Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 September 2016 to 20 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial Reverse Mutation Test", 21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Regulation (EC) No. 440/2008, B.13/14. "Mutagenicity: Reverse Mutation Test Using Bacteria", 30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
EPA Health Effects Test Guidelines, OPPTS 870.5100 "Bacterial Reverse Mutation Test", EPA 712-C-98-247, August 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

Method

Target gene:
The purpose of this study was to evaluate the mutagenic potential of the test item by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli WP2 uvrA strain in the presence and absence of activated rat liver S9 fraction.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Concentration Range Finding Test (Informatory Toxicity Test).
In the Initial Mutation Test and Confirmatory Mutation Test different concentrations were used. Furthermore, a Complementary Confirmatory Mutation Test was also performed based on the results of the Confirmatory Mutation Test using a modified concentration range.
Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 µg/plate and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 µg/plate.
Examined concentrations in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA98 and TA1535 strains without metabolic activation were 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate.
Examined concentrations in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA1535 strain with metabolic activation were 5000, 2500, 1581, 1000, 500, 158.1, 50 and 15.81 µg/plate.
Vehicle / solvent:
Dimethyl sulfoxide (DMSO):
Supplier: Sigma-Aldrich Co.
Batch No.: SZBG1310V
Expiry date: 25 April 2019
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-1,2-phenylenediamine (NPD); Methyl-methanesulfonate (MMS); 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
DESCRIPTION OF THE TEST PROCEDURE
The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test, a Confirmatory Mutation Test and a Complementary Confirmatory Mutation Test. In the Preliminary Concentration Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test, the pre-incubation method was used.

Preliminary Compatibility Test
The solubility of the test item was examined using Distilled water, N,N-Dimethylformamide (DMF) and Dimethyl sulfoxide (DMSO). The test item was insoluble in Distilled water at 100 mg/mL concentrations. However, clear solution was observed at 100 mg/mL concentration using DMF and DMSO as vehicles. Due to the better biocompatibility, DMSO was selected as vehicle (solvent) for the study. The obtained stock formulation (50 µL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension to examine the formulation compatibility (Preliminary Compatibility Test).

Preliminary Concentration Range Finding Test (Informatory Toxicity Test)
Based on the available information and the solubility and compatibility test, 100 mg/mL stock solution was prepared in DMSO, which was diluted in 6 steps by factors of 2, 2.5 and approximately v10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate of the test item. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.

Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test, Confirmatory Mutation Tests and Complementary Confirmatory Mutation Test)
Based on the results of the preliminary test, 100 mg/mL stock solution was prepared in DMSO, which was diluted by serial dilutions in at least five steps to obtain at least six dosing formulations for lower doses. The maximum test concentration was 5000 µg test item/plate.
Examined concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 µg/plate.
Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 µg/plate.
Examined concentrations in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA98 and TA1535 strains without metabolic activation were 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate.
Examined concentrations in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA1535 strain with metabolic activation were 5000, 2500, 1581, 1000, 500, 158.1, 50 and 15.81 µg/plate.

Control Groups Used in the Tests
Strain-specific positive and negative (vehicle/solvent) controls, both with and without metabolic activation were included in each test. In addition, untreated control was used demonstrating that the chosen vehicle induced no deleterious or mutagenic effects.

Procedure for Exposure in the Initial Mutation Test
A standard plate incorporation procedure was performed as an Initial Mutation Test.
Bacteria (cultured in Nutrient Broth No.2.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45°C. The equivalent number of minimal glucose agar plates (three plates per concentration and for each control) was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes:
top agar: 2000 µL
vehicle (solvent) or test item solution (or reference controls): (50) µL
overnight culture of test strain: 100 µL
phosphate buffer (pH 7.4) or S9 mix: 500 µL
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls.
After preparation, the plates were incubated at 37°C for 48 hours.

