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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation (OECD 471): negative

Cytogenicity/chromosome aberration in mammalian cells (OECD 473): negative without metabolic activation, positive with metabolic activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 Dec 2014 - 13 Jan 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted in 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted in 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
adopted in 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Gyógyszerészeti és Egészségügyi Minőség- és Szervezetfejlesztési Intézet (National Institute for Quality- and Organizational Development in Healthcare and Medicines), Budapest, Hungary
Type of assay:
other: in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Cell proliferation: doubling time 12 - 14 h
- Type and identity of media:
DME medium supplemented with:
- L-glutamine (2mM)
- 1% of Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphoptericin-B)
- heat-inactivated bovine serum (final concentration 10 %)
During the 3 and 20 h treatments the serum content was reduced to 5%.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding study:
3 h treatment: 0.5, 1, 2, 4, 8, 16 and 32 µg/mL without S9 mix
3 h treatment: 5, 10, 15, 30, 60, 120 and 240 µg/mL with S9 mix
20 h treatment: 0.5, 1, 2, 4, 8, 16 and 32 µg/mL without S9 mix

Experiment 1:
3 h treatment: 0.5, 1, 2, 4 and 4.5 µg/mL without S9 mix
3 h treatment: 10, 20, 40, 80 and 90 µg/mL with S9 mix

Experiment 2:
3 h treatment: 10, 20, 40, 80 and 90 µg/mL with S9 mix
20 h treatment: 0.5, 1, 2, 4 and 4.5 µg/mL without S9 mix

4.5 µg/mL was tested but not evaluated due to sufficient cytotoxicity at the next lower concentration and sufficient number of concentrations.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h and 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 3 h treatment: 20 h and 28 h; 20 h treatment: 20 h and 28 h

SPINDLE INHIBITOR (cytogenetic assays): colchicine, 0.2 µg/mL
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: Relative Increase in Cell Counts (RICC)

OTHER EXAMINATIONS
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
Validity criteria for the test:
The chromosome aberration assay is considered acceptable if it meets the following criteria:
- the number of aberrations found in the negative and/or solvent controls falls within the range of historical laboratory control data.
- the positive control items produce biologically relevant increases in the number of cells with structural chromosome aberrations.

Evaluation of test results:
The test item is regarded as non-clastogenic if:
- the number of metaphases with structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data
- and/or no significant increase in the number of metaphases with structural chromosome aberration is observed.

A test item is classified as clastogenic if it meets the following criteria:
- increase in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (above the range of our historical control data).
- the increase is reproducible between replicate cultures and between tests (when the treatment conditions are the same).
- the increase is statistically significant.
Both, biological and statistical significance should be considered together.
Statistics:
For statistical analysis, Fisher exact and CHI2 tests were utilized. The parameters evaluated for statistical analysis were as follows: number of aberration and number of cells with aberration (with and without gaps).
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 80 µg/mL and higher following the 3 h treatment
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 4 µg/mL following the 3 h and 20 h treatment
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No significant differences between test item treatment and control groups were observed.
- Effects of osmolality: No significant differences between test item treatment and control groups were observed.
- Precipitation: A clear solution was obtained up to a concentration of 10 mg/mL. There was no precipitation in the medium at any concentration tested.

RANGE-FINDING/SCREENING STUDIES: In order to determine the treatment concentrations of test item in the cytogenetic study a dose selection was performed. The cells were treated using increasing concentrations of test item in the absence or presence of S9 mix and were incubated at 37 °C for 3 h. Cell counts were performed after 20 h. Additional groups of cells were treated for 20 h without metabolic and for 3 h with metabolic activation, with cell counts conducted after 20 h (without S9 mix only) and 28 h (without and with S9 mix).

COMPARISON WITH HISTORICAL CONTROL DATA: In the concurrent negative control group the percentage of cells with structural aberration(s) without gap was equal or less than 5 %, confirming the suitability of the cell line used.

Table 1. Results of chromosomal aberration test.

Test item

Concentration

RICC

Aberrant cells per 200 metaphase chromosome spreads

 

in µg/mL

in %

No. of cells with aberrations
(+ gaps)

No. of cells with aberrations
(- gaps)

Exposure period 3 h, sampling time 20 h, without S9 mix

DMSO

0.45 µL

100

4

2

EMS

1.0 µL

- 

26**

21**

Test substance

0.5

97

6

2

1

86

5

2

2

77

5

2

4

50

7

2

Exposure period 3 h, sampling time 20 h, with S9 mix

DMSO

9 µL

100

6

3

CP

5.0

 -

35**

30**

Test substance

10

99

6

3

20

86

7

4

40

80

10

4

80

50

9

6

90

44

11

7

Exposure period 20 h, sampling time 20 h, without S9 mix

DMSO

0.45 µL

100

4

2

EMS

0.4 µL

 

