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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test article was not mutagenic in guideline compliant OECD 471 and OECD 476 studies. furthermore, no clastogenic or aneugenic potential was evident in an OECD 487 compliant study.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan - Feb 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Jul 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: test substance was weighed and topped up with DMSO and shaken thoroughly
- Final dilution of a dissolved solid, stock liquid or gel: further concentrations were diluted from the stock solution according to the planned doses

FORM AS APPLIED IN THE TEST: solution
Target gene:
his and trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : rat liver
- method of preparation of S9 mix: At least 5 male Wistar rats [Crl:WI(Han)] received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days. Twenty-four hours after the last administration, rats were sacrificed, and livers were prepared
using sterile solvents and glassware at a temperature of +4°C. Livers were weighed and
washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three
volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at
+4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.
- concentration or volume of S9 mix and S9 in the final culture medium : 0.5 ml
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): yes
Test concentrations with justification for top dose:
- 1st and second experiment: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available
Untreated negative controls:
yes
Remarks:
omitting tester strains
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: • +S9: 2-aminoanthracene (2.5 µg/plate for S. typhimurium, 60 µg/plate for E. coli) • -S9: N-methyl-N'-nitro-N-nitrosoguanidine (5 µg/plate for TA 1535, TA 100) • -S9: 4-nitro-o-phenylenediamine (10 µg/plate for TA 98)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2
- 1st experiment: Standard plate test with and without S9 mix
- 2nd experiment: Preincubation test with and without S9 mix

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): approximately 10^9 cells per mL
- Test method: preincubation and plate incorporation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes
- Exposure duration/duration of treatment: 48-72 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition; decrease in the number of revertants (factor ≤ 0.6)

METHODS FOR MEASUREMENTS OF GENOTOXICIY :
- Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments.

OTHER:
- Precipitation
Evaluation criteria:
Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 2500 µg/plate (slight decrease in the number of his+ revertants) at plate incorporation test only (1st experiment)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True 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
Remarks:
at and above 2500 µg/plate and at 5000 µg/plate with and without S9, respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1000 µg/plate and 5000 µg/plate without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Solubility:
Test substance precipitation was observed at and above 2500 μg/plate with and without S9 mix.TEST-SPECIFIC CONFOUNDING FACTORS

Cytotoxicity:
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was observed only using tester strain TA 1535 with and without S9 mix at and above 2500 μg/plate. In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ revertants) was occasionally observed depending on the strain and test conditions at and above 1000 μg/plate.

Experimental Results:

Standard Plate Test:

Mean revertants per plate
Strain TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
DMSO 18.7 18.0 115.0 116.0 9.0 11.0 22.3 32.7 31.3 30.0
33 15.0 19.0 114.3 123.0 6.7 14.3 21.0 28.7 26.3 30.3
100 14.0 19.0 111.7 117.7 7.0 7.7 19.0 35.3 26.7 34.0
333 15.0 15.3 119.3 129.3 10.7 12.0 16.0 30.3 31.7 28.0
1000 21.7 12.3 125.7 120.3 6.7 12.7 19.0 33.7 24.3 30.3
2500 10.7 12.0 109.7 112.3 8.3 8.7 17.3 27.0 22.7 27.3
5000 11.7 9.7 101.7 112.3 8.0 7.7 16.7 23.3 22.3 24.0
positive control 5536.3 284.0 3517.7 2227.0 943.3 162.7 871.3 2520.3 1276.7 226.3

Preincubation test:

Mean revertants per plate
Strain TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
DMSO 13.7 12.3 114.7 115.0 9.3 8.3 27.7 28.3 30.0 29.7
33 14.0 9.3 111.7 104.0 6.3 6.7 23.0 22.7 25.7 26.3
100 15.7 8.7 109.3 109.7 11.0 8.0 22.3 29.0 29.0 25.0
333 12.7 9.3 108.3 108.3 9.7 9.7 22.7 32.0 32.0 26.0
1000 12.7 14.7 109.3 104.3 11.0 10.7 17.0 29.3 26.0 28.0
2600 15.7 10.3 102.7 104.3 5.7 6.0 18.3 26.7 26.0 24.3
5200 11.0 9.0 101.3 98.0 4.0 4.7 16.3 23.0 24.0 20.7
positive control 4773.0 126.7 3041.3 1307.7 756.0 105.0 876.0 1128.0 647.0 240.3
Conclusions:
Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. Strains used were TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The dose range was 33 μg - 5000 μg/plate (SPT) 33 μg - 5000 μg/plate (PIT). Test conditions chosen were the standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was observed at and above 2500 μg/plate with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at and above 1000 μg/plate. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system. Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Apr - Oct 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:
Jul 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: test substance was weighed and topped up with DMSO and shaken thoroughly
- Final dilution of a dissolved solid, stock liquid or gel: further concentrations were diluted from the stock solution according to the planned doses

