Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): positive with and without activation in TA 100, TA 1535 and WP2 uvrA strains (OECD TG 471) (BioReliance, 2000).
Cytogenicity in mammalian cells: positive with and without metabolic activation in human blood lymphocytes (OECD TG 473) (SafePharm, 2004cb).

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:
1999-09-02 to 1999-11-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
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:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
100, 333, 1000, 3333, 5000 μg/ plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
All salmonella strains + WP2 uvrA (with activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 (without activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 100, TA 1535 (without activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 (without activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA (without activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar ; preincubation

ACTIVATION: The S9 mix contained 10 % S9, 5 mM glucose-6-phosphate, 4 mM β-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCl2 and 33 mM KCl in 100 mM phosphate buffer at pH 7.4.

DURATION
- Preincubation period: 60 minutes

- Expression time (cells in growth medium): 48 to 72 hours

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn analysis

Evaluation criteria:
A result is positive if the number of revertants is significantly increased compared with the solvent control to at least 2-fold of the solvent control for TA 98, TA 100 and WP2 uvrA and 3-fold of the solvent control for TA 1535 and TA 1537.

Cytotoxicity is defined as a reduction in the number of colonies by >50% compared with the solvent control and/or a sparse background lawn.

Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
reduced background lawn3333 - 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
2.1 - 3.1-fold increase in revertants
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
reduced background lawn 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
4.1 - 9.5-fold increase in revertants
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
reduced background lawn 3333 - 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
13.1 - 17-fold increase in revertants
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
10.4 - 21.8-fold increase in revertants
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
reduced background lawn 3333 - 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
reduced background lawn 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
reduced background lawn 3333 - 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
3.2 - 4.4-fold increase in revertants
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
4.7 - 5.2-fold increase in revertants
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 2:  Preliminary toxicity assay for all strains with and without metabolic activation

 

TA98

TA100

TA1535

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

0*

14

26

No

171

160

No

23

15

No

6.7

15

NC**

No

183

147

No

20

17

No

10

14

12

No

167

187

No

25

19

No

33

12

26

No

173

204

No

24

14

No

67

22

24

No

205

175

No

22

20

No

100

16

19

No

209

190

No

26

22

No

333

12

18

No

325

193

No

36

22

No

667

12

22

No

374

186

No

40

22

No

1000

18

22

No

614

188

No

50

27

No

3333

17

18

No

862

324

No

237

37

No

5000

17

20

No

928

462

No

418

97

No

*solvent control with DMSO

**NC = No count due to procedural error in which plate did not receive an aliquot of tester strain

Table 2:  Preliminary toxicity assay for all strains with and without metabolic activation

 

TA1537

WP2 uvrA

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

0*

13

13

No

22

17

No

6.7

12

15

No

17

11

No

10

12

19

No

14

13

No

33

11

14

No

14

11

No

67

12

13

No

13

17

No

100

10

6

No

18

13

No

333

15

15

No

21

22

No

667

11

12

No

14

17

No

1000

13

10

No

27

23

No

3333

24

10

No

40

24

No

5000

20

12

No

38

31

No

*solvent control with DMSO

Table 3: Experiment 1 Preincubation Number of revertants per plate (mean of 3 plates)

 

TA98

TA100

TA1535

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

 MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

0*

32

36

No

100

118

No

14

10

No

100

26

32

No

149

110

No

26

12

No

333

24

33

No

211

108

No

54

22

No

1000

22

28

No

453

127

No

87

28

No

3333

27**

35

Yes

705**

249

Yes

145**

104

Yes

5000

28**

26

Yes

948**

363

Yes

120**

170

Yes

Positive control

640

544

No

410

522

No

248

78

No

*solvent control with DMSO

**moderately reduced background lawn

***extremely reduced background lawn

Table 3: Experiment 1 Preincubation Number of revertants per plate (mean of 3 plates)

 

TA1537

WP2 uvrA

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

0*

13

19

No

12

13

No

100

13

16

No

10

10

No

333

13

22

No

15

15

No

1000

16

9

No

26

18

No

3333

9**

18

No

62

22

No

5000

6***

20

Yes

58

42

No

Positive control

829

89

No

188

54

No

*solvent control with DMSO

**moderately reduced background lawn

***extremely reduced background lawn

Table 4: Experiment 2 Preincubation Number of revertants per plate (mean of 3 plates)

 