Procedure for Exposure in the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test
A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed. The same method was used in the Complementary Confirmatory Mutation Test.
For the pre-incubation method, bacteria (cultured in Nutrient Broth No.2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
Before the overlaying, 50 µL of test item formulations or its vehicle (or positive reference controls or their solvent), 100 µL of the overnight culture of bacterial cells and the 0.5 mL of S9 mix (activated test conditions) or phosphate buffer pH 7.4 (nonactivated test conditions) were added into the appropriate tubes to provide direct contact between bacteria and the test item. These tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37°C in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.
Rationale for test conditions:
The experimental methods were conducted according to the methods described Ames et al. and Maron and Ames, Kier et al., Venitt and Parry, OECD Guideline No. 471, 1997, Commission Regulation (EC) No. 440/2008, 2008, EPA Guidelines, OPPTS 870.5100, 1998, 1996 and according to the relevant SOPs of CiToxLAB Hungary Ltd.
Evaluation criteria:
The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.
Statistics:
no

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test and Complementary Mutation Test, the pre-incubation method was used.
The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA strain in the presence and absence of a metabolic activation system (±S9 mix) with appropriate untreated, negative (vehicle/solvent) and positive controls.
The Complementary Confirmatory Mutation Test was carried out using Salmonella typhimurium TA98 and TA1535 strains in the presence and/or absence of a metabolic activation system (S9 mix) with appropriate untreated, negative (vehicle/solvent) and positive controls. In the main tests, each sample (including the controls) was tested in triplicate.
Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 µg/plate and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 µg/plate.
Based on the results of the Confirmatory Mutation Test, the examined test concentrations in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA98 and TA1535 strains without metabolic activation were 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate and in Salmonella typhimurium TA1535 strain with metabolic activation were 5000, 2500, 1581, 1000, 500, 158.1, 50 and 15.81 µg/plate.
In the Initial Mutation Test (using the plate incorporation method), there was no relevant increase of mutation factor with and without metabolic activation, there was no clear dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range. Indeed, this first assay was considered to be negative.
In the Confirmatory Mutation Test using the pre-incubation method, negative results were obtained with Salmonella typhimurium strains (TA98, TA100 and TA1537) and Escherichia coli WP2 uvrA strain with and without metabolic activation, and with TA1535 strain without metabolic activation. Positive effect of the test item was obtained in Salmonella typhimurium TA1535 bacterial strains with metabolic activation as the calculated mutation factor values were over the biologically relevant threshold value of 3 (at the two highest tested doses) and dose dependence was also observed.
The observed positive effect was confirmed in the Complementary Confirmatory Mutation Test (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 at 2500 µg/plate concentration with metabolic activation (the observed mutation factor value was 2.97). This borderline result demonstrated the slightly positive effect was reproducible and dose dependent.
In the Complementary Confirmatory Mutation Test, in Salmonella typhimurium TA98 bacterial strain without metabolic activation the highest revertant rate was observed at 158.1 µg/plate concentration. The observed mutation factor value was 2.48. Higher numbers of revertant colonies compared to the DMSO control were observed at several other concentrations in this strain. However, no dose-dependent relationship was observed. The number of revertant colonies was within the historical control range in each case. Furthermore, higher number of revertant colonies compared to the DMSO control were detected for untreated control (MF: 1.55) also in this strain without metabolic activation, indicating a higher than usual variability in this case. These results were also examined for reproducibility, but no increased revertant counts were detected in the first main test (using the same experimental method) in this strain. Thus, these values were considered as having no real biological relevance.
Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.
No precipitate was observed in the Initial Mutation Test, in the Confirmatory Mutation Tests and in the Complementary Confirmatory Mutation Test.
Inhibitory, cytotoxic effect of the test item (reduced background lawn development) was observed in the Confirmatory Mutation Test in all strains without metabolic activation at 5000 and/or 1581 and/or 500 µg/plate concentrations and a similar cytotoxic effect (reduced background lawn development/ reduced colony number) was seen in salmonella typhimurium TA1537 strains with metabolic activation at 5000 µg/plate concentration. The effect was excessive in Salmonella typhimurium TA98 and TA1535 strains without metabolic activation at 5000, 1581 and 500 µg/plate concentrations, thus a complementary experiment was performed to ensure validity.
In the Complementary Confirmatory Mutation Test the background inhibition (reduced/slightly reduced background lawn development) was still observed in Salmonella typhimurium TA98 and TA1535 strains without metabolic activation at 500 µg/plate concentrations.