FORM AS APPLIED IN THE TEST: solution
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: rat liver
- method of preparation of S9 mix: At least 5 male Wistar rats [Crl:WI(Han)] received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days. Twenty-four hours after the last administration, rats were sacrificed, and livers were prepared using sterile solvents and glassware at a temperature of +4°C. Livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.
- concentration or volume of S9 mix and S9 in the final culture medium : 0.5 ml
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): yes
Test concentrations with justification for top dose:
The doses/concentrations tested in this study were selected in accordance with the requirements set forth in the test guidelines and based on the results of a preliminary range finding test (experimental conduct with records and documentation in general accordance with the GLP principles).
Based on a pre-test, the following concentrations were chosen for the main experiment; 4.69, 9.38, 18.75, 37.50, 75.00, 150.00 µg/ml both with and without metabolic activation. Due to the low rate of mutant colonies in the concurrent vehicle control groups the mutant frequencies in the test groups 18.75 μg/mL in the absence and presence of S9 mix and 37.50 μg/mL only in the absence of S9 mix showed a statistically significant increase in the mutant frequencies. For clarification of the results, a 2nd Experiment was performed in the absence and presence of S9 mix using the following concentrations; 9.38, 12.50, 18.75, 25.00, 37.50, 75.00, 150.00 µg/ml.
- 3rd experiment: 9.38, 12.50, 18.75, 25.00, 37.50, 75.00, 150.00 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in culture medium, dimethyl sulfoxide (DMSO) was selected as the vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical data are available.

- Justification for percentage of solvent in the final culture medium: The final concentration of the vehicle DMSO in culture medium was 1% (v/v).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
- Number of independent experiments: three

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20-24 h after seeding
- Exposure duration/duration of treatment: 4 h
- Cells seeded: 0.8 - 1x106 cells were seeded per flask (175 cm²)

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): Entire expression period of 7-9 days
- Selection time (if incubation with a selective agent): 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: flasks
- Selective agent used: 6-thioguanine (10 µg/ml)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency (CE), relative survival (RS)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- mutant frequency. The number of colonies in each flask was counted and recorded.

OTHER:
- pH
- Osmolality
- Solubility
- Cell morphology
Evaluation criteria:
Assessment criteria
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).
Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit)
Statistics:
- An appropriate statistical trend test (MS EXCEL function RGP) was performed. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0.
- Fisher's exact test with Bonferroni-Holm correction. Calculation was performed using R.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the first experiment in the absence of metabolic activation, values in dose group 18.75 µg/ml and in dose group 37.50 µg/ml were statistically significantly increased compared to the concurrent vehicle control value. However, values were within the range of the 95% control limit and a concentration-response relationship was absent. In the first experiment in the presence of metabolic activation, values in dose group 9.38 µg/ml and 18.75 µg/ml were statistically significantly increased compared to the concurrent vehicle control value. However, values were within the range of the 95% control limit and a concentration-response relationship was absent.
The relevance of the observed statistical significance was assessed in a 2nd Experiment.
In the second experiment in the presence of metabolic activation, one value in dose group 25.00 μg/mL exceeded the range of the 95% vehicle control limit. However, this increase was not statistically significant. A concentration related increase in the mutant frequencies was not observed and no statistically significant increase in the mutant frequencies was determined in any of the tested concentrations. The relevance of the observed increase in the mutant frequency was assessed in a 3rd Experiment.
In the 3rd Experiment in the presence of metabolic activation, obtained values were within the range of the 95% vehicle control limit. A concentration related increase in the mutant frequencies was not observed and no statistically significant increase in the mutant frequencies was determined.

Osmolality and pH values were not influenced by test substance treatment. In the 1st Experiment in the presence and absence of S9 mix, test substance precipitation was observed macroscopically in culture medium at the end of treatment at 75.00 μg/mL and above. In the 2nd and 3rd Experiment precipitation occurred from 37.50 μg/mL onward. After 4 hours treatment either in the absence or presence of metabolic activation, the cell morphology and attachment of the cells was not adversely influenced (grade > 2) in any test group tested for gene mutations.