TA98

TA100

TA1535

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

 MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

0*

12

18

No

125

138

No

9

9

No

33

11

19

No

135

190

No

10

6

No

100

13

18

No

139

121

No

13

12

No

333

12

17

No

196

121

No

32

14

No

1000

11

15

No

350

145

No

45

19

No

3333

14**

13

Yes

383**

270

Yes

141

67

No

5000

6***

18**

Yes

518***

288**

Yes

196**

118

Yes

Positive control

527

597

No

378

622

No

165

32

No

*solvent control with DMSO

**moderately reduced background lawn

***extremely reduced background lawn

Table 4: Experiment 2 Preincubation Number of revertants per plate (mean of 3 plates)

 

TA1537

WP2 uvrA

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

0*

6

6

No

11

13

No

33

4

8

No

11

9

No

100

5

5

No

10

11

No

333

4

4

No

9

8

No

1000

2

5

No

16

19

No

3333

4**

6

Yes

50

33

No

5000

5***

6**

Yes

52

57

No

Positive control

396

59

No

207

134

No

*solvent control with DMSO

**moderately reduced background lawn

***extremely reduced background lawn

Conclusions:
Under test conditions, there was a positive, dose related increase in the number of revertant colonies in Salmonella typhimurium TA 100, TA 1535 and E. coli WP2 uvrA, with and without metabolic activation. The test substance is mutagenic in these tester strains.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-04-26 to 2004-2004-05-24
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)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Human blood
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone and naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
9.7, 19.41, 38.81, 77.63, 155.25*, 310.5*, 621*, 1242, 1863, 2484 µg/ml (* analysed concentrations)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
(with activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
(without activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

ACTIVATION: 1ml of 20 % S9 mix ( ie 2 % final concentration of S9 in standard co-factors) was added to the cultures.

DURATION

- Exposure duration: 4 hours
- Expression time (cells in growth medium): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Gurrs Giemsa


NUMBER OF REPLICATIONS: 2 cultures per dose level


NUMBER OF CELLS EVALUATED: 950 in total. (150 for positive control, 200 for treatments and vehicle control)


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, relative total growth


OTHER EXAMINATIONS:
- Determination of polyploidy: mean frequency % for experiment 1
Evaluation criteria:
A positive response was recorded if the % of cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, with or
without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate
statistical tests may be applied in order to record a positive response
Key result
Species / strain:
lymphocytes: human blood
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
50% mitotic inhibition at 310.5 μg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: human blood
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
73% mitotic inhibition at 2484 μg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: Results were within range of historical control data

Table 3: Results of chromosome analysis Experiment 1, 4h treatment without activation (total count from 2 cultures)

 

Solvent*

Control**

Positive

Control***

621 μg/ml**

310.5 μg/ml**

155.25 μg/ml**

Cytotoxicity

No

Yes

No

Yes

Yes

 

Mean

Chromatid aberrations

gaps

2

20

33

14

8

breaks

1

25

36

10

5

interchanges

1

33

28

1

0

Chromosome

aberrations

gaps

NR

NR

NR

NR

NR

breaks

0

2

4

3

1

interchanges

0

1

0

0

0

Mitotic index (%)

100

49

16

50

80

Polyploidy (% mean freq.)

0.5

0

0

0.5

0

Endo reduplication

NR

NR

NR

NR

NR

 *Solvent control with DMSO

** Per 200 cells

*** Per 150 cells

NR not reported

Table 4: Results of chromosome analysis Experiment 1, 4h treatment with activation (total count from 2 cultures)

 

Solvent*

Control**

Positive

Control***

621 μg/ml**

1242 μg/ml**

1863

μg/ml**

2484

μg/ml***

Cytotoxicity

No

Yes

No

No

No

Yes

 

Mean

Chromatid aberrations

gaps

0

39

8

8

26

27

breaks

0

47

1

4

25

42

interchanges

0

33

0

3

7

14

Chromosome aberrations

gaps

NR

NR

NR

NR

NR

NR

breaks

1

3

1

3

5

5

interchanges

0

0

0

0

0

1

Mitotic index (%)

100

19

117

70

57

27

Polyploidy (% mean freq.)

0

0

0

0

1.5

0

Endo reduplication

NR

NR

NR

NR

NR

NR

 *Solvent control with DMSO

** Per 200 cells

*** Per 100 cells

NR not reported

Conclusions:
The test material induced a statistically significant dose-related increase in the frequency of cells with chromosome aberrations in both the absence and presence of metabolic activation. The test material was therefore considered to be clastogenic to human lymphocytes in vitro.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Micronucleus assay in mouse (oral gavage): Negative (OECD TG 474) (BioReliance, 2001 and Shin-Etsu. 2009).

