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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471; Mutagenecity = negative; Thompson P., 2011.

OECD 473; Chromosomal abberations = negative; Bowles A, 2011

OECD 476; Mutagenecity = negative; Flanders L, 2011

Link to relevant study records

Referenceopen allclose all

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:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.
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)
Remarks:
UK GLP Monitoring authority
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and Tryptophan synthesis genes
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared from the livers of male rats treated with 80/100 mg Phenobarbitone/B-naphthoflavone
Test concentrations with justification for top dose:
Preliminary test: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate for Salmonella strains TA100, TA1535, TA98 (presence of S9) and E. Coli strain WP2uvrA- (Absence and presence of S9)

Definitive test: 5, 15, 50, 150, 500, 1500, 5000ug/plate for Salmonella strains TA100, TA1535, TA98 (absence of S9) and TA1537 (Absence and presence of S9)
Vehicle / solvent:
Sterile distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
2 ug/plate for WP2uvrA- without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
3 ug/plate for TA100 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
5 ug/plate for TA1535 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 ug/plate for TA1537 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 ug/plate for TA98 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Remarks:
2-Aminoanthracene 1ug/plate for TA100 with S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Remarks:
2-Aminoanthracene 2ug/plate for TA1535 and TA1537 with S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Remarks:
2-Aminoanthracene 10ug/plate for WP2uvrA- with S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5 ug/plate for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
no
Positive control substance:
other:
Remarks:
Water
Details on test system and experimental conditions:
Summary
Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay and ranged between 5 and 5000 μg/plate, depending on bacterial strain type and presence or absence of S9-mix. The experiment was repeated on a separate day (pre-incubation method) using a similar dose range to Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test material.

Tester strains
The following strains were used:

Salmonella typhimurium TA1535, TA1537, TA98 and TA100
Escherichia coli WP2uvrA

The four strains of Salmonella used in the test were obtained either from the University of California, Berkeley, on culture discs, on 04 August 1995 or from Syngenta CTL, Alderley Edge, as frozen vials, on 20 March 2007. E. coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately ¯196°C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate (5). In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 757012 03/14) and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates

Preparation of Test and Control Materials
The test material was fully miscible in sterile distilled water at 50 mg/ml in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle. The test material was accurately weighed and approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated impurity content (ethylene glycol at 38.5%) of the test material. Analysis for concentration, homogeneity and stability of the test material 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.

Vehicle and positive controls were used in parallel with the test material. A solvent treatment group was used as the vehicle control and the positive control materials used in the series of plates without S9-mix were as follows:

N-ethyl-N'-nitro-N-nitrosoguanidine (without S9) abbreviated to ENNG
9-Aminoacridine (without S9) abbreviated to 9AA
4-Nitroquinoline-1-oxide (without S9) abbreviated to 4NQO
2-Aminoanthracene (with S9) abbreviated to 2AA
Benzo(a)pyrene (with S9) abbreviated to BP

Preliminary Toxicity test
In order to select appropriate dose levels for use in the main test, a preliminary test wascarried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA-), 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of test material formulation and 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test material formulation and a vehicle control (sterile distilled water) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn

Mutation test - Experiment 1
Up to seven concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method. In the initial test, the toxicity of the test material yielded results that differed slightly from the preliminary toxicity test and consequently, there was an insufficient number of non-toxic dose levels for the Salmonella strains (predominantly in the absence of S9-mix). Therefore, part of the experiment was repeated using additional dose levels and expanded dose range. Dose ranges were allocated as follows:

Salmonella strains TA100, TA1535, TA98 (presence of S9-mix) and E.coli strain WP2uvrA- (absence and presence of S9-mix): 50, 150, 500, 1500, 5000 μg/plate.
Salmonella strains TA100, TA1535, TA98 (absence of S9-mix) and TA1537 (absence and presence of S9-mix): 5, 15, 50, 150, 500, 1500, 5000 μg/plate.

Additional dose levels and an expanded dose range were selected (where applicable) in order to achieve both four non-toxic dose levels and the toxiclimit of the test material.

Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.

All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter

Mutation test - Experiment 2
The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was amended slightly, following the results of Experiment 1, as follows:

Salmonella strains TA100, TA1535, TA98 (presence of S9-mix): 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
E.coli strain WP2uvrA- (absence and presence of S9-mix): 15, 50, 150, 500, 1500, 5000 μg/plate.
Salmonella strains TA100, TA1535, TA98 (absence of S9-mix) and TA1537 (absence and presence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 μg/plate

Additional dose levels and an expanded dose range were again selected in order to achieve both four non-toxic dose levels, the toxic limit of the test material and to account for the change in test methodology.

The test material formulations and vehicle control were dosed using the pre incubation method as follows: Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.1 ml of the vehicle or test material formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.

The positive and untreated controls were dosed using the standard plate incorporation method previously described..

All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter






Evaluation criteria:
The reverse mutation assay may be considered valid if the following criteria are met:

1. All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls within historic norms for the laboratory
2. The appropriate characteristics for each tester strain have been confirmed, eg rfa cellwall mutation and pKM101 plasmid R-factor etc.
3. All tester strain cultures should be in the range of 1 to 9.9 x 109 bacteria per ml.
4. Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9 mix.
4. There should be a minimum of four non-toxic test material dose levels.
5. There should be no evidence of excessive contamination.