Table 1: Summary of results - experimental parts without S9 mix

Exp.

Exposure period [h]

Test groups [µg/ml]

S9 mix

Prec.*

Genotoxicity** MFcorr. [per 106cells]

Cytotoxicity***

 

 

 

 

 

 

RS [%]

CE2 [%]

1

4

Vehicle control1

-

n.d.

0.60

100.0

100.0

 

 

4.69

-

-

n.c.

156.7

n.c.

 

 

9.38

-

-

1.56

151.9

97.0

 

 

18.75

-

-

4.78s

229.5

94.9

 

 

37.50

-

-

3.27s

187.2

92.4

 

 

75.00

-

+

0.75

167.7

81.0

 

 

150.00

-

+

n.c.1

n.c.1

n.c.1

 

 

Positive control2

-

n.d

438.29s

123.9

52.9

2

4

Vehicle control1

-

n.d.

2.92

100.0

100.0

 

 

9.38

-

-

0.00

96.6

97.4

 

 

12.50

-

-

3.10

102.8

94.2

 

 

18.75

-

-

2.00

64.7

97.4

 

 

25.00

-

-

3.07

112.9

84.7

 

 

37.50

-

+

2.83

105.7

91.9

 

 

75.00

-

+

n.c.1

n.c.1

n.c.1

 

 

150.00

-

+

n.c.1

n.c.1

n.c.1

 

 

Positive control2

-

n.d.

121.57s

108.9

82.8

* Macroscopically visible precipitation in culture medium at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per 106 cells corrected with the CE2 value

*** Cloning efficiency related to the respective vehicle control

s Mutant frequency statistically significantly higher than corresponding control values (p ≤ 0.05)

n.c. Culture was not continued since a minimum of only four analysable concentrations is required

n.c.1 Culture was not continued since only one concentration beyond the solubility limit is required

n.d. Not determined

1 DMSO 1% (v/v) 2 EMS 400 μg/mL 3 DMBA 1.25 μg/mL

Table 2: Summary of results - experimental parts with S9 mix

Exp.

Exposure period [h]

Test groups [µg/ml]

S9 mix

Prec.*

Genotoxicity** MFcorr. [per 106cells]

Cytotoxicity***

 

 

 

 

 

 

RS p%[

CE2[%]

1

4

Vehicle control1

+

n.d.

0.50

100.0

100.0

 

 

4.69

+

-

n.c.

102.9

n.c.

 

 

9.38

+

-

3.89s

86.9

84.1

 

 

18.75

+

-

6.21s

86.5

77.1

 

 

37.50

+

-

1.93

75.3

91.2

 

 

75.00

+

+

1.79

68.3

98.5

 

 

150.00

+

+

n.c.1

n.c.1

n.c.1

 

 

Positive control3

+

n.d.

143.59s

67.3

78.6

2

4

Vehicle control1

+

n.d.

5.05

100.0

100.0

 

 

9.38

+

-

1.74

130.5

116.2

 

 

12.50

+

-

5.19

112.9

103.7

 

 

18.75

+

-

0.55

109.0

122.2

 

 

25.00

+

-

8.72

95.5

100.3

 

 

37.50

+

+

1.68

88.8

100.0

 

 

75.00

+

+

n.c.1

n.c.1

n.c.1

 

 

150.00

+

+

n.c.1

n.c.1

n.c.1

 

 

Positive control3

+

n.d.

79.67s

105.9

102.7

3

4

Vehicle control1

+

n.d.

6.23

100.0

100.0

 

 

9.38

+

-

n.c.

82.7

n.c.

 

 

12.50

+

-

1.92

93.3

94.5

 

 

18.75

+

-

1.24

85.9

83.9

 

 

25.00

+

-

1.00

92.3

77.7

 

 

37.50

+

+

2.75

77.6

75.6

 

 

75.00

+

+

n.c.1

n.c.1

n.c.1

 

 

150.00

+

+

n.c.1

n.c.1

n.c.1

 

 

Positive control3

+

n.d.