Mammalian alkaline comet assay in rat (oral gavage): Ambiguous result. Negative result for cells of glandular stomach and liver. The mean tail intensity of cells from the duodenum at the highest dose tested was above the historic control limit and was statistically significant compared to the concurrent negative control. Toxicity: no abnormalities were observed at necropsy and there were no histological alterations detected in any tissue (OECD TG 489) (BSL Bioservice, 2019).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-06-09 to 2009-07-20
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
Remarks:
(compliant with 1997 guideline; number of cells scored not compliant with 2014 revision)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: CrljOri : CD1 (ICR)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Orient Bio Inc, Korea
- Age at study initiation: 7 weeks old
- Weight at study initiation: 30.3 - 33.8 g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: 10 or 6 animals were housed together in polycarbonate cages with bedding
- Diet: pelleted food, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 50 - 60 %
- Air changes (per hr): 10 - 20 times/hour
- Photoperiod (hrs dark / hrs light): 12/ 12
Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: The test item was soluble in corn oil according to the solubility test prior to the study.
- Concentration of test material in vehicle: the test item was soluble in 200 mg/mL
- Lot/batch no. (if required): 065K0077
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose-range finding study: 250, 500, 1000 and 2000 mg/kg were administered orally to 4 males and 4 female mice for 2 consecutive days. Throughout the study, all the animals were observed daily for signs of toxicity.
Main study: based on the findings from the dose-range finding study the chosen doses for the main study were 500, 1000 and 2000 mg/kg.
The study report uses the phrase "oral injection"; it is clearly stated that test substance was administered orally. It is considered by the reviewer that gavage was used.

Duration of treatment / exposure:
The test item was administered twice
Frequency of treatment:
24 hours
Post exposure period:
24 hours
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6 males
Control animals:
yes
Positive control(s):
cyclophosphamide monohydrate dissolved in saline
- Justification for choice of positive control(s): no data
- Route of administration: injected intraperitoneally
- Doses / concentrations: 70 mg/kg
Tissues and cell types examined:
Bone marrow erythrocytes
Number of plychromatic erythrocytes (PCE) examined per animal: 2000
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
During the dose range finding test, clinical signs of toxicity were the criteria for dose selection .

TREATMENT AND SAMPLING TIMES:
The test item was orally administered twice with intervals of 24 hours between each dose administration. 24 hours after the last administration the animals were sacrificed by CO2 gas inhalation. Bone marrow preparations were made according to Schmid and two slides of cell suspension per animal were made.

DETAILS OF SLIDE PREPARATION:
Bone marrow cells were collected in foetal bovine serum using a disposable syringe with 23G needle, centrifuged at about 1000 rpm for 5 minutes. After removing the supernatant, a small drop of the viscous suspension was smeared onto microscope slides. Preparations were air-dried and fixed by submerging in absolute methanol for 5 min. Fixed slides were stained as follows: May-Grunwald stain 3 min, May-Grunwald stain (1:1 diluted) 2 min, Giemsa stain (1:6 diluted) 10 min. Stained slides were rinsed with distilled water, dried and mounted with a mountant (Depex, Fluka). Stained slides were examined under 1000x magnification.

METHOD OF ANALYSIS:
Slides, which have a good stain condition were randomly coded and examined by microscopy. Small round or oval shaped bodies within erythrocytes with a size of about 1/5 or 1/20 of the diameter of a PCE, were regarded as micronuclei. Attention was given to discriminate micronuclei from artefacts. The results were expressed as the number of MNPCEs in 2000 PCEs. The mean number of MNPCEs +/- SD was calculated for each treatment group. In addition, the PCE (PCE+NCE) ratio, indication cytotoxicity to the haematopoietic system was calculated by counting 500 cells.