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, statistical methods, 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 or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviation only
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
From 500 ug/plate absence of S9 and from 1500 ug/plate with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
From 500 ug/plate absence of S9 and from 1500 ug/plate with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
From 500 ug/plate absence of S9 and from 1500 ug/plate with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
From 500 ug/plate absence of S9 and from 1500 ug/plate with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Preliminary Toxicity test

S9 Strain Dose ug/plate
  0 0.15 0.5 1.5 5 15 50 150 500 1500 5000
- TA100 93 115 120 110 109 134 118 108 140 129 154
+ TA100 99 70 91 89 87 151 144 130 115 154 135
- WP2uvrA- 42 31 23 27 34 23 37 29 34 19 30
+ WP2uvrA- 42 51 48 41 40 44 49 43 44 36 23

Experiment 1

Experiment One
S9 Mix Concn
(ug/plate)		 Strain
TA100 TA1535 WP2uvrA TA98 TA1537
Count Mean (SD) Count Mean (SD) Count Mean (SD) Count Mean (SD) Count Mean (SD)
- 0 126 111
(13.1)		 11 14
(3.8)		 47 45
(3.8)		 20 20
(1.5)		 12 14
(4.4)
107 18 41 18 11
101 12 48 21 19
- 5 115 103
(10.7)		 18 17
(2.6)		 NT NT 27 22
(5.5)		 7 10
(3.6)
94 14 16 9
101 19 22 14
- 15 110 104
(7.8)		 10 12
(1.5)		 NT NT 19 20
(1.5)		 11 10
(1)
106 13 20 10
95 12 22 9
- 50 87 97
(21.5)		 11 10
(1.5)		 42 44
(2)		 21 21
(3.5)		 7 10
(4.6)
83 8 46 25 7
122 10 44 18 15
- 150 75 94
(16.8)		 12 14
(2.1)		 45 43
(4)		 19 24
(4.2)		 7 8
(1)
107 13 45 25 8
100 16 38 27 9
- 500 90 S 85
(5)		 5 S 7
(2.5)		 42 47
(4.6)		 17 S 17
(4.5)		 4 S 4
(2.5)
86 S 10 S 51 21 S 2 S
80 S 7 S 48 12 S 7 S
- 1500 54 S 47
(5.9)		 7 S 11
(3.6)		 40 42
(4)		 3 S 2
(1)		 1 V 2
(1.2)
45 S 14 S 47 1 S 3 V
43 S 12 S 40 2 S 1 V
- 5000 27 S 38
(9.9)		 6 V 7
(2.1)		 41 41
(3)		 4 S 4
(1)		 0 V 0
(0)
45 S 9 V 44 3 S 0 V
43 S 5 V 38 5 S 0 V
- Name ENNG ENNG ENNG 4NQO 9AA
Concn
(ug/plate)		 3 5 2 0.2 80
 Results 553 469
(73.9)		 198 190
(14.7)		 285 280
(9.5)		 152

154
(14.6)

 1154 1078
(66.1)
438 173 286 140 1038
415 199 269 169 1041

Where S = Sparse bacterial background lawn, V= Very sparse bacterial background lawn

Experiment One
S9 Mix Concn
(ug/plate)		 Strain
TA100 TA1535 WP2uvrA TA98 TA1537
Count Mean (SD) Count Mean (SD) Count Mean (SD) Count Mean (SD) Count Mean (SD)
+ 0 97 99
(7.6)		 9 13
(3.8)		 48 48
(3.5)		 29 32
(3.6)		 15 12
(3.1)
92 15 44 36 11
107 16 51 31 9
+ 5 NT NT NT NT NT NT NT NT 12 13
(3.1)
16
10
+ 15 NT NT NT NT NT NT NT NT 12 13
(4.6)
18
9
+ 50 108 95
(13)		 13 12
(1.2)		 35 42
(6.2)		 31 31
(4.5)		 12 11
(1.2)
96 11 47 27 10
82 13 44 36 10
+ 150 89 96
(6.5)		 13 13
(1.5)		 46

49
(2.5)

 31 30
(3.1)		 12 11
(3.2)
96 15 49 27 13
102 12 51 36 7
+ 500 93 86
(6.6)		 12 13
(1.2)		 59 47
(11.1)		 41 37
(4.6)		 9 11
(2.1)
85 14 37 38 13
80 12 45 32 12
+ 1500 55 67
(10.6)		 16 13
(2.5)		 43 42
(4.6)		 31 25
(5.3)		 3 S 3
(1)
75 11 37 23 4 S
71 13 46 21 2 S
+ 5000 40 S 40
(11.5)		 8 S 10
(3.8)		 48 48
(3)		 11 S 8
(4.2)		 3 S 3
(1.5)
52 S 7 S 51 9 S 1 S
29 S 14 S 45 3 S 4 S
- Name 2AA 2AA 2AA BP 2AA
Concn
(ug/plate)		 1 2 10 5 2
 Results 1656 1207
(431.7)		 236 236
(13)		 223 235
(13.9)		 229 238
(27.6)		 246 246
(9.5)
1169 223 250 216 236
795 249 231 269 255