87.86s

74.8

72.7

* Macroscopically visible precipitation in culture medium at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per 106 cells corrected with the CE2 value

*** Cloning efficiency related to the respective vehicle control

s Mutant frequency statistically significantly higher than corresponding control values

n.c. Culture was not continued since a minimum of only four analysable concentrations is required

n.c.1 Culture was not continued since only one concentration beyond the solubility limit is required

n.d. Not determined

1 DMSO 1% (v/v) 2 EMS 400 μg/mL 3 DMBA 1.25 μg/mL

Conclusions:
Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
Executive summary:

The substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out.

The 1st and 2nd Experiment in the absence and presence of liver S9 mix and the 3rd Experiment only in the presence of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following concentrations were tested. The highest tested concentration (150 μg/mL) was based on the test substance precipitation in culture medium at the end of test substance treatment. Test concentrations ranged from 4.69 µg/ml to 150 µg/ml. Test groups ranging from 9.38 µg/ml to 75 µg/ml were evaluated for gene mutations.

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line.

Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]- anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

In this study, in both experiments in the absence and the presence of metabolic activation no relevant cytotoxicity (relative survival below 20%) was observed up to the highest concentrations evaluated for gene mutations.

Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in three experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb 2019 - Feb 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
Jul 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 735/2017; B.49
Version / remarks:
Feb 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution.
To achieve a solution of the test substance in the vehicle, the test substance preparation was treated with shaken thoroughly.
The further concentrations were diluted from according to the planned doses.
All test substance solutions were prepared immediately before administration.

FORM AS APPLIED IN THE TEST: solution

OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: At the beginning of the treatment period, the pH was measured at least for the top concentration and for the vehicle control, each. At the beginning of the treatment period, the osmolality was measured at least for the top concentration and for the vehicle control, each. Test substance precipitation was checked immediately after start of treatment of the test cultures (macroscopically) and at the end of treatment (macroscopically and using a microscope).
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Human lymphocytes

For lymphocytes:
- Sex, age and number of blood donors: 1st, 3rd, 4th and 5th Experiment blood from male donors (29, 33, 34 and 29 years old, respectively) was used. In the 6th Experiment blood from a 23 year old female donor was used.
- Whether whole blood or separated lymphocytes were used: Whole blood cultures

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: In this study all incubations were performed at 37°C with a relative humidity of ≥ 90% in a 5% [v/v] CO2 atmosphere.
Cytokinesis block (if used):
For the Cytochalasin B treatment the medium was supplemented with: 30 μL Cytochalasin B (Cyt B, stock solution: 2 mg/mL in DMSO, final concentration: 6 μg/mL)
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : rat liver
- method of preparation of S9 mix: At least 5 male Wistar rats received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days. Twenty-four hours after the last administration, rats were sacrificed, and induced livers were prepared using sterile solvents and glassware at a temperature of +4°C. Livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution (1 mL ≙ 1 g wet liver), then homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5-mL portions of the supernatant (S9 fraction) was stored at -70°C to -80°C.
- concentration or volume of S9 mix and S9 in the final culture medium : 6 ml
Test concentrations with justification for top dose:
The concentrations were selected in accordance with the requirements set forth in the test guidelines and based on the results of two preliminary solubility tests (experimental conduct with records and documentation in general accordance with the GLP principles).
- 1st: 2.04, 3.68, 6.62, 11.91, 21.43, 38.58, 69.44, 125.00 µg/ml (with and without S9)
- 3rd (without S9): 0.34, 0.62, 1.11, 2.01, 3.61, 6.50, 8.00, 12.00 µg.ml; (with S9): 0.19, 0.34, 0.62, 1.11, 2.01, 3.61, 6.50, 12.00 µg/ml
- 4th (without S9): 0.11, 0.21, 0.37, 0.67, 1.20, 2.16, 3.89, 7.00 µg/ml
- 5th (with S9): 0.06, 0.11, 0.21, 0.37, 0.67, 1.20, 2.16, 2.89, 7.00 µg/ml
- 6th (wothout S9): 0.91, 1.63, 2.94, 5.29 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in culture medium, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the in vitro micronucleus test and for which historical control data are available.
- Justification for percentage of solvent in the final culture medium: The final concentration of the vehicle DMSO in culture medium was 1% (v/v).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive control substance:
colchicine
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 6