32**

26**

Test substance

0.5

94

5

3

1

82

4

2

2

67

4

2

4

49

6

3

Exposure period 20 h, sampling time 28 h, without S9 mix

DMSO

0.45 µL

100

4

2

EMS

0.4 µL

- 

33**

27**

Test substance

0.5

98

4

2

1

81

4

2

2

74

4

2

4

50

5

2

Exposure period 3 h, sampling time 28 h, with S9 mix

DMSO

9 µL

100

3

1

CP

5.0

 -

34**

29**

Test substance

10

97

4

2

20

92

4

2

40

79

5

3

80

49

7

4

90

43

11*

7*

* (p ≤ 0.05); ** (p ≤ 0.01)

DMSO: Dimethyl sulfoxide

EMS: Ethylmethane sulphonate

CP: Cyclophosphamide

RICC: Relative Increase in Cell Counts

In Experiment 1, the test item caused a moderate increase in the number of cells with structural chromosome aberrations in the presence of metabolic activation, up to and including cytotoxic concentrations. This increase was dose associated and biologically important.

In Experiment 2, the test item caused an increase in the number of cells with structural chromosome aberrations without gaps in the presence of S9 mix at concentration of 90 µg/mL following 3 h treatment. This increase was biologically and statistically significant.

No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of the test item.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 - 25 Mar 2003
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:
adopted in 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted in 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Landwirtschaft und Forsten, Wiesbaden, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strain)
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:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone (80 mg/kg bw)
Test concentrations with justification for top dose:
Pre-experiment
3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation.

Experiment 1
33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation.

Experiment 2
33, 100, 333, 1000, 2500 and 5000 µg/plate with metabolic activation.
10, 33, 100, 333, 1000, 2500 and 5000 µg/plate without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)
Remarks:
+S9: 2-AA (2.5 µg/plate, TA1535, TA1537, TA98, TA100; 10 µg/plate, WP2 uvrA); -S9: sodium azide (10 µg/plate, TA1535 and TA100); 4-NOPD (50 µg/plate, TA1537; 10 µg/plate, TA98); methyl methane sulfonate (4 µL/plate, WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (experiment 1); preincubation (experiment 2)

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies or clearing of the background lawn
Statistics:
Mean values and standard deviation were calculated.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
2500 and 5000 µg/plate in Experiment I and II, without S9; 5000 µg/plate in Experiment II, with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1000 – 5000 µg/plate in Experiment I, 2500 and 5000 µg/plate in Experiment II, without S9; 5000 µg/plate in Experiment I and II, with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/plate in Experiment I, 2500 and 5000 µg/plate in Experiment II, without S9; 5000 µg/plate in Experiment II, with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/plate in Experiment I, 2500 and 5000 µg/plate in Experiment II, without S9; 5000 µg/plate in Experiment II, with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to the highest investigated concentration.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as part of experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA: The historical range of positive controls was exceeded without metabolic activation in strains TA1535 and TA100 (experiment I and II) and with metabolic activation in strains TA1537 and TA98 (experiment II). This effect indicates the sensitivity of the strains rather than comprising the assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in all strains used, except WP2 uvrA.

Table 1. Test results of experiment 1 (plate incorporation).

With or without S9 mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2 uvrA

TA98

TA1537

-

0

124 ± 9.1

10 ± 1.7

35 ± 1.7

21 ± 1.0

6 ± 1.7

-

0 (DMSO)

117 ± 2.3

11 ± 1.5

28 ± 2.1

20 ± 2.6

9 ± 2.5

-

33

120 ± 11.1

10 ± 4.0

24 ± 1.5

22 ± 4.7

5 ± 1.5

-

100

112 ± 9.3

10 ± 1.5

33 ± 4.5

23 ± 5.8

5 ± 1.5

-

333

121 ± 9.9

7 ± 4.0

30 ± 3.8

22 ± 4.6

4 ± 1.5

-

1000

121 ± 6.9

6 ± 0.6

34 ± 1.5

18 ± 3.2

2 ± 1.0

-

2500

103 ± 16.0

4 ± 2.9

35 ± 8.7

12 ± 5.6

3 ± 1.5

-

5000

0

1 ± 1.2

32 ± 4.7

0

1 ± 1.0

Positive controls,
-S9 mix

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentrations

(μg/plate)

10

10

4 μL

10

50

Mean No. of colonies/plate

(average of 3 ± SD)