Where S = Sparse bacterial background lawn

Experiment 2

Experiment Two
S9 Mix Concn
(ug/plate)		 Strain
TA100 TA1535 WP2uvrA TA98 TA1537
Count Mean (SD) Count Mean (SD) Count Mean (SD) Count Mean (SD) Count Mean (SD)
- 0 130 117
(11.6)		 19 17
(3.5)		 32 31
(2.1)		 18 21
(3.1)		 11 14
(3.8)
109 13 29 22 12
111 19 33 24 18
- 1.5 128 117
(13.6)		 19 18
(1.7)		 NT NT 18 18
(0.6)		 12 13
(2.6)
102 16 18 16
122 19 19 11
- 5 107 114
(7)		 21 15
(6)		 NT NT 20 21
(1)		 15

13
(2.5)
114 9 22 13
121 16 21 10
- 15 111 101
(8.5)		 14 17
(3.1)		 36 33
(5.2)		 21 21
(4.5)		 12 14
(1.5)
98 16 27 25 14
95 20 36 16 15
- 50 95 88
(5.9)		 16 18
(2.9)		 29 29
(2.5)		 20 21
(1)		 9 12
(2.6)
84 16 31 21 13
86 21 26 22 14
- 150 101 109
(7.2)		 18 17
(3.6)		 31 30
(2.3)		 20 19
(3.6)		 9 11
(2.5)
114 13 27 15 11
113 20 31 22 14
- 500 98 S 99
(0.6)		 16 15
(1.2)		 24 26
(1.7)		 20 16
(3.5)		 8 S 6
(1.7)
99 S 14 27 16 5 S
99 S 16 27 13 5 S
- 1500 47 S 55
(9.1)		 4 S 4
(0.6)		 23 26
(2.1)		 4 S

5
(1.2)

 2 S 3
(2.1)
54 S 5 S 27 6 S 5 S
65 S 4 S 29 4 S 1 S
- 5000 NT NT NT NT 29 33
(4)		 NT NT NT NT
37
32
- Name ENNG ENNG ENNG 4NQO 9AA
Concn
(ug/plate)		 3 5 2 0.2 80
 Results 503 497
(13.7)		 64 162
(85.3)		 262 283
(22.6)		 114 116
(5.3)		 2297 2530
(315.3)
481 216 280 122 3889
506 207 307 112 2405

Where S = Sparse bacterial background lawn

Experiment Two
S9 Mix Concn
(ug/plate)		 Strain
TA100 TA1535 WP2uvrA TA98 TA1537
Count Mean (SD) Count Mean (SD) Count Mean (SD) Count Mean (SD) Count Mean (SD)
+ 0 117 109
(6.8)		 18 18
(0.6)		 36 36
(4)		 26 27
(1.7)		 13 12
(2.1)
107 19 32 26 10
104 18 40 29 14
+ 1.5 NT NT NT NT NT NT NT NT 14 14
(0.6)
14
15
+ 5 114 102
(12)		 18 16
(2.1)		 NT NT 31 27
(3.6)		 12 12
(0.6)
90 15 24 12
102 14 26 13
+ 15 118 112
(5.1)		 15 14
(1.2)		 38 39
(2.1)		 28 25
(3.5)		 11 12
(2.6)
111 13 37 21 10
108 15 41 25 15
+ 50 103 112
(10.7)		 21 19
(1.5)		 23 34
(10.2)		 25 25
(2)		 11 12
(1.5)
124 19 36 27 14
110 18 43 23 12
+ 150 125 112
(11.4)		 21 18
(3.1)		 38 32
(5.6)		 30 27
(3.5)		 12 12
(1.5)
107 15 31 23 11
104 19 27 27 14
+ 500 99 112
(12.1)		 14 13
(2.6)		 37 35
(3.2)		 23 25
(4.4)		 14 13
(1.2)
123 15 31 22 12
113 10 36 30 12
+ 1500 97 S 108
(9.8)		 14 18
(4)		 42 36
(5.5)		 14 15
(2.1)		 5 S 8
(3)
114 S 18 33 17 8 S
114 S 22 32 13 11 S
+ 5000 57 S 54
(3)		 13 S 12
(0.6)		 31 32
(1.5)		 4 S 4
(1.5)		 NT NT
54 S 12 S 32 6 S
51 S 12 S 34 3 S
+ Name 2AA 2AA 2AA BP 2AA
Concn
(ug/plate)		 1 2 10 5 2
 Results 629 801
(157.7)		 288 292
(15.4)		 143 130
(11.9)		 180 189
(18.8)		 100 95
(5.5)
939 309 120 177 89
834 279 126 211 95

Where S = Sparse bacterial background lawn

Note History profile for vehichle (Distilled water) and positive controls for 2008 and 2009 are available and indicate that the results reported here for these controls are within historic norms for the laboratory.

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

The test material (PR-4758) was considered non-mutagenic under the conditions of the assay. The sensitivity of the assay and the efficacy of the S9-mix were validated as the vehicle (sterile distilled water) 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.
Executive summary:

At the request of Nalco Limited UK, Harlan laboratories performed a Bacterial reverse mutation assay on PR-4758. The study was conducted in accordance with Good laboratory practise.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 MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonized guidelines.


