Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471 the test item did not show a mutagenic potential.

No information on chromosome aberration or gene mutation potential of the test item is available. To cover these endpoints, a read-across with a suitable source substance (Blue Sema, CAS No. 1040873 -93 -5) was performed.

In an in vitro chromosome aberration assay in mammalian cells according to OECD guideline 473, the source substance was not clastogenic and did not induce numerical chromosomal aberrations.

In an in vitro mutagenicity assay (HPRT) in mammalian cells according to OECD guideline 476, the source substance was not mutagenic.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 20 - June 12, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
In addition to histidine and tryptophan mutation, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2uvrA) is also defective in DNA excision repair.


Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/b-naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Based on the results of the preliminary concentration range finding tests (informatory toxicity tests) the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 5000; 1600; 500; 160; 50; 16 and 5 µg/plate.
Vehicle / solvent:
- Solvents used: Dimethyl sulfoxide (DMSO), Ultrapure water
- Justification for choice of solvents: Based on the results of these tests, dimethyl sulfoxide (DMSO) was found to be the most appropriate solvent for preparing the test item stock formulations and dilutions. The solvent was chosen due to its non-toxicity to the bacteria and the compatibility to the S9 activity based on the available laboratory’s historical control database.
The positive control substances sodium acide and methyl methanesulfonate were solved in Ultrapure water

Name: Dimethyl sulfoxide (DMSO)
Supplier: SIGMA-ALDRICH
Batch Numbers: SZBG0830V
Retest date: 08 March 2019
Storage: Room temperature

Name: Ultrapure water
Supplier: Prepared in the laboratory of TOXI-COOP ZRT.
Type: MILLIPORE, DIRECT Q5, No.: F0DA13956K
Batch Number: 20161213; 20170117; 20170321 and 20170329
Expiry date: 20 December 2016; 24 January 2017;
28 March 2017 and 05 April 2017
Storage: Room temperature

Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylenediamine
Remarks:
without S9 mix, TA 98, 4 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix; TA 100, TA 1535; 2 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix, TA 1538, 50 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix, E.coli WP2 uvrA, 2 µL/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9 mix, all tested S. typhimurium strains, 2 µg/plate; E.coli WP2 uvrA, 50 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS:
1

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: revertant colony numbers and the inhibition of the background lawn

- OTHER:
Origin of the Bacterial Strains
Tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were obtained from:
Supplier: Trinova Biochem GmbH; Rathenau Str. 2; D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA.
Frozen stock cultures were prepared from the disc cultures.

Storage of Tester Strains
The strains are stored at -80 ± 10ºC in the Laboratory of TOXI-COOP ZRT. in the form of lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains are prepared from fresh, overnight cultures to which DMSO (8 % (v/v)) is added as a cryoprotective agent.

Confirmation of Phenotypes of Tester Strains
The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. [1][2]. Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture and raw data and reports of phenotype confirmation are stored in the Laboratory of TOXI-COOP ZRT.

Spontaneous Reversion of Tester Strains
Each tester strain reverts spontaneously at a frequency that is characteristic for the strain. Spontaneous reversions of the test strains to histidine or tryptophan prototrophs are measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.

Procedure for Bacterial Cultures
The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 10-14 hours in a 37 °C Benchtop Incubator Shaker.

Viability and the Cell Count of the Testing Bacterial Cultures
The viability of each testing culture was determined by plating 0.1 mL of the 10-5, 10-6, 10-7 and 10-8 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by manual colony counting.

The Minimal Glucose Agar (MGA) Plates
Ready-to-use minimal glucose agar (MGA) plates were used in the study. The origin of the ready-to use MGA plates:
Supplier: VWR International;
Manufacturer: Merck Life Science GmbH, Germany.
Certificates of Analysis1) were obtained from the supplier.
Typical composition (g/1000 mL) of MGA plates:
Glucose 20.0 g
Magnesium sulfate 0.2 g
Citric acid 2.0 g
di-Potassium hydrogenphosphate 10.0 g
Sodium ammonium hydrogenphosphate 3.5 g
Agar agar 13.0 g

Nutrient broth No. 2. 25.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121˚C in an autoclave.

Nutrient Agar 20.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121˚C in an autoclave.

Top Agar for Salmonella typhimurium Strains
Agar solution:
Agar Bacteriological 4.0 g
NaCl 5.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121˚C in an autoclave.