953 ± 47.8

1038 ± 26.4

776 ± 117.8

235 ± 20.6

56 ± 5.5

+

0

141 ± 7.1

12 ± 3.5

31 ± 0.6

28 ± 3.1

10 ± 1.2

+

0 (DMSO)

135 ± 20.8

12 ± 2.5

40 ± 7.2

25 ± 4.2

8 ± 3.2

+

33

134 ± 19.6

13 ± 3.1

39 ± 4.7

22 ± 3.5

9 ± 4.0

+

100

145 ± 16.7

15 ± 3.5

39 ± 4.0

20 ± 1.5

13 ± 2.6

+

333

155 ± 4.0

18 ± 4.6

38 ± 8.6

27 ± 3.1

14 ± 2.1

+

1000

137 ± 12.3

15 ± 4.4

37 ± 7.1

29 ± 2.5

8 ± 1.5

+

2500

148 ± 5.0

10 ± 2.5

29 ± 4.5

26 ± 2.5

7 ± 2.5

+

5000

142 ± 11.2

10 ± 1.0

33 ± 3.8

21 ± 1.2

3 ± 2.3

Positive controls, +S9 mix

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

2.5

2.5

10

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

1436 ± 74.3

275 ± 15.7

196 ± 9.8

768 ± 82.4

125 ± 5.1

DMSO: Dimethyl sulfoxide

2-AA: 2-aminoanthracene

MMS: Methyl methane sulfonate

NaN3: Sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

Table 2. Test results of experiment 2 (preincubation).

With or without S9 mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2 uvrA

TA98

TA1537

-

0

111 ± 2.9

16 ± 1.2

30 ± 4.6

32 ± 10.0

6 ± 1.2

-

0 (DMSO)

92 ± 19.3

16 ± 6.0

36 ± 9.3

23 ± 2.0

6 ± 1.5

-

10

87 ± 9.5

16 ± 2.0

26 ± 4.9

22 ± 2.5

8 ± 1.5

-

33

92 ± 12.4

16 ± 2.1

29 ± 4.2

24 ± 1.5

6 ± 1.2

-

100

85 ± 11.6

16 ± 1.5

33 ± 1.5

24 ± 4.5

6 ± 3.1

-

333

67 ± 10.7

13 ± 3.6

32 ± 5.6

23 ± 1.7

6 ± 0.6

-

1000

75 ± 26.0

8 ± 1.0

41 ± 9.8

18 ± 0.6

6 ± 2.6

-

2500

25 ± 13.3

5 ± 1.2

41 ± 4.0

8 ± 2.3

2 ± 2.1

-

5000

14 ± 1.5

0

29 ± 3.5

2 ± 0.6

0

Positive controls,
-S9 mix

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentrations

(μg/plate)

10

10

4 μL

10

50

Mean No. of colonies/plate

(average of 3 ± SD)

1182 ± 45.0

1034 ± 50.4

499 ± 36.1

326 ± 40.7

63 ± 6.1

+

0

135 ± 14.4

17 ± 3.1

39 ± 5.7

30 ± 6.1

12 ± 4.0

+

0 (DMSO)

120 ± 2.3

15 ± 4.9

40 ± 1.2

27 ± 5.5

9 ± 1.5

+

33

108 ± 12.3

18 ± 7.0

38 ± 1.0

33 ± 4.2

12 ± 1.2

+

100

114 ± 21.5

19 ± 4.5

41 ± 6.5

31 ± 1.7

8 ± 2.6

+

333

122 ± 3.8

17 ± 3.1

39 ± 5.3

31 ± 4.0

8 ± 1.5

+

1000

132 ± 8.6

19 ± 2.1

28 ± 5.5

37 ± 5.2

8 ± 3.5

+

2500

150 ± 11.8

10 ± 2.0

32 ± 4.9

37 ± 5.7

9 ± 3.5

+

5000

27 ± 10.4

6 ± 1.7

29 ± 6.4

6 ± 1.7

3 ± 1.2

Positive controls, +S9 mix

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

2.5

2.5

10

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

1355 ± 131.6

227 ± 29.3

230 ± 6.1

1338 ± 30.1

192 ± 12.3

DMSO: Dimethyl sulfoxide

2-AA: 2-aminoanthracene

MMS: Methyl methane sulfonate

NaN3: Sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

Table 3. Reduction in the number of revertants at following concentrations (µg/plate).