Any other information on results incl. tables

 Summary Table of the Preliminary Concentration range Finding Test

Concentrations

(µg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

TA 98

TA 100

-S9

+S9

-S9

+S9

Untreated control

Mean

26.7

23.0

94.7

103.0

MF

1.38

1.05

0.94

1.04

DMSO control

Mean

19.3

22.0

100.3

98.7

MF

1.00

1.00

1.00

1.00

Distilled water control

Mean

--

--

96.7

--

MF

--

--

0.96

--

5000

 

23.0

25.0

92.3

86.7

 

1.19

1.14

0.92

0.88

2500

Mean

22.3

20.7

102.3

105.3

MF

1.16

0.94

1.02

1.07

1000

Mean

24.7

29.3

98.7

107.0

MF

1.28

1.33

0.98

1.08

316

Mean

25.7

28.0

98.7

113.7

MF

1.33

1.27

0.98

1.15

100

Mean

21.0

36.0

97.3

110.0

MF

1.09

1.64

0.97

1.11

31.6

Mean

26.0

34.0

101.0

118.3

MF

1.34

1.55

1.01

1.20

10

Mean

22.0

28.3

109.7

117.0

MF

1.14

1.29

1.09

1.19

NPD (4µg)

Mean

289.3

--

--

--

MF

20.14

--

--

--

2AA (2µg)

Mean

--

2357.3

--

2325.3

MF

--

107.15

--

23.57

SAZ (2µg)

Mean

--

--

1168.0

--

MF

--

--

12.08

--

 

Summary Table of the Initial Mutation Test (Plate Incorporation Method)

Concentrations (µg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

20.0

25.0

109.7

119.7

13.0

8.7

6.3

9.3

32.7

33.0

MF

1.05

0.99

0.98

1.04

1.39

0.74

0.76

1.00

1.01

0.93

DMSO control

Mean

19.0

25.3

112.3

114.7

9.3

11.7

8.3

9.3

32.3

35.7

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

Distilled water control

Mean

--

--

104.3

--

11.3

--

--

--

35.0

--

MF

--

--

0.93

--

1.21

--

--

--

1.08

--

5000

Mean

21.0

20.0

78.3

77.0

13.3

15.0

7.0

6.7

23.0

26.7

MF

1.11

0.79

0.70

0.67

1.43

1.29

0.84

0.71

0.71

0.75

1581

Mean

21.0

27.7

101.0

100.0

10.0

22.3

9.3

5.3

28.7

22.0

MF

1.11

1.09

0.90

0.87

1.07

1.91

1.12

0.57

0.89

0.62

500

Mean

24.7

26.0

96.3

102.3

9.0

20.3

5.3

6.7

25.3

24.0

MF

1.30

1.03

0.86

0.89

0.96

1.74

0.64

0.71

0.78

0.67

158.1

Mean

24.3

28.0

91.3

100.7

11.7

17.0

10.7

9.0

22.3

30.0

MF

1.28

1.11

0.81

0.88

1.25

1.46

1.28

0.96

0.69

0.84

50

Mean

20.3

29.7

83.0

94.7

11.3

11.3

11.7

8.3

24.0

26.7

MF

1.07

1.17

0.4

0.83

1.21

0.97

1.40

0.89

0.74

0.75

15.81

Mean

24.0

28.3

86.3

95.3

10.7

14.3

7.7

13.0

23.7

25.0

MF

1.26

1.12

0.77

0.83

1.14

1.23

0.92

1.39

0.73

0.70

NPD (4µg)

Mean

404.0

--

--

--

--

--

--

--

--

--

MF

21.26

--

--

--

--

--

--

--

--

--

2AA (2µg)