TREATMENT SCHEDULE:
Day 1: Activation of the cells with Phytohemagglutinin
Day 3: Test substance incubation (approx. 48 hours after activation); removal of test substance by intense washing (only for 4 hours exposure period)
Day 4: Removal of test substance by intense washing (only for 20 hours exposure period); treatment with Cyt B
Day 5: Preparation of the slides
- Stimulating time: 48 h
- Exposure time: 4/20 h
- Recovery time: 16 h
- Harvest time: 20 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: cytochalasin B, 6 µg/ml
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): After the hypotonic treatment, cells were fixed. The fixation step will be repeated twice. After the last fixation step, cells can be spread on slides.The slides were dipped in deionized water, the cells were pipetted on the slide and fixed by passing through a flame. Cells were stained with May-Grünwald (3 min) and 10% [v/v] Giemsa (in Titrisol, pH 7.2, 10 min) and mounted.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): As a rule, at least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The diameter of the micronucleus was less than 1/3 of the main nucleus, The micronucleus was not linked to the main nucleus and was located within the cytoplasm of the cell, Only binucleated cells were scored.
- The cytokinesis-block proliferation index (CBPI) is a direct measure of the proliferative activity of the cells and it was determined in 500 cells per culture (1000 cells per test group).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index

Evaluation criteria:
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).
A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).
Statistics:
one-sided Fisher's exact test, statistical trend test (SAS procedure REG), significance level p ≤ 0.05)
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1st exp.: with and without S9 decreased cell proliferation at 2.04 µg/ml and 6.62 μg/mL, resp. 5th exp.: Slight cytostasis after 4h with 2.16 μg/mL . 6th exp.: After 20 h without S9, decreased cell proliferation at 17.15 μg/mL and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the 2nd Experiment in the absence of S9 mix after 4 hours treatment precipitation was observed down to 5.00 μg/mL. Only one dose group was dissolved, and therefore, this experimental part did not fulfill the acceptance criteria of the current OECD guideline. In the 2nd Experiment with S9 mix due to a technical error the slides were not scorable. Therefore, this experimental part was repeated in the 3rd Experiment. In the 4th Experiment in the presence of S9 mix the positive control substance CPA did not cause the expected distinct increase in the number of micronucleated cells. Due to this, the requirements of the current OECD guidelines were not fulfilled, this experiment was repeated in the 5th Experiment. In the 5th Experiment in the absence of metabolic activation, after 20 hours continuous treatment, no test substance precipitation in culture medium was observed. According to the previous experimental parts without S9 mix no cytotoxicity was expected. Therefore, this experimental part was discontinued and repeated in the 6th Experiment.

The results for the valid experiments are described below. In the absence of S9 mix three valid experiments with 4 hours pulse treatment (experiment 1, 3 and 4) and one experiment valid with 20 hours continuous treatment (experiment 6) were performed. In the presence of S9 mix three valid experiments with 4 hours exposure (experiment 1, 3 and 5) were performed.