Histidine – Biotin solution (0.5 mM):
D-Biotin 122.2 mg
L-Histidine•HCl H2O 104.8 mg
Ultrapure water ad 1000.0 mL
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete Top Agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100.0 mL
Agar solution 900.0 mL

Top Agar for Escherichia coli Strain
Tryptophan solution (2 mg/mL):
L-Tryptophan 2000.0 mg
Ultrapure water ad 1000.0 mL
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete Top Agar for Escherichia coli strain:
Nutrient Broth by 5.4.2 50.0 mL
Tryptophan solution (2 mg/mL) 2.5 mL
Agar solution by 5.4.4 947.5 mL

Metabolic Activation System
The test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which
is a cofactor-supplemented post-mitochondrial fraction (S9).

Rat Liver S9 Fraction
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH
(Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).

The Quality Control & Production Certificate of each lot of S9 was obtained from the supplier. The original Quality Control & Production
Certificates of rat liver S9 are stored in the Laboratory of TOXI-COOP ZRT.
The following lots of the S9 were applied:
1) Lot Number: 3593; Expiry date: February 25, 2018; Protein content: 36.4 mg/mL
(used in the Informatory Toxicity Tests);
Lot Number: 3650; Expiry date: June 16, 2018; Protein content: 36.5 mg/mL
(used in the first Informatory Toxicity Test);
Lot Number: 3662; Expiry date: July 07, 2018; Protein content: 40.5 mg/mL
(used in the Informatory Toxicity Tests and in the Initial Mutation Test);
Lot Number: 3712; Expiry date: November 03, 2018; Protein content: 34.3 mg/mL
(used in the Confirmatory Mutation Test);
Lot Number: 3727; Expiry date: December 01, 2018; Protein content: 33.7 mg/mL
(used in the Confirmatory Mutation Test).

The S9 Mix (with Rat Liver S9)
Salt solution for S9 mix Final concentration in S9 mix
NADP Na 7.66 g 4 mM
D-glucose-6 phosphate Na 3.53 g 5 mM
MgCl2 1.90 g 8 mM
KCl 6.15 g 33 mM
Ultrapure water ad 1000 mL
Sterilized by filtration through a 0.22 µm membrane filter.

The complete S9 mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 mL
Rat liver homogenate (S9) 100 mL
Salt solution for S9 mix 400 mL
The S9 mix (containing 10 % S9) was kept in an ice bath before it was added to the culture medium.

Sodium Phosphate Buffer (0.2 M, pH 7.4)
Solution A:
Na2HPO4 x 12H2O 71.63 g
Ultrapure water ad 1000 mL
Solution B:
NaH2PO4 x H2O 27.6 g
Ultrapure water ad 1000 mL

Solution A 880 mL
Solution B 120 mL*
* The components were mixed in the above ratio; thereafter the pH was checked and corrected.
The correction was performed with admixture of the solution A or B.
After the pH setting the sterilization was performed by filtration through a 0.22 µm membrane filter.






Rationale for test conditions:
Justification of concentrations:
Selection of the concentration range was done on the basis of solubility tests and concentration range finding tests (informatory toxicity tests).

Based on the solubility tests, stock suspensions with a concentration of 50 mg/mL were prepared in ultrapure water and dimethyl sulfoxide (DMSO), respectively and diluted accordingly. In the informatory toxicity tests any correction factor, based on the active component of the test item (88 %) was not taken into consideration; therefore the 50 mg/mL stock suspension concentration corresponded to 44 mg active component/mL. 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 in both tests. In the informatory toxicity tests the revertant colony numbers of solvent control plates with and without S9 mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected biological relevant increases in induced revertant colonies in both tester strains.
Evaluation criteria:
The colony numbers on the untreated, solvent control, positive control and the test item treated plates were determined
visually by manual counting, and the mean values, standard deviations and the mutation rates were calculated.

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the
dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the solvent control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at
least three times higher than the reversion rate of the solvent control.