 


Salmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay and ranged between 5 and 5000 μg/plate, depending on bacterial strain type and presence or absence of S9-mix.


 


The experiment was repeated on a separate day (pre-incubation method) using a similar dose range to Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test material.


 


The vehicle (sterile distilled water) 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. The test material caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains, initially from 500 and 1500 μg/plate, in the absence and presence of S9-mix, respectively. No toxicity was noted for Escherichia coli strain WP2uvrA- at any test material dose level. The sensitivity of the tester strains to the toxicity of the test material varied slightly between strain type, exposures with or without S9-mix and experiment number. The test material was, therefore, either tested up to the maximum recommended dose level of 5000 μg/plate or the toxic limit, depending on bacterial strain type and experiment number. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. PR-4758 was considered non-mutagenic under the conditions of the assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Monitoring authority
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: Human, peripheral blood
Details on mammalian cell type (if applicable):
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started. Volunteers were screened to ensure had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.

Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal calf serum (FBS), at 37ºC with 5% CO2 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared from livers of male rats induced with three consecutive daily doses of phenobarbitone/beta-naphthoflavone (80/100 mg.kg per day).
Test concentrations with justification for top dose:
Experiment 1
With S9-mix, 4hr exposure; 20 hr culture: 0, 80, 160, 320, 640, 960 and 1280 µg/mL
Without S9-mix, 4hr exposure; 20 hr culture: 0, 40, 80, 160, 320 and 480 µg/mL
Experiment 2
With S9-mix, 4hr exposure; 20 hr culture: 0, 80, 160, 320, 480, 640 and 900 µg/mL
Without S9-mix, 24hr exposureand culture: 0, 40, 80, 160, 320, 480 and 640 µg/mL
Vehicle / solvent:
Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and
10% foetal calf serum (FBS)
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9 Migrated to IUCLID6: 0.4 and 0.2 ug/l in Minimal essential medium for experiment 1 and 2 respectively
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Minimal essential medium
True negative controls:
no
Positive controls:
no
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9 Migrated to IUCLID6: 5 ug/l in DMSO for experiment 1 and 2
Details on test system and experimental conditions:
Preparation of Test and Control Materials
The test material was accurately weighed, dissolved in MEM and serial dilutions prepared. The test material was considered to be a complex mixture, therefore the maximum dose level was 5000 μg/ml, the maximum recommended dose level. The purity of the test material was 61.5% and was accounted for in the formulations.

The test material was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation because it is not a requirement of the guidelines.

Culture Conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:

8.05-9.05 ml MEM, 10% (FBS)
0.1 ml Li-heparin
0.1 ml phytohaemagglutinin
0.75 ml heparinised whole blood

With Metabolic Activation (S9) Treatment
In Preliminary Toxicity Test and of Experiment 1, after approximately 48 hours incubation at 37ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 ml of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 ml of the appropriate solution of vehicle control or test material was added to each culture. For the positive control, 0.1 ml of the appropriate solution was added to the cultures. 1 ml of 20% S9¯mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures.

In Experiment 2, 1 ml of 10% S9-mix (i.e. 1% final concentration of S9 in standard co-factors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 ml. After 4 hours at 37ºC, 5% CO2 in humidified air , the cultures were centrifuged, the treatment medium 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 original culture medium. The cells were then re-incubated for a further 20 hours at 37ºC in 5% CO2 in humidified air.

Without Metabolic Activation (S9) Treatment
In Experiment 1, after approximately 48 hours incubation at 37ºC with 5% CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 ml of the culture medium was removed and reserved. The cells were then re-suspended in the required volume of fresh MEM (including serum) and dosed with 1.0 ml of the appropriate vehicle control, test material solution or 0.1 ml of positive control solution. The total volume for each culture was a nominal 10 ml. After 4 hours at 37ºC, 5% CO2 in humidified air, the 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. The cells were then returned to the incubator for a further 20 hours.

In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.0 ml. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 ml of vehicle control, test material dose solution or 0.1 ml of positive control solution. The nominal final volume of each culture was 10 ml. The cultures were then incubated at 37ºC, 5% CO2 in humidified air, for 24 hours.

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/ml) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4ºC for at least four hours to ensure complete fixation.

Preparation of Metaphase Spreads
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. Each slide was permanently labelled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Assessment

The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation if any of the test material. These observations were used to select the dose levels for the mitotic index evaluation.

Mitotic index
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in
metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. The classification and evaluation criteria are outlined in Appendix 1.