After 4 hours treatment without S9 mix in the 1st Experiment compared to the concurrent vehicle control group (0.7% micronucleated cells) no statistically significant increase in the number of micronucleated cells was observed. In the dose group treated with 3.68 and 11.91 μg/mL the values (0.3 and 0.2% micronucleated cells, respectively) were below the 95% upper control limit of the historical negative control data range (0.9% micronucleated cells). In the dose group 6.62 μg/mL the value (1.3% micronucleated cells) exceeded the maximum of the historical negative control data range (1.0% micronucleated cells). However, the increase in the number of micronucleated cells could be observed in only one of two cultures (culture A) and a dose-response as assesd by trend analysis could not be observed. In the 3rd Experiment without S9 mix the values of all test groups (0.3 – 0.4% micronucleated cells) were as high as or below the value of the concurrent vehicle control group (0.4% micronucleated cells) and below the 95% upper control limit of the historical negative control data 0.9% micronucleated cells). In the 4th Experiment the values of all test groups (0.5 – 0.9% micronucleated cells) were below or as high as the 95% upper control limit of the historical negative control data (0.9% micronucleated cells). A statistical significance compared to the concurrent vehicle control group (0.6% micronucleated cells) could not be observed. Neither of the two repeat experiments described above (experiment 3 and 4) showed a positive dose reponse as assesd by a trend analysis.
After 20 hours continuous treatment in the 6th Experiment the values of all test groups (0.4 – 0.7% micronucleated cells) were below the value of the concurrent vehicle control group and below the 95% upper control limit of the historical negative control data range (both 0.9% micronucleated cells). No positive trend in the response was observed.
In the presence of S9 mix in the 1st Experiment the values of the test groups treated with 3.68 and 6.62 μg/mL (0.5 and 0.6% micronucleated cells, respectively) were below or as high as the value of the concurrent vehicle control group (0.6% micronucleated cells), in the test group treated with 2.04 μg/mL the value was statistically significant increased (1.4% micronucleated cells) and exceeded the 95% upper control limit of the historical negative control data range (1.2% micronucleated cells). However, this value ist not dose related increased as assesd by a trend analysis and the increase could be observed in only one of two cultures (culture A). To clarify these finding two repeat experiments (experiment 3 and 5 were performed). In the 3rd Experiment no dose related, or statistically significant increase compared to the concurrent vehicle control group (0.4% micronucleated cells) was observed. The values of all test groups (0.4 – 0.6% micronucleated cells) were below the 95% upper control limit of the historical negative control data (1.2% micronucleated cells). In the 5th Experiment with S9 mix compared to the concurrent vehicle control group (0.3% micronucleated cells) a statistically significant increase in the microncueleus frequency could not be observed. All values (0.3 – 0.5%
micronucleated cells) were below the 95% upper control limit of the historical negative control data (1.2% micronucleated cells). A dose-related increase assesd by trend analysis was, however, observed.
The positive control substances MMC (without S9 mix 0.04 and 0.31 μg/mL), Colchicine (without S9 mix: 0.05 μg/mL) and CPA (with S9 mix; 2.50 μg/mL) induced statistically significantly increased micronucleus frequencies in all independently performed experiments. In this study, in the absence and presence of metabolic activation the frequencies of micronucleated cells (4 h without S9 mix: 8.2 – 14.0 % micronucleated cells; 20 h without S9 mix: 4.4% and 2.9% micronucleated cells (MMC and Col, respectively); with S9 mix 2.6 – 4.5% micronucleated cells) were compatible to the historical positive control data range (Appendix 7).

TREATMENT CONDITIONS
The Osmolality and pH values were not relevantly influenced by test substance treatment. In the absence of S9 mix after four hours of treatment test substance precipitation in culture medium was observed at 11.91, 3.61 or 3.89 μg/mL and above in the 1st, 3rd and 4th Experiment, respectively. After 20 hours continuous treatment without metabolic activation in the 6th Experiment test substance precipitation in culture medium occurred at 55.56 μg/mL and above. At the end of treatment in the presence of S9 mix test substance precipitation in culture medium was observed at 6.62, 0.62 or 3.89 μg/mL and above in the 1st, 3rd or 5th Experiment, respectively.

CYTOTOXICITY
In the 1st Experiment without metabolic activation decreased cell proliferation could be observed at 6.62 μg/mL (CBPI cytostasis: 35.9%). However, in this test group the cell proliferation was only in culture A decreased, both cultures were evaluable for the occurrence of micronuclei. In the 3rd Experiment without S9 mix no relevantly reduced cell proliferation was observed. In the 4th Experiment slight cytostasis was observed after 4 hours treatment with 2.16 μg/mL (CBPI cytostasis: 21.6%). After 20 hours continuous treatment without S9 mix in the 6th Experiment decreased cell proliferation occurred at 17.15 μg/mL and above (CBPI cytostasis: 19.0 – 40.0%). In the presence of S9 mix in the 1st Experiment cytotoxicity occurred in the test group 2.04 μg/mL (CBPI cytostasis 31.9%). However, in this test group the cell proliferation was only
in culture A decreased, both cultures were evaluable for the occurrence of micronuclei. In the repeat experiments with S9 mix (3rd and 5th Experiment), no relevantly reduced cell proliferation was observed.

Table 1: Summary of results - experimental parts without S9 mix

 

 

Exp.

 

Exposure/Recovery/Preparation interval

 

Test groups [µg/mL]

 

S9

mix

 

Prec.*

 

Micro- nucleatedcells**[%]

 

Cytotoxicity Proliferationindexcytostasis[%]

1

4/16/20

Vehicle control1

-

n.d

0.7

0.0

 

 

2.04

-

-

n.d.

-5.1

 

 

3.68

-

-

0.3

-2.4

 

 

6.62

-

-

1.3

35.9

 

 

11.91

-

+

0.2

16.7

 

 

21.43

-

+

n.p.

n.p.

 

 

38.58

-

+

n.p.

n.p.