OTHER:
-Clinical observations: the mortality and external appearance of the test animals was checked and recorded daily during the study period. Following the final dose administration on the final day of the treatment period, observations were made three times.
-Body weight measurement: body weight of each test animal was recorded on the day of reception, grouping, dosing and autopsy.
Evaluation criteria:
The result was considered positive when there was a statistically significant and dose-related increase or a reproducible increase in the frequency of MNPCEs at least at one dose level.
Statistics:
-Kruskal-Wallis H-test and Dunn's Rank Sum test: test for differences of numbers of MNPCEs between treated and vehicle control group.
-Mann-Whitney U-test: test for differences of numbers of MNPCEs between positive and vehicle control group.
-ANOVA test and Dunnett's test: test for the differences of PCE/(PCE+NCE) ratio between treated and vehicle control group
-Student's t-test: test for the differences of PCE/(PCE+NCE) ratio between positive and vehicle control group
-Bartlett's test, followed by ANOVA test, if p value < 0.05: for comparison of body weight of animals
-Cochran-Armitage trend test: used for evaluation of dose-responsiveness
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 500, 1000 and 2000 mg/kg
- Clinical signs of toxicity in test animals: There were no clinical signs of toxicity

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): At all sampling times, mice treated with the test material showed no significant increase in the frequency of micronucleated polychromatic erythrocytes.
- Ratio of PCE/NCE (for Micronucleus assay): The test substance did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes.
- Cyclophosphamide monohydrate positive control: There was a statistically significant increase in the number of micronucleated polychromatic erythrocytes (p < 0.05)
- Ration of PCE/(PCE+NCE) (indicator of cytotoxicity): Showed no significant difference between the vehicle control and test item-treated group.

Table 1: Results from in vivo micronucleus assay in mice

Micronucleus test of KBE-402

Chemical treated

Dose (mg/kg)

No. of animals

MNCPE/2000 PCE (Mean)

PCE/(PCE+NCE) (Mean)

Vehicle

0

6

1.17

0.55

Test item

500

6

0.50

0.52

Test item

1000

6

0.50

0.49

Test item

2000

6

0.50

0.54

CPA

70

6

46.67

0.44

PCE: polychromatic erythrocyte

NCE: normochromatic erythrocyte

MNPCE: polychromatic erythrocyte with one or more micronuclei

CPA: cyclophosphamide monohydrate (positive control substance)

Conclusions:
[3-(2,3-epoxypropoxy)propyl]diethoxy(methyl)silane has been tested for the induction of micronuclei in mice according to OECD TG 474 and in compliance with GLP. No evidence for a test substance-induced increase in the incidence of micronucleated polychromatic erythrocytes in mice bone marrow was observed when tested up to limit concentration. No clinical signs of toxicity were observed following administration of 500, 1000 and 2000 mg/kg of test material. Appropriate positive and vehicle controls were included and gave the expected results. It is concluded that the test substance does not cause damage to chromosomes under the conditions of the test.
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 June 2018 to 20 December 2019, histopathology completed 29 March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
Rats have been used for many years as standard experimental animals in genotoxicity investigations. In addition, they have been used as experimental animal in the toxicological safety characterisation of the test item.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany.
- Age at study initiation: approximately 7-9 weeks.
- Weight at study initiation: 258 ± 11.5 g.
- Assigned to test groups randomly: yes.
- Fasting period before study: no.
- Housing: 2-3 animals per cage, IVC cages (type III, polysulphone cages) on Altromin saw fibre bedding.
- Diet: ad libitum access to Altromin (Batch 1030) maintenance diet for rats and mice.
- Water: ad libitum access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals).
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

IN-LIFE DATES: From: 2nd March 2018 To: 5th July 2018
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: none.
Details on exposure:
Dose volume was based on test item density of 0.980 g/cm³
Duration of treatment / exposure:
2 days
Frequency of treatment:
Daily
Post exposure period:
No
Dose / conc.:
0 mg/kg bw/day
Remarks:
Physiological saline (0.9 % NaCl) 2.04 ml/kg bw
Dose / conc.:
400 mg/kg bw/day
Remarks:
0.41 ml/kg bw
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
1.02 ml/kg bw
Dose / conc.:
2 000 mg/kg bw/day
Remarks:
2.04 ml/kg bw
No. of animals per sex per dose:
5 males per dose
Control animals:
yes
Positive control(s):
- ethylmethanesulphonate
- Justification for choice of positive control(s): selected according to the JaCVAM validation trial.
- Route of administration: oral, single dose 4 hours before sacrifice
- Doses / concentrations: 300 mg/kg bw
Tissues and cell types examined:
liver, glandular stomach, duodenum
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on pre-experiment to determine maximum tolerated dose

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Animals were treated at 0 and 24 hours. Tissues were sampled after euthanasia on day two. Organs were rinsed with cold mincing buffer to remove residual blood and stored in mincing buffer on ice until further processing.

