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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Sponsor's identification: CAS No 160901-14-4 Fatty acids, tall oil, oligomeric products with maleic acid and rosin, Calcium, magnesium, zinc salt
Fatty acids, tall oil, oligomeric products with maleic acid and rosin, Calcium, magnesium, zinc salt

Description : Brown Solid
Date received : 15 October 2009
Storage conditions: Approximately -20 °C in the dark

Method

Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: The test material dose range was 15 to 5000 µg/plate. An additional dose level (15 µg/plate) was selected in Experiment 2 in order to allow for potential toxicity following the change in test methodology.
Vehicle / solvent:
The test material was insoluble in dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml but was fully soluble in tetrahydrofuran at 200 mg/ml in solubility checks performed in house. Distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested that the test material was insoluble in this solvent vehicle. Tetrahydrofuran was therefore selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 1 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 10 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix

Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix

Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix

Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate
Details on test system and experimental conditions:

Tester Strains
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 numbers 747600 02/2014 and 757012 03/2014) 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 insoluble in dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml but was fully soluble in tetrahydrofuran at 200 mg/ml in solubility checks performed in house. Distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested that the test material was insoluble in this solvent vehicle. Tetrahydrofuran was therefore selected as the vehicle.
The test material was accurately weighed and approximate half-log dilutions prepared in tetrahydrofuran by mixing on a vortex mixer and sonication for 5 minutes at 40°C on the day of each experiment. Tetrahydrofuran is toxic to the bacterial cells at and above 50 µl, therefore all of the formulations were prepared at concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 25 µl aliquots. Tetrahydrofuran is an acceptable vehicle for use in this test system (6). Analysis for concentration, homogeneity and stability of the test material formulations is not a requirement of the test guidelines and was, therefore, not determined. Prior to use, the solvent was dried to remove water using molecular sieves ie 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x 10-4 microns.

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 (ENNG): 2 µg/plate for WP2uvrA-
N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 3 µg/plate for TA100
N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 5 µg/plate for TA1535
9-Aminoacridine (9AA): 80 µg/plate for TA1537
4-Nitroquinoline-1-oxide (4NQO): 0.2 µg/plate for TA98
In addition, 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP), which are non mutagenic in the absence of metabolising enzymes, were used in the series of plates with S9-mix at the following concentrations:
2-Aminoanthracene (2AA): 1 µg/plate for TA100
2-Aminoanthracene (2AA): 2 µg/plate for TA1535 and TA1537
2-Aminoanthracene (2AA): 10 µg/plate for WP2uvrA-
Benzo(a)pyrene (BP): 5 µg/plate for TA98

Microsomal Enzyme Fraction
S9 was prepared in-house on 07 June 2009 (Preliminary Toxicity Test only) and 13 September 2009 from the livers of male rats weighing ~ 200g. These had each orally received three consecutive daily doses of phenobarbitone/ b­naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on Day 4. Before use, each batch of S9 was assayed for its ability to metabolise appropriate indirect mutagens used in the Ames Test. The S9 was stored at approximately ­196ºC.

S9-Mix and Agar
The S9-mix was prepared immediately before use using sterilised 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.
Top agar was prepared using 0.6% Bacto agar (lot number 8301157 09/2013) and 0.5% sodium chloride with 5 ml of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 ml of top agar. Vogel-Bonner Minimal agar plates were purchased from ILS Limited (lot numbers 1089823-02 05/2014 preliminary toxicity test and Experiment 1 and 1098679 08/2014 Experiment 2).

Test Procedure
Preliminary Toxicity Test
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 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, 0.025 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 (tetrahydrofuran) were tested. In addition, 0.025 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. Manual counts were performed at 5000 µg/plate because of excessive test material precipitation.

Mutation Test - Experiment 1
Five concentrations of the test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.025 ml of the vehicle or test material formulation or 0.1 ml of 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. Manual counts were performed at 5000 µg/plate because of excessive test material precipitation.

Mutation Test - Experiment 2
The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was 15 to 5000 µg/plate. An additional dose level (15 µg/plate) was selected in Experiment 2 in order to allow for potential toxicity following 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 0.025 ml of the vehicle or test material formulation and incubated for 20 minutes at 37°C 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 described in Section "Mutation Test - Experiment 1". Manual counts were performed at and above 1500 µg/plate because of excessive test material precipitation.

Evaluation criteria:
Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method section 3 with historical control ranges for 2007 and 2008 in Appendix 1.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the range of 1 to 9.9 x 109 bacteria per ml.
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. The historical control ranges for 2007 and 2008 are presented in Appendix 1.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

Evaluation Criteria
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (7) 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

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.
The numbers of revertant colonies for the toxicity assay were: please see table "The numbers of revertant colonies for the toxicity assay were" in the section "Overall remarks"

Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9 mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