Mean

--

2357.3

--

2417.3

--

222.7

--

201.3

--

--

MF

--

93.05

--

21.08

--

19.09

--

21.57

--

--

2AA (50µg)

Mean

--

--

--

--

--

--

--

--

--

233.7

MF

--

--

--

--

--

--

--

--

--

6.55

SAZ (2µg)

Mean

--

--

1049.3

--

1178.7

--

--

--

--

--

MF

--

--

10.06

--

104.00

--

--

--

--

--

9AA (50µg)

Mean

--

--

--

--

--

--

494.7

--

--

--

MF

--

--

--

--

--

--

59.36

--

--

--

MMS (2µL)

Mean

--

--

--

--

--

--

--

--

1066.7

--

MF

--

--

--

--

--

--

--

--

30.48

--

 

Summary Table of the Confirmatory Mutation Test (Pre-Incorporation Method)

Concentrations (µg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

--

44.3

89.7

102.7

--

12.7

7.3

9.7

31.3

39.3

MF

--

1.01

0.93

0.98

--

1.27

1.47

0.81

0.95

0.96

DMSO control

Mean

--

44.0

96.3

105.0

--

10.0

5.0

12.0

33.0

41.0

MF

--

1.00

1.00

1.00

--

1.00

1.00

1.00

1.00

1.00

Distilled water control

Mean

--

--

96.7

--

--

--

--

--

39.0

--

MF

--

--

1.00

--

--

--

--

--

1.18

--

5000

Mean

--

38.7

25.7

43.7

--

30.3

3.0

11.3

29.3

39.3

MF

--

0.88

0.27

0.42

--

3.03

0.60

0.94

0.89

0.96

1581

Mean

--

37.3

65.7

85.3

--

37.7

3.3

9.7

32.7

38.3

MF

--

0.85

0.68

0.81

00

3.77

0.67

0.81

0.99

0.93

500

Mean

--

40.7

91.3

115.3

--

21.3

6.3

11.0

31.0

46.7

MF

--

0.92

0.95

1.10

--

2.13

1.27

0.92

0.94

1.14

158.1

Mean

--

36.3

101.7

106.7

--

16.7

10.7

9.7

31.0

41.7

MF

--

0.93

1.06

1.02

--

1.67

2.13

0.81

0.94

1.02

50

Mean

--

42.0

110.3

113.3

--

12.7

9.7

12.3

31.0

35.3

MF

--

0.95

1.15

1.08

--

1.27

1.93

1.03

0.94

0.86

15.81

Mean

--

49.0

108.3

123.7

--

15.0

13.0

10.7

29.0

41.0

MF

--

1.11

1.12

1.18

--

1.50

2.60

0.89

0.88

1.00

5

Mean

--

41.0

139.0

100.3

--

11.0

6.0

11.3

29.3

33.0

MF

--

0.93

1.44

0.96

--

1.10

1.20

0.94

0.89

0.80

NPD (4µg)

Mean

--

--

--

--

--

--

--

--

--

--

MF

--

--

--

--

--

--

--

--

--

--

2AA (2µg)

Mean

--

2345.3

--

2457.3

--

252.7

--

213.7

--

--

MF

--

53.30

--

23.40

--

25.27

--

17.81

--

--

2AA (50µg)

Mean

--

--

--

--

--

--

--

--

--

199.0

MF

--

--

--

--

--

--

--

--

--

4.85

SAZ (2µg)

Mean

--

--

1064.0

--

--

--

--

--

--

--

MF

--

--

11.01

--

--

--

--

--

--

--

9AA (50µg)

Mean

--

--

--

--

--

--

408.7

--

--

--

MF

--

--

--

--

--

--

81.73

--

--

--

MMS (2µL)

Mean

--

--

--

--

--

--

--

--

1009.3

--

MF

--

--

--

--

--

--

--

--

25.88

--

 

Summary Table of the Complementary Confirmatory Mutation Test (pre-Incubation Method)