25 samples of liver, 25 samples of glandular stomach and 25 samples of duodenum were prepared according to the following procedure:

PREPARATION OF SINGLE CELLS
- Isolation of primary hepatocytes:
A portion of the liver was minced with a pair of scissors, to isolate the cells. The cell suspension was filtered through a mesh (70 µm) to yield a single cell suspension. The number of isolated cells was determined by the trypan blue dye exclusion test to adjust the cell number. Approximately 2E+05,cells were pelleted by centrifugation and the supernatant was decanted and the cell pellets were stored on ice.
- Isolation of glandular stomach cells:
The stomach was cut open and washed free from content using cold mincing buffer. The forestomach was removed and discarded. The surface epithelia was gently removed by scraping two times using a scalpel blade. This layer was discarded. A portion of the glandular stomach was minced with a pair of scissors and grated with a pestle to release the cells. The cell suspension was filtered through a mesh (70 µm) to yield a single cell suspension. The number of isolated cells was determined by the trypan blue dye exclusion test to adjust the cell number. Approximately 3E+05 cells were pelleted by centrifugation and the supernatant was decanted and the cells were stored on ice.
- Isolation of duodenum cells:
The duodenum was cut open and washed free from food using cold mincing buffer. The surface epithelia was gently removed by scraping two times using a scalpel blade. This layer was discarded. The duodenum was minced with a pair of scissors, to isolate the cells. The cell suspension was filtered through a mesh (70 µm) to yield a single cell suspension. The number of isolated cells was determined by the trypan blue dye exclusion test to adjust the cell number. Approximately 3E+05 cells were pelleted by centrifugation and the supernatant was decanted and the cells were stored on ice.
For all tissues, the cell pellets were used for preparing comet slides.

COMET ASSAY PROCEDURE

DETAILS OF SLIDE PREPARATION:
The slides used were pre-coated with normal-melting agarose, coded with a random number and the cell pellet (approx. 2E+05 to 3E+05 cells) was directly resuspended in 225 µL low-melting temperature agarose (Trevigen) Approximately 6E+05 to 10E+05 cells/slide were embedded in low-melting temperature agarose on the slides in one layer. Directly after application of the LMA, the slides were covered with a cover slip and cooled for 30 min on ice (3 slides per animal and tissue).
Lysis:
Cover slips were carefully removed and the slides incubated overnight in chilled lysing solution at 2 - 8 °C in the fridge to lyse cellular and nuclear membranes and allow the release of coiled DNA loops during electrophoresis. After completion of lysis, the slides were rinsed in purified water to remove residual detergent and salts.
Unwinding of DNA and electrophoresis:
Prior to electrophoresis, the slides were incubated in alkaline (pH > 13) electrophoresis buffer for 20 min. After alkali unwinding, the single-stranded DNA was electrophoresed under alkaline conditions to enable the formation of DNA tails. The slides were placed in a horizontal gel electrophoresis chamber, positioned close to the anode and covered with 600 mL electrophoresis buffer. The electrophoretic conditions were 21 V and approximately 300 mA, with the DNA being electrophoresed for 30 min.
Neutralisation and dehydration of slides:
After electrophoresis, the slides were neutralised by rinsing with neutralisation buffer three times for 5 min each. The slides were incubated for approximately 10 – 20 min in ice-cold ethanol and air-dried afterwards.
DNA staining:
Following dehydration, the cells were stained by applying 75 µL gel red staining solution on top of the slides and covering with a cover slip.

METHOD OF ANALYSIS:
Comet slides were analysed for potential DNA damage using a fluorescence microscope with magnification (200x) coupled to a camera and the Comet Software ‘Comet Assay IV’ (Perceptive Instrument, software version 2.1.2). The slides were coded so that the evaluator was not aware of which dose group was evaluated.
Each slide was screened for cells in a meandering pattern in the unfrosted area of the slide by an evaluator. The calculation of the different parameters was done automatically by the Comet Software, but the set front, middle and back lines of the comet might have been adjusted manually if they were not set correctly automatically. All cells of the visual field were scored, except of e.g. overlapping cells, cells with an atypical nucleus, cells with a strong background or “hedgehogs” (cells that exhibit a microscopic image consisting of a small or non-existent head and a large diffuse tail, are considered to be heavily damaged cells). Therefore, cells were classified into three categories: scorable, non-scorable and “hedgehog”. To avoid artefacts only scorable cells (defined round to oval nucleus) and at least 150 cells per sample on two slides (75 cells per slide) were scored. The %-tail intensity was the parameter for evaluation and interpretation of DNA damage, and was determined by the DNA staining intensity present in the tail region expressed as a percentage of the cell's total staining intensity including the nucleus.