A history profile of vehicle and positive control values for 2007 and 2008 is presented in Appendix 1.
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level in Experiment 1 (plate incorporation method). However, in Experiment 2 (pre-incubation method) the test material did cause a visible reduction in the growth of the bacterial background lawn at and above 1500 µg/plate for all of the strains dosed in the absence of S9 and to tester strains TA100 and TA1535 only, dosed in the presence of S9. The toxicity caused by the test material was of insufficient severity to prevent testing up to the maximum recommended dose level of 5000 µg/plate.
An off-white, fibrous precipitate was noted at and above 1500 µg/plate and from 500 µg/plate in Experiment 1 and Experiment 2, respectively. These observations did not prevent the scoring of revertant colonies.
No toxicologically 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.
A small increase in revertant colony frequency for tester strain TA98 was noted, in the presence of S9, at 150 µg/plate in Experiment 2 only. However, this increase was non reproducible in two separate experiments and the individual counts at 150 µg/plate were within the in-house historical control range for the strain. The increase was, therefore, considered to be of no biological consequence.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table1              Spontaneous Mutation Rates (Concurrent Negative Controls)

EXPERIMENT 1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

117

 

23

 

27

 

24

 

14

 

128

(117)

20

(22)

24

(22)

16

(18)

4

(8)

107

 

22

 

14

 

14

 

7

 

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

139

 

19

 

36

 

20

 

7

 

114

(123)

16

(17)

35

(35)

20

(20)

6

(6)

116

 

16

 

34

 

21

 

6

 

 

Table2              Test Results: Experiment 1 – Without Metabolic Activation

Test Period

From: 06 December 2009

To: 09 December 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

0

124

121

125

(123)

2.1#

22

31

21

(25)

5.5

18

27

26

(24)

4.9

34

22

42

(33)

10.1

16

11

19

(15)

4.0

-

50

142

132

135

(136)

5.1

27

22

27

(25)

2.9

33

25

26

(28)

4.4

27

38

40

(35)

7.0

12

16

23

(17)

5.6

-

150

136

132

121

(130)

7.8

22

15

13

(17)

4.7

26

33

30

(30)

3.5

41

34

44

(40)

5.1

10

10

18

(13)

4.6

-

500

131

117

115

(121)

8.7

26

25

34

(28)

4.9

24

33

25

(27)

4.9

40

41

31

(37)

5.5

23

14

14

(17)

5.2

-

1500

125 P

110 P

108 P

(114)

9.3

19 P

27 P

CP

(23)

5.7

26 P

38 P

29 P

(31)

6.2

32 P

36 P

36 P

(35)

2.3

20 P

25 P

19 P

(21)

3.2

-

5000

102 P

123 P

125 P

(117)

12.7

19 P

24 P

29 P

(24)

5.0

26 P

22 P

20 P

(23)

3.1

28 P

19 P

23 P

(23)

4.5

13 P

6 P

13 P

(11)

4.0

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

426

566

576

(523)

83.9

236

296

263

(265)

30.0

636

697

754

(696)

59.0

145

144

136

(142)

4.9

648

751

785

(728)

71.3

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

C        Contaminated

P        Precipitate

#        Standard deviation

Table3              Test Results: Experiment 1 – With Metabolic Activation

Test Period

From: 06 December 2009

To: 09 December 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

+

0

104

92

125

(107)

16.7#

10

18

15

(14)

4.0

29

29

30

(29)

0.6

26

23

24

(24)

1.5

9

19

9

(12)

5.8

+

50

124

100

112

(112)

12.0

8

15

9

(11)

3.8

37

23

22

(27)

8.4

20

16

29

(22)

6.7

11

11

9

(10)

1.2

+

150

129

150

113

(131)

18.6

15

12

12

(13)

1.7

29

34

23

(29)

5.5

40

27

37

(35)

6.8

11

8

9

(9)

1.5

+

500

112

124

114

(117)

6.4

25

10

16

(17)

7.5

25

35

35

(32)

5.8

35

31

30

(32)

2.6

11

14

9

(11)

2.5

+

1500

104 P

115 P

103 P

(107)

6.7

15 P

15 P

16 P

(15)

0.6

31 P

46 P

40 P

(39)

7.5

34 P

30 P

33 P

(32)

2.1

8 P

13 P

18 P

(13)

5.0

+

5000

98 P

129 P

104 P

(110)

16.4

16 P

11 P

15 P

(14)

2.6

26 P

29 P

27 P

(27)

1.5

39 P

23 P

30 P

(31)

8.0

10 P

7 P

5 P

(7)

2.5

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1492

1587

1618

(1566)

65.7

145

134

151

(143)

8.6

313

360

404

(359)

45.5

201

186

236

(208)

25.7

169

213

241

(208)

36.3

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

P        Precipitate

#        Standard deviation

Table 4              Test Results: Experiment 2 – Without Metabolic Activation

Test Period

From: 18 December 2009

To: 21 December 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

0

90

124

121

(112)

18.8#

25

22

27

(25)

2.5

32

44

33

(36)

6.7

21

18

16

(18)

2.5

13

9

10

(11)

2.1

-

15

106

100

100

(102)

3.5

20

20

22

(21)

1.2

38

34

34

(35)

2.3

15

18

22

(18)

3.5

8

10

9

(9)

1.0

-

50

102

117

110

(110)

7.5

30

25

26

(27)

2.6

37

35

33

(35)

2.0

14

24

14

(17)

5.8

8

9

8

(8)