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Criteria for a negative response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:
The mean values and appropriate standard deviations and mutation rates were calculated by EXCEL software.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the preliminary phase of this study two pre-experiments were performed to find the most appropriate solvent for the main experiments. Based on the solubility tests, stock suspensions with a concentration of 50 mg/mL were prepared in ultrapure water and dimethyl sulfoxide (DMSO), respectively and diluted accordingly. In the informatory toxicity tests any correction factor, based on the active component of the test item (88 %) was not taken into consideration; therefore the 50 mg/mL stock suspension concentration corresponded to 44 mg active component/mL. 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 in both tests. In the informatory toxicity tests the revertant colony numbers of solvent control plates with and without S9 mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected biological relevant increases in induced revertant colonies in both tester strains.
In the informatory toxicity tests a cytotoxic effect of the test item on the bacterial strains was not observed. The colony and background lawn development was not affected in any case. All of the observed slight revertant colony number decreases or increases (compared to the revertant colony numbers of the solvent control) remained within the biological variability range of the applied test system. Test item precipitates were observed by the unaided eye on the plates at concentrations of 5000, 1600 and 500 µg/plate in the absence and presence of an exogenous metabolic activation (±S9 mix) when using water as the solvent. When using DMSO as the solvent, precipitation of the test item on the plates was observed at concentration 5000 µg/plate (±S9 mix). In the informatory toxicity tests any correction factor, based on the active component of the test item (88 %) was not taken into consideration; therefore the 5000, 1600 and 500 µg/plate concentrations corresponded to 4400, 1408 and 440 µg active component/plate.


Table 4: Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)

Concentrations (µg/plate)

Salmonella typhimurium tester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

24.0

1.57

26.7

1.25

96.3

1.11

101.3

1.07

12.7

1.09

11.0

0.89

7.0

1.00

8.0

0.96

22.7

1.03

32.3

0.87

DMSO Control

15.3

1.00

21.3

1.00

87.0

1.00

95.0

1.00

11.7

1.00

12.3

1.00

7.0

1.00

8.3

1.00

22.0

1.00

37.0

1.00

Ultrapure Water Control

85.3

1.00

9.7

1.00

25.0

1.00

5000

14.7

0.96

15.7

0.73

92.7

1.07

104.7

1.10

8.7

0.74

11.0

0.89

2.0

0.29

9.7

1.16

22.7

1.03

30.0

0.81

1600

17.0

1.11

19.7

0.92

85.3

0.98

100.3

1.06

6.0

0.51

8.7

0.70

3.0

0.43

9.7

1.16

28.0

1.27

23.7

0.64

500

14.0

0.91

34.7

1.63

83.3

0.96

91.3

0.96

5.7

0.49

12.7

1.03

5.7

0.81

6.7

0.80

30.0

1.36

34.0

0.92

160

17.0

1.11

25.0

1.17

78.0

0.90

92.7

0.98

13.7

1.17

9.3

0.76

7.7

1.10

11.3

1.36

25.7

1.17

34.0

0.92

50

17.3

1.13

30.7

1.44

80.7

0.93

78.0

0.82

8.7

0.74

9.3

0.76

10.0

1.43

13.3

1.60

22.3

1.02

33.3

0.90

16

13.0

0.85

30.7

1.44

80.7

0.93

92.3

0.97

7.7

0.66

10.0

0.81

9.0

1.29

9.3

1.12

29.7

1.35

30.0

0.81

5

15.7

1.02

29.0

1.36

88.7

1.02

98.7

1.04

11.0

0.94

11.7

0.95

6.7

0.95

9.3

1.12

25.7

1.17

24.3

0.66

NPD (4 µg)

240.3

15.67

SAZ (2 µg)

1154.7

13.53

962.7

99.59

9AA (50 µg)

318.0

45.43

MMS (2 µL)

644.0

25.76

2AA (2 µg)

1325.3

62.13

1429.3

15.05

146.7

11.89

95.7

11.48

2AA (50 µg)

204.0

5.51

MR: Mutation Rate;         NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks:           DMSO was applied as solvent of the test item and positive control substances: NPD, 9AA and 2AA and the ultrapure water was applied as solvent for the SAZ and MMS. The mutation rate of the test item and the untreated control is given referring to the DMSO. The mutation rate of the NPD, 9AA and 2AA is given referring to the DMSO and the mutation rate of the SAZ and MMS positive control is given referring to the ultrapure water.