In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%)reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant increase in the number of cells with chromosome aberrations.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Fisher’s Exact test
Species / strain:
lymphocytes: Human, peripheral blood
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Osmolarity & pH

Ug/ml 0 19.53 39.06 78.13 156.25 312.5 625 1250 2500 5000
pH 7.33 7.34 7.36 7.39 7.40 7.39 7.41 7.39 7.38 7.33
mOsm 283 280 279 282 282 276 286 292 309 338

Mitotic Index - Preliminary Toxicity Test

Concentration
(ug/ml)		 4(20)h without S9 4(20)h with S9 24 hr without S9
Mitotic index % Control Mitotic index % Control Mitotic index % Control
0 7.45 100 6.60 100 5.65 100
19.53 - - - - 5.60 99
39.06 9.05 121 - - 6.90 122
78.13 6.80 91 6.90 105 8.35 148
156.25 4.45 H 60 4.65 70 5.80 103
312.5 3.35 H 45 3.60 55 2.70 H 48
625 1.35 H 18 3.20 H 48 NM H -
1250  H - - 2.65 H 40 NM H -
2500 NM H - NM H - NM H -
5000 NM H - NM H - NM H -

Where

- = Not assessed for mitotic index

NM = No metaphases suitable for scoring

H = Haemolysis

Mitotic Index - Experiment 1

Dose level
(ug/ml		 4 hour treatment without S9
A B Mean % of control
0 6.15 6.20 6.18 100
40 - - - -
80 4.05 4.70 4.38 71
160 4.85 2.20 3.53 57
240 4.30 2.85 3.58 58
320 2.65 2.40 2.53 41
480 NM NM NM 0
MMC 0.4 1.80 1.70 1.75 28

Where

MMC = Mitomycin C

Dose level
(ug/ml		 4 hour treatment with S9
A B Mean % of control
0 5.90 5.00 5.45 100
80 - - - -
160 4.25 3.25 3.75 69
240 2.80 3.30 3.05 56
640 2.75 1.85 2.30 42
960 2.60 0.70 1.65 30
1280 NM NM NM 0
CP 5 0.09 0.85 0.88 16

Where CP = Cyclophosphamide

Mitotic Index - Experiment 2

Dose level
(ug/ml		 24 hour treatment without S9
A B Mean % of control
0 10.35 8.80 9.58 100
40 10.40 8.10 9.25 97
80 5.60 8.85 7.23 75
160 8.20 7.25 7.73 81
320 2.60 2.40 2.50 26
480 0.95 0.40 0.68 7
640 NM NM NM 0
MMC 0.2 2.30 3.15 2.73 28

Dose level
(ug/ml		 4 hour treatment with S9
A B Mean % of control
0 9.45 8.30 8.88 100
80 8.15 8.80 8.48 95
160 6.95 3.35 5.15 58
320 4.60 5.80 5.20 59
480 5.55 5.00 5.28 59
640 4.05 4.05 4.05 46
800 - - - -
CP 5 2.95 2.70 2.83 32

MMC = Mitomycin C

CP = Cyclophosphamide

NA = Not applicable

- = Not assessed for mitotic index

NM = No metaphases suitable for scoring

Results of Chromosome Aberration Test - Experiment 1 With and Without Metabolic Activation (S9)

Treatment period
(hr)		 S9 mix Concentration
(ug/ml)		   Number and percent of cells showing structural chromosome Aberrations (%) g Cell growth index Number and percentage of cells showing numerical aberrations (%)
Observed ctb cte csb cse Others Total Mitotic index (%) Observed Polyploids Others Total
4 - Negative control (MEM)
0		 A 100 0 0 0 0 0 0 0 6.15 100 0 0 0
B 100 0 0 0 0 0 0 1 6.20 100 0 0 0
Total 200 0 0 0 0 0 0 1 - 200 0 0 0
% 100 0 0 0 0 0 0 0.5 100 - - - 0
- 80 A 100 0 0 0 0 0 0 2 4.05 100 0 0 0
B 100 2 0 1 0 0 2 0 4.70 100 0 0 0
Total 200 2 0 1 0 0 2 2 - 200 0 0 0
% 100 1 0 0.5 0 0 1 1 71 - - - 0
- 160 A 100 0 0 0 0 0 0 2 4.85 100 0 0 0
B 100 0 0 0 0 0 0 1 2.20 100 0 0 0
Total 200 0 0 0 0 0 0 3 - 200 0 0 0
% 100 0 0 0 0 0 0 1.5 57 - - - 0
- 240 A 100 1 0 0 0 0 1 1 4.30 100 0 0 0
B 100 1 0 0 0 0 1 0 2.85 100 0 0 0
Total 200 2 0 0 0 0 2 1 - 200 0 0 0
% 100 1 0 0 0 0 1 0.5 58 - - - 0
- 320 A 100 0 0 0 0 0 0 3 2.65 100 0 0 0
B 100 1 0 2 0 0 3 3 2.40 101 1 0 0
Total 200 1 0 2 0 0 3 6 - 201 1 0 1
% 100 0.5 0 1 0 0 1.5 3 41 - - - 0.5
- Positive control (MMC)
0.4		 A 50 a 16 19 8 0 0 30 10 1.80 50 0 0 0
B 50 a 20 16 7 0 1 31 11 1.70 50 0 0 0
Total 100 36 35 15 0 1 60
P <0.001		 21 - 100 0 0 0
% 100 36 35 15 0 1 60 21 28 - - - 0