0.6

-

150

114

101

92

(102)

11.1

26

26

26

(26)

0.0

31

36

36

(34)

2.9

18

16

21

(18)

2.5

8

10

9

(9)

1.0

-

500

107 P

109 P

106 P

(107)

1.5

22 P

26 P

20 P

(23)

3.1

42 P

38 P

37 P

(39)

2.6

20 P

18 P

23 P

(20)

2.5

10 P

11 P

10 P

(10)

0.6

-

1500

107SP

96 SP

93 SP

(99)

7.4

0 VP

0 VP

0 VP

(0)

0.0

44 SP

38 SP

33 SP

(38)

5.5

22 SP

18 SP

18 SP

(19)

2.3

0 VP

0 VP

0 VP

(0)

0.0

-

5000

84 SP

94 SP

90 SP

(89)

5.0

0 VP

0 VP

0 VP

(0)

0.0

39 SP

34 SP

37 SP

(37)

2.5

24 SP

18 SP

17 SP

(20)

3.8

0 VP

0 VP

0 VP

(0)

0.0

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

758

643

641

(681)

67.0

985

961

874

(940)

58.4

925

844

797

(855)

64.7

118

124

113

(118)

5.5

1302

1329

1383

(1338)

41.2


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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

S        Sparse bacterial background lawn

V        Very weak bacterial background lawn

#        Standard deviation

Table 5              Test Results: Experiment 2 – With Metabolic Activation

Test Period

From: 18 December 2009

To: 21 December 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

+

0

93

114

89

(99)

13.4#

12

13

12

(12)

0.6

46

43

32

(40)

7.4

24

22

18

(21)

3.1

11

12

14

(12)

1.5

+

15

106

106

96

(103)

5.8

11

10

15

(12)

2.6

32

35

36

(34)

2.1

22

19

19

(20)

1.7

9

8

12

(10)

2.1

+

50

93

101

88

(94)

6.6

10

10

7

(9)

1.7

47

45

38

(43)

4.7

21

21

23

(22)

1.2

9

8

10

(9)

1.0

+

150

102

89

96

(96)

6.5

9

12

10

(10)

1.5

40

40

45

(42)

2.9

27

26

27

(27)

0.6

*

12

10

9

(10)

1.5

+

500

107 P

88 P

87 P

(94)

11.3

12 P

14 P

12 P

(13)

1.2

42 P

43 P

42 P

(42)

0.6

24 P

21 P

23 P

(23)

1.5

15 P

13 P

13 P

(14)

1.2

+

1500

75 SP

82 SP

87 SP

(81)

6.0

14 SP

10 SP

12 SP

(12)

2.0

46 P

38 P

43 P

(42)

4.0

23 P

19 P

24 P

(22)

2.6

13 P

9 P

10 P

(11)

2.1

+

5000

82 SP

82 SP

90 SP

(85)

4.6

9 SP

9 SP

12 SP

(10)

1.7

41 P

39 P

41 P

(40)

1.2

22 P

25 P

21 P

(23)

2.1

9 P

10 P

8 P

(9)

1.0

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1215

1350

1327

(1297)

72.2

348

386

378

(371)

20.0

495

481

474

(483)

10.7

159

183

160

(167)

13.6

287

336

338

(320)

28.9


2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

P        Precipitate

S        Sparse bacterial background lawn

*         p 0.05

#        Standard deviation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based on an absence of genotoxic/mutagenic effects in a bacterial reverse mutation test with Salmonella typhimurium strains TA 98, TA 100, TA 1535 or TA 1537, or in E. coli strain WP2, with or without metabolic activation, Fatty acids, tall oil, oligomeric reaction products with maleic anhydride and rosin, calcium magnesium zinc salts is not classifiable for Germ Cell Mutagenicity according to Directive 67/548/EEC, the UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) or the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
Executive summary:

The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It alsoets the requirents of 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 harmonised guidelines.

Methods.

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 six 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 was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. 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. 

An additional dose level (15 µg/plate) was selected in Experiment 2 in order to allow for potential toxicity following the change in test methodology.

Results.

The vehicle (tetrahydrofuran) 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 no visible reduction in the growth of the bacterial background lawn at any dose level in Experiment 1 (plate incorporation method). However, in Experiment 2 (pre-incubation method) the test material did cause a visible reduction in the growth of the bacterial background lawn at and above 1500 µg/plate for all of the strains dosed in the absence of S9 and to tester strains TA100 and TA1535 only, dosed in the presence of S9. The toxicity caused by the test material was of insufficient severity to prevent testing up to the maximum recommended dose level of 5000 µg/plate.

An off-white, fibrous precipitate was noted at and above 1500 µg/plate and from 500 µg/plate in Experiment 1 and Experiment 2, respectively. These observations did not prevent the scoring of revertant colonies.

No toxicologically 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.

A small increase in revertant colony frequency for tester strain TA98 was noted, in the presence of S9, at 150 µg/plate in Experiment 2 only. However, this increase was non-reproducible in two separate experiments and the individual counts at 150 µg/plate were within the in-house historical control range for the strain. The increase was, therefore, considered to be of no biological consequence.

Conclusion.

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