HISTOPATHOLOGY:
Duodenum from all animals were preserved in 10% neutral buffered formalin and transferred to AnaPath GmbH, Department AnaPath Services Liestal, Switzerland for preparation for histopathology.
Samples were trimmed, embedded in paraffin wax, cut to approximately 4 micrometres and stained with hematoxylin and eosin.
Histopathological examination occurred at AnaPath GmbH, Oberbuchsiten, Switzerland. Slides were evaluated by light microscope.
Evaluation criteria:
Increases in DNA damage in the presence of a clear evidence for cytotoxicity during e.g. clinical observations should be interpreted with caution. A positive response should minimally yield a statistically significant increase in the %-tail DNA in at least one dose group at a single sampling time in comparison with the negative control value. The positive control should produce a positive response, and the vehicle control a negative response and if not, the study data will not be acceptable.
Providing all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
- at least one of the test doses exhibits a statistically significant increase in tail intensity compared with the concurrent negative control, and
- this increase is dose-related when evaluated with an appropriate trend test,
- any of these results are outside the distribution of the historical negative control data
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if:
- none of the test concentrations exhibits a statistically significant increase in tail intensity compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data,
- direct or indirect evidence supports exposure of, or toxicity to, the target tissue(s).
To assess the biological relevance of a positive or equivocal result, information on cytotoxicity at the target tissue can be required. Where positive or equivocal findings are observed solely in the presence of a clear evidence for cytotoxicity, the study should be concluded as equivocal for genotoxicity unless there is enough information supporting a more definitive conclusion.
Statistics:
All slides, including those of positive and negative controls were independently coded before microscopic analysis and subsequently scored blinded. The median %-tail DNA for each slide was determined and the mean of the median values was calculated for each of the tissue types from each animal.
For each tissue type, the mean of the individual animal means was then determined to give a group mean. Statistical analysis was done using a variety of approaches
Key result
Sex:
male
Genotoxicity:
ambiguous
Remarks:
the mean tail intensity of cells from the duodenum at the highest dose tested was above the historic control limit and was statistically significant compared to the concurrent negative control
Toxicity:
yes
Remarks:
Systemic toxicity: slight toxic effects were observed after dosing. Cytotoxicity to target tissue: histological analysis of duodenum samples from all animals showed no evidence of cytotoxicity
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
no increase in tail intensity was observed in the liver and glandular stomach
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw for two consecutive days
- Solubility: no information
- Clinical signs of toxicity in test animals: Slight signs of toxicity such as moving the bedding, piloerection and salivation after the second application were noted in each gender, but no gender specific differences were determined.
- Evidence of cytotoxicity in tissue analysed: not applicable
- Rationale for exposure: limit dose

RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: The route of exposure and dose levels were appropriate according to the guideline.
- Statistical evaluation: Normality was tested according to Kolmogorov-Smirnov-test. For the determination of statistical significances, the mean values of each animal per dose group were evaluated with one-way ANOVA (Dunnett’s test) at the 5 % level (p<0.05). The p value was used as a limit in judging for significance levels in comparison with the corresponding negative control.
A statistically significant increase was determined for the positive control group of each organ (Table 3). Moreover, a significance compared to the negative control was determined in duodenum cells at the maximum tolerated dose. This significance was regarded as biologically relevant.
- Necropsy and histopathology: No gross lesions were observed at necropsy. No histological alterations were noted in any specimen from any group.

Table 2 Summary of Mean Tail intensities [%] in Liver, Glandular Stomach and Duodenum

Dose

Group

Mean Tail Intensity [%]

 

 

 

 

Liver

Glandular Stomach

Duodenum

Signs of Toxicity

PC

14.20**

16.30**

21.35**

None

NC

2.52

4.23

5.61

None

400 mg/kg bw

2.21

5.47

7.10

None

1000 mg/kg bw

2.65

5.88

7.32

None

2000 mg/kg bw

3.28

7.33

10.91*

Slight Toxic Effects

Statistically significant trend

No

No

No

/

Historic           Control Range

NC: 0.00 - 8.70 %

PC: 4.66 – 32.87 %

NC: 1.98 – 9.42 %

PC: 3.29 – 47.23 %

NC: 0.00 - 8.41 %

PC: 5.15 – 37.18 %

/

*       significantly increased (One-way ANOVA with Dunnett’s test after normality test by Kolmogorov-Smirnov)