Where

MEM = Eagles minimal essential medium with HEPES Buffer

MMC = Mitomycin C

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed

Results of Chromosome aberration test - Experiment 1 with metabolic activation

Treatment period
(hr)		 S9 mix Concentration
(ug/ml)		   Number and percent of cells showing structural chromosome Aberrations (%) g Cell growth index Number and percentage of cells showing numerical aberrations (%)
Observed ctb cte csb cse Others Total Mitotic index (%) Observed Polyploids Others Total
4 + Negative control (MEM)
0		 A 100 0 0 0 0 0 0 2 5.90 100 0 0 0
B 100 0 0 0 0 0 0 2 5.00 100 0 0 0
Total 200 0 0 0 0 0 0 4 - 200 0 0 0
% 100 0 0 0 0 0 0 2 100 - - - 0
+ 160 A 100 2 0 1 0 0 3 0 4.25 100 0 0 0
B 100 1 0 0 0 0 1 0 3.25 100 0 0 0
Total 200 3 0 1 0 0 4 0 - 200 0 0 0
% 100 1.5 0 0.5 0 0 2 0 69 - - - 0
+ 320 A 100 1 0 0 0 0 1 0 2.80 100 0 0 0
B 100 0 0 0 0 0 0 1 3.30 100 0 0 0
Total 200 1 0 0 0 0 1 1 - 200 0 0 0
% 100 0.5 0 0 0 0 0.5 0.5 56 - - - 0
+ 640 A 100 1 0 0 0 0 1 3 2.75 100 0 0 0
B 100 0 0 0 0 0 0 0 1.85 100 0 0 0
Total 200 1 0 0 0 0 1 3 - 200 0 0 0
% 100 0.5 0 0 0 0 0.5 1.5 42 - - - 0
+ Positive control (CP)
5		 A 50 a 19 8 2 0 0 24 8 0.90 50 0 0 0
B 50 a 16 6 3 0 1 23 5 0.85 50 0 0 0
Total 100 35 14 5 0 1 47
p <0.001		 13 - 100 0 0 0
% 100 35 14 5 0 1 47 13 16 - - - 0

Where CP = Cyclophosphamide

Result of Chromosome Aberration test - Experiment 2 without metabolic activation

Treatment period
(hr)		 S9 mix Concentration
(ug/ml)		   Number and percent of cells showing structural chromosome Aberrations (%) g Cell growth index Number and percentage of cells showing numerical aberrations (%)
Observed ctb cte csb cse Others Total Mitotic index (%) Observed Polyploids Others Total
24 - Negative control (MEM)
0		 A 100 0 0 0 0 0 0 0 10.35 100 0 0 0
B 100 0 0 1 0 0 1 2 8.80 100 0 0 0
Total 200 0 0 1 0 0 1 2 - 200 0 0 0
% 100 0 0 0.5 0 0 0.5 1 100 - - - 0
- 40 A 100 0 0 0 0 0 0 3 10.40 100 0 0 0
B 100 2 0 1 0 0 3 1 8.10 100 0 0 0
Total 200 2 0 1 0 0 3 4 - 200 0 0 0
% 100 1 0 0.5 0 0 1.5 2 97 - - - 0
- 80 A 100 1 0 0 0 1 2 3 5.60 100 0 0 0
B 100 0 0 1 0 0 1 1 8.85 100 0 0 0
Total 200 1 0 1 0 1 3 4 - 200 0 0 0
% 100 0.5 0 0.5 0 0.5 1.5 2 75 - - - 0
- 160 A 100 2 0 1 0 0 2 2 8.20 100 0 0 0
B 100 0 0 0 0 0 0 3 7.25 100 0 0 0
Total 200 2 0 1 0 0 2 5 - 200 0 0 0
% 100 1 0 0.5 0 0 1 2.5 81 - - - 0
- 320 A 100 1 0 0 0 0 1 1 2.60 100 0 0 0
B 100 2 0 0 0 0 2 1 2.40 100 0 0 0
Total 200 3 0 0 0 0 3 2 - 200 0 0 0
% 100 1.5 0 0 0 0 1.5 1 26 - - - 0
- Positive control (MMC)
0.2		 A 50 a 13 18 2 0 0 25 5 2.30 50 0 0 0
B 50 a 19 16 3 0 0 29 0 3.15 50 0 0 0
Total 100 32 34 5 0 0 54
p <0.001		 5 - 100 0 0 0
% 100 32 34 5 0 0 54 5 28 - - - 0