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

2500, 5000

/

2500, 5000

5000

TA 1537

1000 - 5000

5000

2500, 5000

5000

TA 98

5000

/

2500, 5000

5000

TA 100

5000

/

2500, 5000

5000

WP2 uvrA

/

/

/

/

/ = no toxic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Mammalian Erythrocyte Micronucleus Test (OECD 474): Negative.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From: 26 Apr 2021 to 03 Aug 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Remarks:
According to Chem VwV-GLP Nr. 5.3/OECD Guidance
Type of assay:
mammalian erythrocyte micronucleus test
Strain:
Wistar
Remarks:
Species: Han
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
Research Models and Services Germany GmbH
Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 6-10 weeks
- Weight at study initiation: 164-201 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Group housed in Makrolon Type IV, with wire mesh top
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum; Supplier: Envigo Teklad Diets, Madison, Wisconsin, United States of America
- Water: tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24°C
- Humidity (%): 45-65%
- Air changes (per hr): at least 8
- Photoperiod (hrs dark / hrs light): 12 /12

IN-LIFE DATES: From: 26 April 2021To: 03 August 2021
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: The vehicle was chosen due to its relative non-toxicity for the animals and ability to form a suitable dosing formulation
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was freshly formulated in corn oil. The formulations were prepared at room temperature, and applied to the animals within 15 minutes
after preparation
Duration of treatment / exposure:
Not applicable
Frequency of treatment:
Single dose
Post exposure period:
Pre-Experiment on Toxicity: The animals were treated once orally with the test item and examined for acute toxic symptoms at intervals of approx. 0-1 h, 2-4 h, 5-6 h, 24 h, 30 h, and 48 h after administration of the test item.
Main study: The animals of all dose groups, except the positive control group, were examined for acute toxic symptoms at intervals of around 0-1 h, 2-4 h, 5-6 h, 24 h, and/or 48 h after
administration of the test item. Sampling of the bone marrow was conducted at low, mid and high doses at 24 h and then at the high dose only at 48 h
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
Dose / conc.:
2 000 mg/kg bw (total dose)
No. of animals per sex per dose:
Pre experiment on toxicity: 2 males/2 females
Main study: 6 males per dose
Control animals:
yes, concurrent vehicle
other:
Positive control(s):
Cyclophosphamide dissolved in sterile water
- Justification for choice of positive control(s): Identified as a suitable positive control in the guidelines
- Route of administration: oral
- Doses / concentrations: 20 mg/kg bw
Tissues and cell types examined:
The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The nucleated cells were separated from the erythrocytes using the method of Romagna et. al.
Details of tissue and slide preparation:
Tissue preparation: The cell suspensions were passed through a column consisting of α-cellulose and cellulose. The columns were then washed with Hank’s buffered saline. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded.

Slide preparation: A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 4000 polychromatic erythrocytes (PCE) per animal were analysed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per total erythrocytes. The analysis was performed with coded slides.

Bioanalysis was performed (report number S21-02411-L1) by Eurofins Agroscience Services EAG Laboratories GmbH.
Six additional male rats were assigned to 2 groups in order to be treated as satellite animals for plasma sampling. These animals were treated once with the test item at the maximum tolerated dose level, as determined within the range finding experiment. All animals were sampled twice by retroorbital puncture (eyes alternating). A maximum blood volume of 1.5 mL was withdrawn during the first sampling time point. The first sampling was performed under light isoflurane anaesthesia. The second, terminal sampling was performed under deep CO2 anaesthesia, and the animals were humanely euthanized after sampling by cervical dislocation whilst they were are in deep anaesthesia.
Blood sampling scheme:
Group 1: 1st sampling before first treatment '2nd sampling and termination 1 hour after application
Group 2: 1st sampling 0.5 hours after application, 2nd sampling and termination 4 hours after application.
The blood of the animals was collected in tubes containing K3-EDTA. The blood samples were centrifuged at 10’000 rpm for about 5 minutes to obtain plasma samples. The obtained plasma was divided in duplicates of about 0.2 mL each.


Evaluation criteria:
The test substance is classified as positive in the assay if:
a) At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent
negative control,
b) This increase is dose-related at least at one sampling time when evaluated with an
appropriate trend test, and
c) Any of these results are outside the distribution of the historical negative control data
(e.g., Poisson-based 95% control limits)