**     significantly increased (unpaired t-test with Welch’s correction after normality test by Kolmogorov-Smirnov)

NC: Negative Control (Physiological Saline)

PC: Positive Control (Ethyl methansulfonate): 300 mg/kg bw

MTD: Maximum Tolerated Dose

Table 3 Incidence of induced DNA-strand breaks: Statistical significance at the 5 % level (p < 0.05) evaluated by means with one-way ANOVA (Dunnett’s test)

Negative Control vs.       Test Group

Liver Cells

Glandular Stomach Cells

Duodenum Cells

 

male

male

male

PC

Significance

p-value

+

0.0039

+

0.0160

+

0.0400

0.2 MTD

Significance

p-value

-

0.9564

-

0.8796

-

0.7532

0.5 MTD

Significance

p-value

-

0.9955

-

0.7661

-

0.6722

MTD

Significance

p-value

-

0.6220

-

0.3390

+

0.0266

Conclusions:
[3-(2,3-Epoxypropoxy)propyl]diethoxy(methyl)silane has been tested in an in vivo mammalian alkaline comet assay conducted according to OECD TG 489 and in compliance with GLP. The undiluted test substance was administered orally by gavage at doses of 400, 1000 and 2000 mg/kg bw for two consecutive days. A negative control, physiological saline, was administered according to the same schedule, and a positive control was administered orally 4 hours before sacrifice. The positive control item, ethyl methanesulphonate, induced a statistically significant increase in DNA damage for all evaluated organs, as shown by an increase in mean tail intensity relative to the negative control. The test item did not result in any significant positive signal in the liver and glandular stomach. However, increased comet formation was detected in duodenum cells only after application of the highest tested limit-dose of 2000 mg/kg bw. Neither a dose-response relationship nor any significant signal outside the historical control interval at the lower doses was detected. Duodenum samples were preserved for histopathological analysis; no abnormalities were observed at necropsy and there were no histological alterations detected in any tissue.

Additional information

The registered substance was tested in a valid bacterial reverse mutation assay according to OECD TG 471 and under GLP, using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2uvrA (BioReliance, 2000). There was a positive, dose related increase in the number of revertant colonies in TA 100, TA 1535 and WP2 uvrA, with and without metabolic activation. The test substance is mutagenic in these tester strains. Appropriate positive and solvent controls were added and gave expected results. It is concluded that the test substance is positive for mutagenicity to bacteria under the conditions of the test. This study was selected as key as it used a more appropriate solvent than the more recent study.

The registered substance has been tested for mutagenicity to bacteria in a study conducted according to OECD TG 471, and in compliance with GLP, in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (RCC, 2008). The registered substance produced a biologically relevant increase in the number of revertants in strains TA 100, TA 1535 and WP2 uvr, with and without a metabolic activation system. No toxic effects, evident as a reduction in the condition of the background lawn, occurred in the test groups. Appropriate positive, solvent and negative controls were used and gave the expected results. It is concluded that the test substance is positive for mutagenicity to bacteria under the conditions of the study. The solvent used in this study, ethanol, is theoretically able to react with the substance, and therefore is considered by the reviewer not to be an appropriate solvent, so the study was not selected as key study. The study is still considered valid, since the results are in line with the earlier study which used DMSD, which is considered to be an appropriate solvent.

The registered substance was tested for ability to cause chromosome aberrations in cultured human blood lymphocytes in a study conducted according to OECD 473, and in compliance with GLP (SafePharm 2004c). The test material induced a statistically significant dose-related increase in the frequency of cells with chromosome aberrations in both the absence and presence of metabolic activation. Appropriate positive and solvent controls were used and gave the expected results. The test material was therefore considered to be clastogenic to human lymphocytes in vitro.

The registered substance has been tested for the induction of micronuclei in mice according to OECD TG 474, and in compliance with GLP (Shin-Etsu, 2009). No evidence for a test substance induced increase in the incidence of micronucleated polychromatic erythrocytes in mice bone marrow was observed when tested up to limit concentration. No clinical signs of toxicity were observed following oral administration of 500, 1000 and 2000 mg/kg of test material to mice. Appropriate positive and vehicle controls were included and gave the expected results. It is concluded that the test substance does not cause damage to chromosomes under the conditions of the test.