 

 

69.44

-

+

n.p.

n.p.

 

 

125.00

-

+

n.p.

n.p.

 

 

Positive control2

-

n.d

14.0S

21.0

3

4/16/20

Vehicle control1

-

n.d

0.4

0.0

 

 

0.34

-

-

n.d.

0.5

 

 

0.62

-

-

n.d.

2.1

 

 

1.11

-

-

0.4

7.2

 

 

2.01

-

-

0.3

8.1

 

 

3.61

-

+

0.3

6.4

 

 

6.50

-

+

n.p.

n.p.

 

 

8.00

-

+

n.p.

n.p.

 

 

12.00

-

+

n.p.

n.p.

 

 

Positive control2

-

n.d

8.2S

21.8

4

4/16/20

Vehicle control1

-

n.d

0.6

0.0

 

 

0.11

-

-

n.d.

-1.7

 

 

0.21

-

-

n.d.

10.7

 

 

0.37

-

-

n.d.

10.7

 

 

0.67

-

-

n.d.

11.3

 

 

1.20

-

-

0.5

8.4

 

 

2.16

-

-

0.9

21.6

 

 

3.89

-

+

0.5

13.5

 

 

7.00

-

+

n.p.

n.p.

 

 

Positive control2

-

n.d

9.5S

52.0

6

20/0/20

Vehicle control1

-

n.d

0.9

0.0

 

 

0.91

-

-

n.d.

2.2

 

 

1.63

-

-

n.d.

3.3

 

 

2.94

-

-

n.d.

2.7

 

 

5.29

-

-

n.d.

6.7

 

 

9.53

-

-

0.5

10.5

 

 

17.15

-

-

n.d.

19.0

 

 

30.86

-

-

0.4

32.1

 

 

55.56

-

+

0.7

40.0

 

 

100.00

-

+

n.p.

n.p.

 

 

Positive control3

-

n.d

4.4S

14.1

 

 

Positive control4

-

n.d

2.9S

17.5

* Precipitation in culture medium at the end of exposure period (macroscopically)

** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group

SFrequency statistically significantly higher than corresponding control values

n.d. Not determined

n.p. Not continued due to strong test substance precipitation

1DMSO 1% (v/v)2MMC 0.31 μg/mL3MMC 0.04 μg/mL4Col 0.05 μg/mL

Table 2: Summary of results - experimental parts with S9 mix

 

 

Exp.

 

Exposure/Recovery/Preparation interval

 

Test groups [µg/mL]

 

S9

mix

 

Prec.*

 

Micro- nucleatedcells**[%]

 

Cytotoxicity Proliferationindexcytostasis[%]

1

4/16/20

Vehicle control1

+

n.d

0.6

0.0

 

 

2.04

+

-

1.4S

31.9

 

 

3.68

+

-

0.5

-0.1

 

 

6.62

+

+

0.6

2.0

 

 

11.91

+

+

n.p.

n.p.

 

 

21.43

+

+

n.p.

n.p.

 

 

38.58

+

+

n.p.

n.p.

 

 

69.44

+

+

n.p.

n.p.

 

 

125.00

+

+

n.p.

n.p.

 

 

Positive control5

+

n.d

4.5S

26.4

3

4/16/20

Vehicle control1

+

n.d

0.4

0.0

 

 

0.19

+

-

0.5

6.3

 

 

0.34

+

-

0.6

-0.5

 

 

0.62

+

+

0.4

4.5

 

 

1.11

+

+

n.p.

n.p.

 

 

2.01

+

+

n.p.

n.p.

 

 

3.61

+

+

n.p.

n.p.

 

 

6.50

+

+

n.p.

n.p.

 

 

12.00

+

+

n.p.

n.p.

 

 

Positive control5

+

n.d

2.6S

15.2

5

4/16/20

Vehicle control1

+

n.d

0.3

0.0

 

 

0.06

+

-

n.d.

-0.5

 

 

0.11

+

-

n.d.

0.7

 

 

0.21

+

-

n.d.

1.6

 

 

0.37

+

-

n.d.

10.9

 

 

0.67

+

-

n.d.

2.9

 

 

1.20

+

-

0.3

2.0

 

 

2.16

+

-

0.4

2.0

 

 

3.89

+

+

0.5

5.6

 

 

7.00

+

+

n.p.

n.p.