The study was considered valid if the following criteria were met:
• the concurrent negative control is considered acceptable for addition to the laboratory historical control database (should ideally be within the 95% control limits of the distribution of the historical negative control database)
• at least 5 animals per group can be evaluated.
• the appropriate number of doses and cells will be analysed.
• PCE to erythrocyte ratio is not less than 20% of the negative control.
• The positive control shows a statistically significant increase of micronucleated PCEs compared to the negative control and is compatible to those in the historical positive control database.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test using the validated statistical program RScript Wilcoxon_2.Rnw. The Holm-Bonferroni Adjustment method was used to correct for the Familywise error rate of multiple comparisons.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: Decreased activity. No substantial differences between sexes in toxicity were observed, so that only male animals were used in the main experiment.
- Evidence of cytotoxicity in tissue analysed: None
- Rationale for exposure: It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg bw as the upper limit for nontoxic test items.
- Harvest times: Three adequately spaced dose levels spaced by a factor of 2 were administered and samples of bone marrow were collected at the central sampling interval 24 h after treatment.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): 4000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. Results are shown in Tables 1, 2, 3, 4, 5 and 6
- Ratio of PCE/NCE (for Micronucleus assay): The ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per total erythrocytes. Results are shown in Tables 1, 2, 3, 4, 5 and 6
- Appropriateness of dose levels and route: As estimated by a pre-experiment at 2000 mg/kg bw (the maximum guideline-recommended dose) was suitable as highest treatment dose. The animals treated with the test item and the vehicle control did not exhibit any clinical symptoms.
- Statistical evaluation: In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test substance were near to the value of the vehicle control group as shown in Table 1, 2, 3, 4, 5 and 6.

A linear regression (least squares, calculated using the validated statistical program RScript
LM_v02.Rnw) was performed to assess a possible dose dependent increase of mean micronuclei values. The mean number of micronuclei obtained for the groups treated with the test item was compared to the vehicle control group. A trend is judged as significant whenever the p-value (probability value) is below 0.05. A p-value of 0.1964 was obtained, demonstrating that there was no dose dependent increase of mean micronuclei values.

A bioanalysis of the test item in plasma (phase number S21-02411-L1) was performed. The method had been successfully validated by procedural recovery samples for determination of the test substance with an LOQ of 0.10 mg/L and up to 10 mg/L in rat plasma according to the guidance document SANTE/2020/12830 rev. 1 of the European Commission. With regard to selectivity, accuracy and precision, the analytical method was applied successfully for the analytical set when analysing the samples of the study. In all plasma samples of animals treated with the test substance at a concentration of 2000 mg/kg bw, however, the residues of the test substance were below the Limit of Detection (i.e., <0.03 mg/L). Rapid hydrolysis of the test substance was suspected as the most probable underlying reason for the missing proof of exposure.

Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE per 4000 erythrocytes scoring 24 hours after treatment

Table 1: Vehicle Control

Test Group

Dose mg/kg b.w.

Animal No.

Micronuclei in Polychromatic Erythrocytes (PCE)

Evaluation 500 PCE in
total Erythrocytes

  
 
 

No. PCE

No. MN/4000 PCE

%
MN

Total No
Ery

NCE per total Ery

Ratio PCE/Total Ery

  
 

Corn Oil

 0

1

4000

11

0.28

771

271

0.649

  

2

4000

2

0.05

934

434

0.535

  

3

4000

8

0.20

707

207

0.707

  

4

4000

12

0.30

812

312

0.616

  

5

4000

7

0.18

823

323

0.608

  

6

4000

4

0.10

845

345

0.592

  

 

 

 

 

 

 

 

  

Mean  

7.3

0.19

815.3

315.3

0.618

  

SD       

3.9

0.10

75.8

75.8

0.058

  

 

Table 2: Test Item - Low Dose Group

Test Group

Dose mg/kg b.w.

Animal No.

Micronuclei in Polychromatic Erythrocytes (PCE)

Evaluation 500 PCE in
 total Erythrocytes

  
 
 

No. PCE

No. MN/4000 PCE

%
MN

Total No
Ery

NCE per total Ery

Ratio PCE/Total Ery

  
 

Dose 1

500

7

4000

5

0.13

801

301

0.624

  

8

4000

6

0.15

691

191

0.724

  

9

4000

9

0.23

686

186

0.729

  

10

4000

9

0.23

725

225

0.690

  

11

4000

10

0.25

737

237

0.678

  

12

4000

6

0.15

718

218

0.696

  

 

 

 

 

 

 

 

  

Mean  

7.5

0.19

726.3

226.3

0.690

  

SD       

2.1

0.05

41.6

41.6

0.038

  

 

 

 

Table 3: Test Item - Medium Dose Group

Test Group

Dose mg/kg b.w.

Animal No.

Micronuclei in Polychromatic Erythrocytes (PCE)

Evaluation 500 PCE in
 total Erythrocytes

  
 
 

No. PCE

No. MN/4000 PCE

%
MN

Total No
Ery

NCE per total Ery

Ratio PCE/Total Ery

  
 

Dose 2

1000

13

4000

7

0.18

682

182

0.733

  

14

4000

9

0.23

782

282

0.639

  

15

4000

5

0.13

730

230

0.685

  

16

4000

12

0.30

751

251

0.666

  

17

4000

15

0.38

790

290

0.633

  

18

4000

9

0.23

719

219

0.695

  

 

 

 

 

 

 

 

  

Mean  

9.5

0.24

742.3

242.3

0.675

  

SD       

3.6

0.09

40.6

40.6

0.037

  

 

Table 4: Test Item - High Dose Group

Test Group

Dose mg/kg b.w.