Results of Chromosome Aberration test - Experiment 2 with Metabolic activation

Treatment period
(hr)		 S9 mix Concentration
(ug/ml)		   Number and percent of cells showing structural chromosome Aberrations (%) g Cell growth index Number and percentage of cells showing numerical aberrations (%)
Observed ctb cte csb cse Others Total Mitotic index (%) Observed Polyploids Others Total
4 + Negative control (MEM)
0		 A 100 0 0 1 0 0 1 0 9.45 100 0 0 0
B 100 2 0 0 0 0 2 0 8.30 100 0 0 0
Total 200 2 0 1 0 0 3 0 - 200 0 0 0
% 100 1 0 0.5 0 0 1.5 0 100 - - - 0
+ 80 A 100 0 0 0 0 0 0 2 8.15 100 0 0 0
B 100 2 0 1 1 0 3 0 8.80 100 0 0 0
Total 200 2 0 1 1 0 3 2 - 200 0 0 0
% 100 1 0 0.5 0.5 0 1.5 1 95 - - - 0
+ 160 A 100 0 0 0 0 0 0 0 6.95 100 0 0 0
B 100 0 0 0 0 0 0 1 3.35 100 0 0 0
Total 200 0 0 0 0 0 0 1 - 200 0 0 0
% 100 0 0 0 0 0 0 0.5 58 - - - 0
+ 320 A 100 0 0 0 0 0 0 0 4.60 100 0 0 0
B 100 0 0 1 0 0 1 0 5.80 101 1 0 1
Total 200 0 0 1 0 0 1 0 - 201 1 0 1
% 100 0 0 0.5 0 0 0.5 0 59 - - - 0.5
+ 640* B 100 0 0 1 0 0 1 1 4.05 105 5 0 5
B 100 0 1 0 0 0 1 0 4.05 100 0 0 0
Total 200 0 1 1 0 0 2 1 - 200 0 0 5
% 100 0 0.5 0.5 0 0 1 0.5 46 - - - 2.4
+ Positive control (CP)
5		 A 50 a 18 8 3 0 0 23 6 2.95 50 0 0 0
B 50 a 11 4 4 1 0 15 6 2.70 50 0 0 0
Total 100 29 12 7 1 0 38
p <0.001		 12 - 100 0 0 0
% 100 29 12 7 1 0 38 12 32 - - - 0

Where

* = Due to the loss of the culture A, 200 cells analysed from culture B

Conclusions:
Interpretation of results:
negative with metabolic activation
negative with metabolic activation

The test material, PR-4758, did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

The study was conducted according to a method that was designed to assess the potential of the test material to cause structural aberration in cultured mammalian cells. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.473 "Genetic Toxicology: Chromosome aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Two main experiments were performed. In these experiments, duplicate cultures of human lymphocytes obtained from health volunteers were treated with the test material at up to four dose levels, together with vehicle (MEM medium) and positive controls. Four treatment groups were used in the study. In experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20 hour expression period and a 4 hour exposure in the absence of metabolic activation (S9) with a 20 hour expression period. In experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration); whilst in the absence of metabolic activation the exposure period was increased to 24 hours.

 

A range of doses were used in experiments 1 and 2

 

Experiment 1 - 4(20) hr without S9 - 40, 80,160, 240, 320, 480 ug/ml

Experiment 1 – 4(20) hr with S9 – 80, 160, 320, 640, 960, 1280 ug/ml

Experiment 2 – 24hr without S9 – 40, 80, 160, 320, 480, 640 ug/ml

Experiment 2 – 4(20) hr with S9 – 80, 160, 320, 480, 640, 800 ug/ml

 

All vehicles (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

 

All the positive control material induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and the activity of the metabolising system.

 

The test material was toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced 50% mitotic inhibition.

 

The test material was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 November 2010 and 22 November 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity crtieria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Remarks:
GLP Statement
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Type and identity of media:
RPMI 1640 (R0)

Properly maintained:
Yes

Periodically checked for Mycoplasma contamination:
Yes

Periodically checked for karyotype stability:
No

Periodically "cleansed" against high spontaneous background:
Yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
The maximum dose level used was limited by test material induced toxicity.

Vehicle and positive controls were used in parallel with the test material. Solvent (R0 medium) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch 0001423147 at 400 µg/ml and 150 µg/ml for the 4-hour and 24-hour exposures, respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0277203 at 2 µg/ml was used as the positive control in the presence of metabolic activation.
Vehicle / solvent:
Vehicle used:
Vehicle (R0 medium) treatment groups were used as the vehicle controls.


Justification for choice of vehicle:
Formed a solution suitable for dosing at the required concentration.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle (R0 medium) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle (R0 medium) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
This study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

The use of cultured mammalian cells for mutation studies may give a measure of the intrinsic response of the mammalian genome and its maintenance process to mutagens. Such techniques have been used for many years with widely different cell types and loci. The thymidine kinase heterozygote system, TK +/- to TK -/-, was described by Clive et al., (1972) and is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). This system has been extensively validated (Clive et al, 1979; Amacher et al, 1980; Jotz and Mitchell, 1981).

The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Test", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008 and the United Kingdom Environmental Mutagen Society (Cole et al, 1990). The technique used was a fluctuation assay using microtitre plates and trifluorothymidine as the selective agent and is based on that described by Cole and Arlett (1984). Two distinct types of mutant colonies can be recognised, i.e. large and small. Large colonies grow at a normal rate and represent events within the gene (base-pair substitutions or deletions) whilst small colonies represent large genetic changes involving chromosome 11b (indicative of clastogenic activity).
Evaluation criteria:
Please see "Any other information on materials and methods incl. tables" section.
Statistics:
Please see "Any other information on materials and methods incl. tables" section.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:

Preliminary Toxicity Test

The dose range of the test material used in the preliminary toxicity test was 7.81 to 2000 µg/ml.

In all three of the exposure groups there were marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle control groups. The toxicity curve of the test material was very steep in all three of the exposure groups. Precipitate of the test material was not observed at any of the dose levels. In the subsequent mutagenicity test the maximum dose was limited by test material-induced toxicity.

Mutagenicity Test

A summary of the results from the test is presented in attached Table 1.