Table 5: Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (µg/plate)

Salmonella typhimurium tester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

16.7

0.77

24.0

1.33

99.3

1.01

115.0

1.27

9.3

1.00

9.7

0.97

8.0

1.09

9.0

1.17

34.3

1.24

37.3

1.05

DMSO Control

21.7

1.00

18.0

1.00

98.7

1.00

90.7

1.00

9.3

1.00

10.0

1.00

7.3

1.00

7.7

1.00

27.7

1.00

35.7

1.00

Ultrapure Water Control

100.7

1.00

11.3

1.00

29.7

1.00

5000

12.0

0.55

28.3

1.57

106.0

1.07

118.3

1.31

2.7

0.29

8.0

0.80

0.0

0.00

7.7

1.00

33.7

1.22

30.0

0.84

1600

10.7

0.49

29.0

1.61

68.3

0.69

116.7

1.29

3.0

0.32

10.7

1.07

0.0

0.00

7.7

1.00

47.3

1.71

35.3

0.99

500

14.0

0.65

32.0

1.78

76.0

0.77

117.3

1.29

4.3

0.46

10.3

1.03

1.3

0.18

15.7

2.04

29.3

1.06

33.0

0.93

160

16.7

0.77

32.7

1.81

98.0

0.99

107.0

1.18

9.0

0.96

8.3

0.83

7.7

1.05

14.0

1.83

25.7

0.93

38.0

1.07

50

47.0

2.17

31.0

1.72

92.0

0.93

95.7

1.06

14.0

1.50

11.7

1.17

9.0

1.23

14.7

1.91

27.3

0.99

34.0

0.95

16

30.7

1.42

30.0

1.67

105.3

1.07

91.7

1.01

11.3

1.21

13.7

1.37

8.7

1.18

7.7

1.00

34.0

1.23

39.7

1.11

5

25.0

1.15

33.0

1.83

95.7

0.97

88.3

0.97

11.7

1.25

11.7

1.17

8.3

1.14

10.3

1.35

24.7

0.89

32.0

0.90

NPD (4 µg)

226.0

10.43

SAZ (2 µg)

1829.3

18.17

525.3

46.35

9AA (50 µg)

273.7

37.32

MMS (2 µL)

1344.0

45.30

2AA (2 µg)

1213.3

67.41

1626.7

17.94

148.3

14.83

155.3

20.26

2AA (50 µg)

162.0

4.54

MR: Mutation Rate;         NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks:           DMSO was applied as solvent of the test item and positive control substances: NPD, 9AA and 2AA and the ultrapure water was applied as solvent for the SAZ and MMS. The mutation rate of the test item and the untreated control is given referring to the DMSO. The mutation rate of the NPD, 9AA and 2AA is given referring to the DMSO and the mutation rate of the SAZ and MMS positive control is given referring to the ultrapure water.

Conclusions:
In an in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471, the test item did not show a mutagenic potential.
Executive summary:

The mutagenic potential of the test item was assessed in an in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats.

The study included preliminary solubility tests, preliminary concentration range finding tests (informatory toxicity tests), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test).

Based on the results of the solubility tests and the concentration range finding tests the test item was dissolved in dimethyl sulfoxide (DMSO). At the formulation of test item solutions correction of concentrations for active component content (88.05 %, with rounding: 88 %) was made in the main experiments.

Based on the results of the preliminary concentration range finding tests (informatory toxicity tests) the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 5000, 1600, 500, 160, 50, 16 and 5 µg/plate. The selection of the concentration range was based on the recommendations in OECD guideline 471. At the concentration choice the non-toxicity of the test item and the precipitation of the test item in the final treatment mixture were taken into consideration. The observations were made by naked eye. When evaluated by naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at the concentrations of 5000 and 1600 µg/plate in absence and in the presence of S9 following the plate incorporation and pre-incubation procedures, and additionally at 500 µg/plate in absence of S9 following the pre-incubation procedure. An inhibitory effect of the test item was observed in the initial mutation test in the S. typhimurium TA1537 strain, in the confirmatory mutation test in the S. typhimurium TA1535 and TA1537 strains in the absence of exogenous metabolic activation. The inhibitory effect was indicated by absent or decreased revertant colony counts (some of them below the corresponding historical control data ranges) and affected background lawn development: reduced or slightly reduced background lawn. In general, 500 µg/plate was considered as lowest concentration showing cytotoxicity. The revertant colony numbers of solvent control (dimethyl sulfoxide (DMSO) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants mostly fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

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

Additional information

The mutagenic potential of the test item was assessed in an in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats. The study included preliminary solubility tests, preliminary concentration range finding tests (informatory toxicity tests), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test).

Based on the results of the solubility tests and the concentration range finding tests the test item was dissolved in dimethyl sulfoxide (DMSO). At the formulation of test item solutions correction of concentrations for active component content (88.05 %, with rounding: 88 %) was made in the main experiments.