Animal No.

Micronuclei in Polychromatic Erythrocytes (PCE)

Evaluation 500 PCE in
 total Erythrocytes

  
 
 

No. PCE

No. MN/4000 PCE

%
MN

Total No
Ery

NCE per total Ery

Ratio PCE/Total Ery

  
 

Dose 3

2000

19

4000

9

0.23

739

239

0.677

  

20

4000

9

0.23

769

269

0.650

  

21

4000

6

0.15

1129

629

0.443

  

22

4000

4

0.10

784

284

0.638

  

23

4000

14

0.35

910

410

0.549

  

24

4000

13

0.33

1184

684

0.422

  

 

 

 

 

 

 

 

  

Mean  

9.2

0.23

919.2

419.2

0.563

  

SD       

3.9

0.10

193.7

193.7

0.110

  

 

 

 

Table 5: Positive Control

Test Group

Dose mg/kg b.w.

Animal No.

Micronuclei in Polychromatic Erythrocytes (PCE)

Evaluation 500 PCE in
 total Erythrocytes

  
 
 

No. PCE

No. MN/4000 PCE

%
MN

Total No
Ery

NCE per total Ery

Ratio PCE/Total Ery

  
 

Positive

20

25

4000

55

1.38

767

267

0.652

  

26

4000

105

2.63

948

448

0.527

  

27

4000

76

1.90

1104

604

0.453

  

28

4000

63

1.58

1115

615

0.448

  

29

4000

63

1.58

1080

580

0.463

  

30

4000

42

1.05

1281

781

0.390

  

 

 

 

 

 

 

 

  

Mean  

67.3

1.69

1049.2

549.2

0.489

  

SD       

21.6

0.54

174.3

174.3

0.091

  

 

 

Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE per 4000 erythrocytes scoring 48 hours after treatment

Table 6: Test Item - High Dose Group

Test Group

Dose mg/kg b.w.

Animal No.

Micronuclei in Polychromatic Erythrocytes (PCE)

Evaluation 500 PCE in
 total Erythrocytes

  
 
 

No. PCE

No. MN/4000 PCE

%
MN

Total No
Ery

NCE per total Ery

Ratio PCE/Total Ery

  
 

Dose 3

2000

31

4000

15

0.38

625

125

0.800

  

32

4000

10

0.25

820

320

0.610

  

33

4000

9

0.23

766

266

0.653

  

34

4000

10

0.25

963

463

0.519

  

35

4000

7

0.18

969

469

0.516

  

36

4000

8

0.20

970

470

0.515

  

 

 

 

 

 

 

 

  

Mean  

9.8

0.25

852.2

352.2

0.602

  

SD       

2.8

0.07

141.3

141.3

0.113

  

 

Animal Weights

Dose Group

Animal No.

Animal weights before treatment

Animal weights before sacrifice

Initial Weight [g]

Mean [g]

SD [g]

Range
[g]

Initial Weight [g]

Mean [g]

SD  [g]

Range
[g]

Vehicle Control
24 h

1

179.0

179.0

5.4

169.3

-

185.2

185.3

186.2

6.3

174.8

-

193.7

2

185.2

193.7

3

169.3

174.8

4

182.6

189.8

5

179.4

186.3

6

178.7

187.2

500 mg/kg b.w.
24 h
Dose 1

7

191.4

180.6

10.1

165.7

-

191.9

199.8

187.1

9.7

171.5

-

199.8

8

181.0

186.6

9

165.7

171.5

10

191.9

194.6

11

178.8

186.0

12

174.5

184.0

1000 mg/kg b.w.
24 h
Dose 2

13

187.0

187.8

9.3

175.6

-

200.6

194.9

193.0

10.8

177.9

-

205.7

14

196.7

203.9

15

200.6

205.7

16

175.6

177.9

17

183.4

184.8

18

183.8

190.8

2000 mg/kg b.w.
24 h
Dose 3

19

169.8

186.6

11.6

169.8

-

196.6

160.8

184.6

16.6

160.8

-

201.9

20

194.6

201.9

21

192.3

187.0

22

192.1

185.8

23

196.6

201.8

24

173.9

170.0

Positive Control
24 h

25

182.6

178.7

4.6

170.4

-

183.5

188.7

184.1

6.8

171.2

-

188.7

26

170.4

171.2

27

178.7

188.2

28

178.4

185.7

29

178.8

182.1

30

183.5

188.5

2000 mg/kg b.w.
48 h
Dose 3

31

193.1

184.8

12.3

164.3

-

198.7

198.5

188.0

11.3

174.6

-

199.4

32

198.7

199.4

33

181.9

174.9

34

179.7

186.4

35

164.3

174.6

36

190.8

194.4

Animal Weights

Dose Group

Animal No.