Concentrations

(µg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

TA 98

TA 1535

-S9

+S9

-S9

+S9

Untreated control

Mean

15.0

--

15.7

14.0

MF

1.55

--

1.09

1.45

DMSO control

Mean

9.7

--

14.3

9.7

MF

1.00

--

1.00

1.00

Distilled water control

Mean

--

--

12.7

--

MF

--

--

0.88

--

5000

Mean

--

--

--

23.3

MF

--

--

--

2.41

2500

Mean

--

--

--

28.7

MF

--

--

--

2.97

1581

Mean

--

--

--

25.0

MF

--

--

--

2.59

1000

Mean

--

--

--

25.0

MF

--

--

--

2.59

500

Mean

17.3

--

14.3

17.0

MF

1.79

--

1.00

1.76

158.1

Mean

24.0

--

11.7

14.7

MF

2.48

--

0.81

1.52

50

Mean

20.3

--

14.3

10.7

MF

2.10

--

1.00

1.10

15.81

Mean

20.0

--

14.0

8.3

MF

2.07

--

0.98

0.86

5

Mean

16.3

--

12.7

--

MF

1.69

--

0.88

--

1.581

Mean

19.7

--

13.3

--

MF

2.03

--

0.93

--

NPD (4µg)

Mean

397.3

--

--

--

MF

41.10

--

--

--

2AA (2µg)

Mean

--

--

--

199.7

MF

--

--

--

20.66

SAZ (2µg)

Mean

--

--

1197.3

--

MF

--

--

94.53

--

 

Applicant's summary and conclusion

Conclusions:
The test item Tris(2-hydroxyethyl) isocyanurate triacrylate had slightly mutagenic activity in Salmonella typhimurium TA1535 bacterial strain with metabolic activation, no mutagenic activity was observed in Salmonella typhimurium TA98, TA100, TA1537 and Escherichia coli WP2 uvrA with and without metabolic activation and in Salmonella typhimurium TA1535 without metabolic activation under the test conditions used in this study.
Executive summary:

The test item Tris(2-hydroxyethyl) isocyanurate triacrylate was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

 

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/ß-naphthoflavone-induced rats.

 

The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Pre-Incubation Method) and a Complementary Mutation Test (Pre-Incubation Method).

 

Based on the results of a solubility tests, the test item was formulated in Dimethyl sulfoxide (DMSO). Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 µg/plate were examined in the Preliminary Concentration Range Finding Test.

Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 µg/plate and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 µg/plate.

Examined concentrations in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA98 and TA1535 strains without metabolic activation were 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate.

Examined concentrations in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA1535 strain with metabolic activation were 5000, 2500, 1581, 1000, 500, 158.1, 50 and 15.81 µg/plate.

 

In the Initial Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the vehicle control and were within the normal biological variability of the test system.

 

In the Confirmatory Mutation Test in the case of Salmonella typhimurium TA1535 bacterial strain, positive effect was obtained using the pre-incubation method. In the Complementary Confirmatory Mutation Test a slightly positive effect was obtained in this bacterial strain using the pre-incubation method.

 

No precipitate was detected on the plates in the Preliminary Concentration Range Finding Test and in the main tests in all examined strains with and without metabolic activation.

 

Inhibitory, cytotoxic effect of the test item was observed in the Confirmatory Mutation Test in all strains without metabolic activation and a similar cytotoxic effect was seen in Salmonella typhimurium TA1537 strains with metabolic activation. The effect was excessive in Salmonella typhimurium TA98 and TA1535 strains without metabolic activation, thus a complementary experiment was performed to ensure validity. In the Complementary Confirmatory Mutation Test the background inhibition was still observed in these strains without metabolic activation.

 

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

 

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item induced gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

In conclusion, the test item Tris(2-hydroxyethyl) isocyanurate triacrylate (Batch Number: F0766724VS) had slightly mutagenic activity in Salmonella typhimurium TA1535 bacterial strain with metabolic activation, no mutagenic activity was observed in Salmonella typhimurium TA98, TA100, TA1537 andEscherichia coliWP2uvrAwith and without metabolic activation and in Salmonella typhimurium TA1535 without metabolic activation under the test conditions used in this study.