 

 

Positive control5

+

n.d

2.6S

6.4

* Precipitation in culture medium at the end of exposure period (macroscopically)

** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group

SFrequency statistically significantly higher than corresponding control values

n.d. Not determined

n.p. Not continued due to strong test substance precipitation

1DMSO 1% (v/v) 5CPA 2.50 μg/mL

Conclusions:
Under the experimental conditions chosen here, the conclusion is drawn that has no potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in primary human lymphocytes in the absence and the presence of metabolic activation.
Executive summary:

The substance was assessed for its potential to induce micronuclei in primary human lymphocytes in vitro (clastogenic or aneugenic activity). Six independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to two range-finding solubility tests for the determination of the experimental doses, concentrations ranging from 0.06 µg/ml to 125.00 µg/ml were tested in six individual experiments. A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group.

In this study, dimethyl sulfoxide (DMSO) was selected as vehicle. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for primary human lymphocytes. The positive control substances, Mitomycin C (MMC), Colchicin and Cyclophosphamide (CPA), led to the expected increase in the number of cells containing micronuclei. In this study, no relevant cytotoxicity indicated by reduced proliferation index (CBPI) of or above 50% was observed up to the highest applied test substance concentration. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system. Thus, under the experimental conditions described, is considered to have no chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in primary human lymphocytes in the absence and the presence of metabolic activation.

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

Additional information

In vitro Gene Mutation - Bacteria Cells

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. Strains used were TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The dose range was 33 μg - 5000 μg/plate (SPT) 33 μg - 5000 μg/plate (PIT). Test conditions chosen were the standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was observed at and above 2500 μg/plate with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at and above 1000μg/plate. A relevant increase in the number of his+ or trp+ revertants (factor≥2: TA 100, TA 98 and E.coli WP2 uvrA or factor≥3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system. Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation (2019).

 

In vitro Gene Mutation - Mammalian Cells

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out. The 1st and 2nd Experiment in the absence and presence of liver S9 mix and the 3rd Experiment only in the presence of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following concentrations were tested. The highest tested concentration (150 μg/mL) was based on the test substance precipitation in culture medium at the end of test substance treatment. Test concentrations ranged from 4.69 µg/ml to 150 µg/ml. Test groups ranging from 9.38 µg/ml to 75 µg/ml were evaluated for gene mutations. Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]- anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations. In this study, in both experiments in the absence and the presence of metabolic activation no relevant cytotoxicity (relative survival below 20%) was observed up to the highest concentrations evaluated for gene mutations. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in three experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation (2019).

 

In Vitro Micronucleus - Mammalian Cells

The test substance was assessed for its mutagenic potential in the in vitro micronuclei assay in several experiments. In none of the valid assay was a biologically relevant mutagenic effect observed. In the first experiment two values were obtained in the presence and absence of metabolic activation, which were above the 95% control limit. However, these increases were not reproduced in the the repeat experiments. Furthermore, the increases were only observed in one of the parallel cultures and were not dose related. Thus, the increases are considered as incidental. The positive trend observed in the 5th Experiment with S9 mix is considered as incidental, since the values of the treated groups is well below the upper limit of the 95% control limit and not statistically significant as compared to the corresponding vehicle control value. The precipitation of the test item in the cultures varied between the experiments. This effect can not be explained. Nevertheless, each experiment as such is considered as valid. According to the results of the present in vitro micronucleus assay, the test substance did not lead to a biologically relevant increase in the number of micronucleated cells either without S9 mix or after the addition of a metabolizing system. The number of micronucleated cells in the vehicle control groups were within the historical negative control data range (95% control limit) and, thus, fulfilled the acceptance criteria of this study. The proficiency of the laboratory to perform the micronucleus test in primary human lymphocytes was demonstrated by the laboratory’s historical control database on vehicle and positive controls and by X-bar chart to identify the variability of the vehicle control data. The increase in the frequencies of micronuclei induced by the positive control substances MMC, Colchicine and CPA clearly demonstrated the sensitivity of the test system and/or the metabolic activity of the S9 mix employed. The values were compatible the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. Thus, under the experimental conditions chosen here, the conclusion is drawn that the test item has no potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in primary human lymphocytes in the absence and the presence of metabolic activation.

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 the purpose of classification under Regulation 1272/2008. Based on the criteria laid down in Regulation (EC) No.1272/2008, classification for genetic toxicity is not warranted.