level, depending on exposure group.


The test item, ST 08 C 19 was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro with and without metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

The registered substance is not classified for genotoxicity according to the criteria of Regulation (EC) 1272/2008 (CLP) and to the GHS.