4-Hour Exposure With and Without Metabolic Activation

The results of the microtitre plate counts and their analysis are presented in attached Tables 2 to 7.

There was evidence of marked test material-induced toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values (Tables 3 and 6). There was evidence of a reduction in viability (%V) in the absence of metabolic activation, therefore indicating that residual toxicity had occurred in this exposure group (Table 3). However, it should be noted that the reduction was only observed at the one dose level that had been excluded from statistical analysis due to excessive toxicity. Optimum levels of toxicity were not achieved in either the absence or presence of metabolic activation (Tables 3 and 6). Whilst optimum levels of toxicity were not achieved in the absence or presence of metabolic activation due to the very steep toxicity curve of the test material, despite using a comparatively narrow dose interval, a dose level that exceeded the usual upper limit of acceptable toxicity in the absence of metabolic activation was plated for viability and 5 TFT resistance as sufficient cells were available at the time of plating. Although this dose level had been excluded from the statistical analysis, there was no evidence of an increase in mutant frequency at this dose level and the mutant frequency value observed was within the acceptable range for vehicle controls. Therefore, with no evidence of any toxicologically significant increases in mutant frequency at any of the dose levels, including dose levels where optimum levels of toxicity were approached in both the absence and presence of metabolic activation, or exceeded in the absence of metabolic activation, or in the 24-hour exposure group in the absence of metabolic activation where optimum levels of toxicity were achieved, the test material was considered to have been adequately tested. The excessive levels of toxicity observed at 225 µg/ml in the absence of metabolic activation, and at 500 µg/ml in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test material did not induce any statistically significant or dose-related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). Precipitate of test material was not observed at any of the dose levels.

The numbers of small and large colonies and their analysis are presented in attached Tables 4 and 7.

24-Hour Exposure Without Metabolic Activation

The results of the microtitre plate counts and their analysis are presented in attached Tables 8 to 10.

As was seen previously, there was evidence of marked test material-induced toxicity following exposure to the test material as indicated by the %RSG and RTG values (Table 9). There was evidence of reductions in viability (%V) when compared to the vehicle control group, therefore indicating that possible residual toxicity may have occurred in this exposure group (Table 9). However, it should be noted that the viability (%V) value of the vehicle control group was elevated, possibly due to a suspected dilution error that resulted in slightly higher viability plate counts than those usually observed. However, the mutant frequency value observed in the vehicle control group was within the acceptable range and with no evidence of any toxicologically significant increases in mutant frequency at any of the dose levels, including a dose level that induced optimum levels of toxicity on this occasion, the viability value was considered acceptable for the purpose of this study. The toxicity observed at 250 µg/ml exceeded the upper acceptable limit of 90%, therefore, this dose level was excluded from the statistical analysis. The positive control induced acceptable levels of toxicity (Table 9).

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a modest effect on the toxicity of the test material.

The vehicle control mutant frequency value was within the acceptable range of 50 to 200 x 10-6 viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily (Table 9).

The test material did not induce any statistically significant or dose-related (linear-trend) increases in the mutant frequency x 10-6 per viable cell (Table 9). Precipitate of test material was not observed at any of the dose levels.

The numbers of small and large colonies and their analysis are presented in attached Table 10.

DISCUSSION

Due to the nature of the toxicity exhibited by the test material it was considered to achieve optimum toxicity in all three exposure groups would be very difficult. In the absence of metabolic activation optimum toxicity was not achieved after 4 hours exposure, although a dose level with high toxicity was plated out for mutant frequency. However, optimum levels of toxicity were achieved after 24 hours exposure using a very similar dose range. In the presence of metabolic activation optimum levels of toxicity were approached. In all three of the exposure groups there was no evidence of a mutagenic response and it was, therefore, considered that the test material had been adequately tested.

Remarks on result:
other: strain/cell type: Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Remarks:
Migrated from field 'Test system'.

Please see Attached "Tables 1 to 10"

Due to the nature and quantity of tables it was not possible to insert them in this section.
Conclusions:
Interpretation of results:
Non-mutagenic

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.
Executive summary:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Test" and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

One main experiment was performed. In this main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (R0 medium) and positive controls. The exposure groups used were as follows: 4‑hour exposures both with and without metabolic activation, and 24–hour exposure without metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and was 15.63 to 225 µg/ml for the 4-hour exposure group in the absence of metabolic activation, 15.63 to 500 µg/ml for the 4-hour exposure group in the presence of metabolic activation, and 7.81 to 250 µg/ml for the 24-hour exposure group in the absence of metabolic activation.

The maximum dose level used was limited by test material induced toxicity. Precipitate of test material was not observed at any of the dose levels. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any statistically significant or dose-related (linear-trend) increases in the mutant frequency at any dose level, either with or without metabolic activation, in any of the three exposure groups.

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Not applicable, no adverse effects observed.

Additional information

Justification for classification or non-classification

A full suite of in vitro mutagenicity studies, as required by Annexes VII and VIII of REACH, are presented (Bowles, 2011; Flanders, 2011 & Thompson, 2011). All studies indicated negative results for mutagenicity.

In accordance with Regulation (EC) No 1272/2008 (CLP), these results are conclusive but not sufficient for classification.