Based on the results of the preliminary concentration range finding tests (informatory toxicity tests) the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 5000, 1600, 500, 160, 50, 16 and 5 µg/plate.The selection of the concentration range was based on the recommendations in OECD guideline 471. At the concentration choice the non-toxicity of the test item and the precipitation of the test item in the final treatment mixture were taken into consideration. The observations were made by naked eye. When evaluated by naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at the concentrations of 5000 and 1600 µg/plate in absence and in the presence of S9 following the plate incorporation and pre-incubation procedures, and additionally at 500 µg/plate in absence of S9 following the pre-incubation procedure. An inhibitory effect of the test item was observed in the initial mutation test in the S. typhimurium TA1537 strain, in the confirmatory mutation test in the S. typhimurium TA1535 and TA1537 strains in the absence of exogenous metabolic activation. The inhibitory effect was indicated by absent or decreased revertant colony counts (some of them below the corresponding historical control data ranges) and affected background lawn development: reduced or slightly reduced background lawn. In general, 500 µg/plate was considered as lowest concentration showing cytotoxicity. The revertant colony numbers of solvent control (dimethyl sulfoxide (DMSO) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants mostly fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

No information on chromosome aberration or gene mutation potential of the test item are available. However, suitable and reliable data of a source substance for read-across are available.

The clastogenic potential of the source substance was assessed in a mammalian chromosome aberration assay according to OECD 473. The source substance suspended in DMSO was tested in mammalian V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test:

Experiment A: 31.3, 62.5, 125 and 250 µg/mL source substance for with 3/20 h treatment/sampling time (without and with metabolic activation using rodent S9 mix)

Experiment B with 3/28 h treatment/sampling time and 7.8, 15.7, 31.3 and 62.5 µg/mL source substance for Experiment B with 20/20 h and 20/28 h treatment/sampling time.

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/mL) for 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). No precipitation of the source substance was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the source substance. Clear cytotoxicity of about 50% was observed after source substance treatment in all experimental parts. No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation. In experiment A in the absence of metabolic activation, two values were slightly above the 95% control limits of the historical control data. However, no statistical significant differences were observed after source substance treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationship was observed and therefore, the findings were not considered as being biologically relevant. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 µL/mL) and cyclophosphamide (5 µg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

In conclusion, the source substance did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the source substance is considered as being non-clastogenic in this system.

In an in vitro mutagenicity assay (HPRT) in mammalian cells according to OECD guideline 476 the mutagenic potential of the source substance was assessed. The source substance suspended in DMSO was tested in mammalian CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver:

Mutation Assay 5-hour treatment period without S9 mix: 62.5, 125, 250, 350 and 450 µg/mL

Mutation Assay 5-hour treatment period with S9 mix: 125, 250, 500, 750 and 1000 µg/mL

Phenotypic expression was evaluated up to 8 days following exposure in the absence and presence of metabolic activation. Clear cytotoxicity (survival approximately 16%) of the source substance was observed at the highest concentration applied (450 µg/mL in the absence and 1000 µg/mL in the presence of S9 mix). In both experimental parts, there were no statistically significant increases in mutation frequency when compared to the concurrent solvent control and the laboratory historical control data at any concentration tested in the absence and presence of metabolic activation. In the absence of S9 mix, in the cultures treated with 350 and 450 µg/mL the mutation frequency exceeded the 95% confidence interval of the historical control data (1 of 4 and 4 of 4 cultures, respectively). In the presence of S9 mix, in the cultures treated with 500 and 750 µg/mL the mutation frequency exceeded the 95% confidence interval of the historical control data (1 of 4 and 2 of 4 cultures, respectively). These findings were not considered to be biologically relevant since no dose-response relationships were noted, all values were within the normal range of mutation frequency and no statistical difference to the concurrent controls and the historical control range were observed. The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulfonate (1.0 µL/mL) and 7, 12-dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data.Therefore, the test is considered ass valid.

It is concluded that the source substance was not mutagenic in this in vitro mammalian cell gene mutation test performed with in Chinese hamster ovary cells.

In conclusion, the test item did not show mutagenic properties in an bacterial reverse mutation assay. A suitable source substance did not induce chromosome aberrations or increased gene mutation in mammalian cell systems.

Therfore, the test item is considered to be non-genotoxic.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.