Animal weights before treatment

Animal weights before sacrifice

Initial Weight [g]

Mean [g]

SD [g]

Range
[g]

Initial Weight [g]

Mean [g]

SD  [g]

Range
[g]

Vehicle Control
24 h

1

179.0

179.0

5.4

169.3

-

185.2

185.3

186.2

6.3

174.8

-

193.7

2

185.2

193.7

3

169.3

174.8

4

182.6

189.8

5

179.4

186.3

6

178.7

187.2

500 mg/kg b.w.
24 h
Dose 1

7

191.4

180.6

10.1

165.7

-

191.9

199.8

187.1

9.7

171.5

-

199.8

8

181.0

186.6

9

165.7

171.5

10

191.9

194.6

11

178.8

186.0

12

174.5

184.0

1000 mg/kg b.w.
24 h
Dose 2

13

187.0

187.8

9.3

175.6

-

200.6

194.9

193.0

10.8

177.9

-

205.7

14

196.7

203.9

15

200.6

205.7

16

175.6

177.9

17

183.4

184.8

18

183.8

190.8

2000 mg/kg b.w.
24 h
Dose 3

19

169.8

186.6

11.6

169.8

-

196.6

160.8

184.6

16.6

160.8

-

201.9

20

194.6

201.9

21

192.3

187.0

22

192.1

185.8

23

196.6

201.8

24

173.9

170.0

Positive Control
24 h

25

182.6

178.7

4.6

170.4

-

183.5

188.7

184.1

6.8

171.2

-

188.7

26

170.4

171.2

27

178.7

188.2

28

178.4

185.7

29

178.8

182.1

30

183.5

188.5

2000 mg/kg b.w.
48 h
Dose 3

31

193.1

184.8

12.3

164.3

-

198.7

198.5

188.0

11.3

174.6

-

199.4

32

198.7

199.4

33

181.9

174.9

34

179.7

186.4

35

164.3

174.6

36

190.8

194.4

 

 

 

 Historical Control Data (Oct 2014 - Dec 2020)

 

 Vehicle Controls (%)

Male animals 

min

0.025

max

0.750

mean

0.255

95% Ctr. Limit

0.001

0.509

SD

0.127

2x SD

0.254

Range of individual animal micronuclei values*

1 - 30

No° indiv. values  

240

 

 Positive Controls (%)

Male animals

min

0.450

max

4.525

mean

1.718

95% Ctr. Limit

-0.086

3.522

SD

0.902

2x SD

1.804

Range of individual animal micronuclei values*

18 - 181

No° indiv. values 

161

 

*per 4000 Polychromatic Erythrocytes

 

 

 Historical Control Data (Oct 2014 - Dec 2020)

 

 Vehicle Controls (%)

Male animals 

min

0.025

max

0.750

mean

0.255

95% Ctr. Limit

0.001

0.509

SD

0.127

2x SD

0.254

Range of individual animal micronuclei values*

1 - 30

No° indiv. values  

240

 

 Positive Controls (%)

Male animals

min

0.450

max

4.525

mean

1.718

95% Ctr. Limit

-0.086

3.522

SD

0.902

2x SD

1.804

Range of individual animal micronuclei values*

18 - 181

No° indiv. values 

161

 

*per 4000 Polychromatic Erythrocytes

 

 

 

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

Additional information

Gene mutation in bacteria


A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (Poth, 2003). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and the Escherichia coli strain WP2 uvrA were exposed to the test substance dissolved in DMSO using either the preincubation or the plate incorporation method. To evaluate the cytotoxicity of the test substance a pre-experiment with eight concentrations in the range of 3 - 5000 µg/plate was performed in the strains TA 98 and TA 100. In the first experiment (plate incorporation), test concentrations of 33, 100, 333, 1000, 2500 and 5000 µg/plate were used with and without metabolic activation. In the second experiment (preincubation method), test concentrations of 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate were used without and 33, 100, 333, 1000, 2500 and 5000 µg/plate were used with metabolic activation. No visible reduction of the background growth was observed in Experiment 1. In Experiment 2, irregular background growth was observed at the highest concentration in strain TA 98 and WP2 uvrA (with S9 mix) and in strain TA 100 (with and without S9 mix). Toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in all strains used, except WP2 uvrA. No substantial increase in the mean number of revertants per plate was observed in any of the test strains compared to the control. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Under the conditions of this experiment, the test substance did not show mutagenicity in the selected S. typhimurium strains and E. coli strain in the presence and absence of metabolic activation.