It should be noted that the PCE / NCE ratio was unchanged in treated animals, so there is no evidence that the test substance reached the target tissue. However, the available information on the toxicokinetics suggests that the registered substance will hydrolyse very rapidly in the acidic milieu of the stomach (hydrolysis half-life at 37.5°C and pH 2 is 5 seconds). The hydrolysis product, [3-(2,3-epoxypropoxy) propyl]methylsilanediol is water soluble (1.0E+06 mg/l) and has a favourable molecular weight (192.28) for absorption so systemic exposure is likely. Although the parent substance has a molecular weight which is not favourable for uptake (248.4) it is water soluble (1200 mg/l), so some systemic exposure is possible if unhydrolysed parent compound is left in the stomach. Evidence for systemic exposure to the substance or its hydrolysis products can be found in the comet assay (BSL Bioservice, 2018) in which slight toxic effects such as moving the bedding and salivation were observed directly after both applications in most animals at the maximum tolerated dose (MTD) of 2000 mg/kg bw on two consecutive days, and reduced body weight was observed in one animal after administration of the second MTD. It is therefore concluded based on the result of the study that the test substance is negative for the induction of micronuclei under the conditions of the test. This study was selected as key because it is the more recent of the available assays.

The registered substance was tested in a reliable, valid in vivo mouse micronucleus assay according to OECD TG 474 and in compliance with GLP (BioReliance 2001). The test substance did not produce treatment-related or statistically significant increases in the incidence of micronuclei in the peripheral blood polychromatic erythrocytes when administered by oral gavage up to limit concentrations. It was not genotoxic under the conditions of the test.

In the information available for [3-(2,3-epoxypropoxy)propyl]diethoxy(methyl)silane, there was evidence for clastogenicity (causing chromosomal aberrations) in the presence and absence of metabolic activation (SafePharm 2004c) as well as for bacterial mutagenicity in vitro (BioReliance, 2000). Two in vivo micronucleus studies did not support these findings (BioReliance, 2001 and Shin Etsu, 2009), so it is concluded that the in vitro result does not reflect an ability to cause chromosome aberrations in vivo.

[3-(2,3-Epoxypropoxy)propyl]diethoxy(methyl)silane has been tested in an in vivo mammalian alkaline comet assay conducted according to OECD TG 489 and in compliance with GLP (BSL Bioservice, 2019). The undiluted test substance was administered orally by gavage at doses of 400, 1000 and 2000 mg/kg bw for two consecutive days. A negative control, physiological saline, was administered according to the same schedule, and a positive control was administered orally 4 hours before sacrifice. The positive control item, ethyl methanesulphonate, induced a statistically significant increase in DNA damage for all evaluated organs, as shown by an increase in mean tail intensity relative to the negative control. The test item did not result in any significant positive signal in the liver and glandular stomach. However, increased comet formation was detected in duodenum cells only after application of the highest tested limit-dose of 2000 mg/kg bw. Neither a dose-response relationship nor any significant signal outside the historical control interval at the lower doses was detected. Duodenum samples were preserved for histopathological analysis; no abnormalities were observed at necropsy and there were no histological alterations detected in any tissue.


Justification for classification or non-classification

The available information for the substance indicates that when tested in vitro [3-(2,3-epoxypropoxy)propyl]diethoxy(methyl)silane, induces mutations in bacterial cells and chromosomal aberrations in vitro in the presence and absence of metabolic activation, but this potential for genetic toxicity was not confirmed in two reliable in vivo micronucleus studies, so it is concluded that [3-(2,3-epoxypropoxy)propyl]diethoxy(methyl)silane is not clastogenic.

The result of an in vivo mammalian alkaline comet assay was ambiguous, with increased comet formation at highest dose observed in cells of duodenum but not in cells of the glandular stomach or liver. Consideration of potential for germ cell mutagenicity. The ambiguous result from the comet assay has been considered. Although effects suggesting DNA damage were observed in cells of the duodenum, there is no evidence of DNA damage to cells of the liver, therefore the comet assay does not support classification for germ cell mutagenicity.

Implications for site of contact mutagenicity: the evidence of DNA damage to cells of the duodenum at a level slightly above the historical control suggests that the substance might be a site of contact mutagen. Under CLP, only germ cell mutagens are classified.

On the basis of the information currently available, [3-(2,3-epoxypropoxy)propyl]diethoxy(methyl)silane does not require classification for germ cell mutagenicity according to Regulation (EC) No 1272/2008.