 


Chromosome aberrations


The clastogenic activity of the test substance was investigated in an in vitro mammalian chromosome aberration test in Chinese Hamster lung fibroblasts (V79) performed according to OECD Guideline 473 and GLP (Béres, 2015). The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study. In two independent experiments, the test substance concentrations 0.5, 1, 2, 4 and 4.5 µg/mL were used for a 3 h-short-term and for a 20 h-continuous exposure period with a 20 and 28 h sampling time without metabolic activation. In addition, the concentrations 10, 20, 40, 80 and 90 µg/mL were used for a 3 h-exposure period with a 20 and 28 h sampling time with metabolic activation (S9 mix). In Experiment 1, no biologically significant increases in the number of cells showing structural chromosome aberrations without gap were observed in the absence of metabolic activation, up to and including cytotoxic concentrations. However, the test substance caused a moderate increase in the number of cells with structural chromosome aberrations in the presence of metabolic activation, up to and including cytotoxic concentrations. This increase was dose associated and biologically important. In Experiment 2, the frequency of the cells with structural chromosome aberrations without gaps did not show significant alterations compared to the concurrent control, when the test substance was examined up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 h with a 20 and 28 h sampling time. Following the 3 h-exposure period with metabolic activation and a 28 h sampling time, the test substance caused an increase in the number of cells with structural chromosome aberrations without gaps at 90 µg/mL. This increase was biologically and statistically significant. No effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9 mix. Positive control chemicals, ethyl methanesulphonate and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9 mix) functioned properly. In conclusion, the test substance did not induce structural chromosome aberrations in Chinese Hamster lung fibroblasts (V79) in the absence of metabolic activation. However, the test substance induced structural chromosome aberrations in the presence of metabolic activation. Therefore, the test substance is considered clastogenic under the conditions of this test system.


In conclusion, the test substance did not show mutagenicity in bacteria both in the presence and absence of metabolic activation while clastogenic effects were observed in an in vitro mammalian chromosome aberration test in the presence of metabolic activation. Thus, an in vivo somatic cell study according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) was conducted to assess the genetic toxicity potential of the test substance.


 


In vivo Micronucleus Assay in Bone Marrow Cells of the Rat


The potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat was assessed. The test substance was dissolved in corn oil, which was also used as vehicle control. The dose volume administered orally was 10 mL/kg bw. The administered volume of the positive control was 10 mL/kg. 24 h and 48 h after a single administration of the test item to six males per test group, the bone marrow cells were collected for micronuclei analysis for the occurrence of micronuclei. 4000 polychromatic erythrocytes per animal were scored for micronuclei. To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per total erythrocytes. The following dose levels of the test item were investigated: 24 h preparation interval: 500, 1000, and 2000 mg/kg bw 48 h preparation interval: 2000 mg/kg bw The highest dose (maximum guideline-recommended dose) was estimated by a pre experiment to be suitable for the main experiment. The animals treated with the test substance and the vehicle control did not exhibit any clinical symptoms. A bioanalysis of the test substance in plasma (report number S21-02411-L1) was performed. The method had been successfully validated by procedural recovery samples for determination of the test substance with an LOQ of 0.10 mg/L and up to 10 mg/L in rat plasma according to the guidance document SANTE/2020/12830 rev. 1 of the European Commission. With regard to selectivity, accuracy and precision, the analytical method was applied successfully for the analytical set when analysing the samples of the study. In all plasma samples of animals treated with the test substance at a concentration of 2000 mg/kg bw., however, the residues of the test substance were below the Limit of Detection (i.e., <0.03 mg/L). Rapid hydrolysis of the test substance was suspected as the most probable underlying reason for the missing proof of exposure. After treatment with the test item the number of PCEs was slightly decreased in the 24 h high dose group (group mean decrease of approximately 9%) as compared to the mean value of PCEs of the vehicle control thus indicating that the test substance may exert a slight, transient cytotoxic effect in the bone marrow at the maximum guideline-recommended dose level of 2000 mg/kg bw. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronulclei at any preparation interval after administration of the test item and with any dose level used. 20 mg/kg bw cyclosphosphamide administered orally was used as positive control which induced a substantial increase in cells with micronuclei. In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce an increased frequency of micronuclei in the bone marrow cells of the rat after single oral administration up to a dose level of 2000 mg/kg bw. Therefore, the test substance was considered to be non-genotoxic in the in vivo micronucleus assay.

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.