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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): not mutagenic in S. typhimurium TA 98, TA 100, TA 1535 and TA 1537, and E. coli WP2 uvrA, with and without metabolic activation

Micronucleus test (OECD 487): clastogenic in cultured peripheral human lymphocytes without metabolic activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Mar - 29 Apr 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Suitability of cells: Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.
- Sex, age and number of blood donors: 1 male donor, aged 21 years (Experiment I); 1 female donor, aged 33 years (Experiment II)
- Whole blood was used
- Methods for maintenance in cell culture: 3 µg/mL phytohemeagglutinine in medium; cells were cultured at 37 °C with 5.5% CO2 in humidified air

MEDIA USED
- Type and identity of media including CO2 concentration: DMEM/F12 (1:1) supplemented with penicillin/streptomycin (100 U/mL and 100 µg/mL, respectively), FBS (10%), HEPES (10 mM) and heparin (125 U.S.P.-U/mL)
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:
Experiment I:
4 h treatment (without metabolic activation): 69.63, 121.9 and 213.2 µg/mL
4 h treatment (with metabolic activation): 39.79, 69.63 and 121.9 µg/mL

Experiment II:
20 h treatment (without metabolic activation): 79.01, 118.5 and 177.8 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: demecolcin (125 ng/mL in deionized water (- S9 mix, 4 h, continuous treatment))
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation method: 48 h
- Exposure duration: 4 and 20 h
- Fixation time: 40 h

STAIN: Giemsa

NUMBER OF REPLICATIONS: Duplicates each in 2 independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The slides were prepared by dropping the cell suspension in fresh fixative onto a cleas microscope slide and thereafter stained with Giemsa.

NUMBER OF CELLS EVALUATED: 500 binucleated cells per culture

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. According to the study director, the criteria of Countryman and Heddle (1976) were taken for the evaluation of micronuclei.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index was determined and cytotoxicity was expressed as %cytostasis.

OTHER EXAMINATIONS:
- Other: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

[Reference: Countryman P.I. and Heddle J.A. (1976): The production on micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.]
Evaluation criteria:
A test item can be classified as non-clastogenic and non-aneugenic if:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% confidence interval).

A test item can be classified as clastogenic and aneugenic if:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data (95% confidence interval).
Statistics:
Statistical significance will be confirmed by the Chi square test (α < 0.05), using the validated R Script CHI2.Rnw for those values that indicate an increase in the number of cells with micronuclei compared to the concurrent solvent control.
Key result
Species / strain:
lymphocytes: lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. I (4 h): ≥ 373.2 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. I (4 h): ≥ 373.2 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
Exp. II (20 h): at 177.8 µg/mL
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. II (20 h): ≥ 400.0 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the presence of S9 mix and a 4-h exposure period, a statistical significant increase (1.05%) in micronucleated cells was observed at the lowest evaluated concentration (39.79 μg/mL). In the absence of S9 mix and a 4-h exposure period, a statistical significant increase (1.10%) in micronucleated cells was observed at the highest evaluated concentration (213.2 μg/mL). However, both values can be declared as biologically irrelevant, because they are well within the range of the historical laboratory control data (0.02 – 1.15% in the absence and 0.08 – 1.20% in the presence of S9 mix).

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH determined in the maximum concentration without metabolic activation was within the same range as in the solvent control.
- Effects of osmolality: The osmolality of the solvent control did not differ from the osmolality of the maximum concentration without metabolic activation.
- Precipitation: The test substance precipitated at the end of the treatment at 213.2 µg/mL and above with and without metabolic activation in Experiment I. In Experiment II precipitation occured at 400.0 µg/mL at the end of treatment without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: In the dose range finding test, blood cultures were exposed to the test substance at 12.99, 22.74, 39.79, 69.63, 121.9, 213.2, 373.2, 653.1, 1143 and 2000 µg/mL culture medium with and without metabolic activation for 4 h exposure time. Strong cytotoxic effects were determined at concentrations of 373.2 µg/mL and above with and without metabolic activation. Precipitation was observed in the culture medium at concentrations of 213.2 µg/mL and above with and without metabolic activation. Since the pre-test fulfilled the requirements for cytogenetic evaluation, it was designated Experiment I. The concentration levels for Experiment II were selected based on the results of the range-finding test (Experiment 1).

CYTOKINESIS BLOCK
- Distribution of mono-, bi- and multi-nucleated cells: the number of mono-, bi- and multi-nucleated cells were determined.

NUMBER OF CELLS WITH MICRONUCLEI
- Binucleated cells per culture were scored for cytogenetic damage.

HISTORICAL CONTROL DATA
- Positive historical control data: The values of the positive control were within the range of the historical control data [ranges: 2.10 - 6.40 (-S9, continuous treatment); 4.15 - 24.00 (-S9; pulse treatment); 2.25 - 11.30 (+S9, pulse treatment)].
- Negative (solvent/vehicle) historical control data: The values of the negative control were within the range of the historical control data [ranges: 0.05 - 1.43 (-S9, continuous treatment); 0.15 - 1.25 (-S9; pulse treatment); 0.15 - 1.35 (+S9, pulse treatment)].

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

Table 1: Cytogenicity in Experiment I and II

  Concentration
[µg/mL]
mono-
nucleated cells
bi-nucleated cells multi-
nucleated
cells
proliferation index
CBPI
mono-
nucleated cells
bi-nucleated cells multi-
nucleated
cells
proliferation index
CBPI
Mean proliferation index
CBPI
Cytostasis [%] *
Experiment I: 4 h without S9 mix
Vehicle control (Ethanol) 0.5 % (v/v) 94 329 77 1.97 107 326 67 1.92 1.94  
Positive control (MMC) 1.0 168 304 28 1.72 144 304 52 1.82 1.77 18.6
Test substance 69.63 160 304 36 1.75 83 350 67 1.97 1.86 8.8
Test substance 121.9 203 278 19 1.63 246 236 18 1.54 1.59 37.6
Test substance 213.2 P 190 286 24 1.67 211 272 17 1.61 1.64 32.1
Experiment I: 4 h with S9 mix
Vehicle control (Ethanol) 0.5 % (v/v) 146 286 68 1.84 162 263 75 1.83 1.82  
Positive control (CPA) 15.0 177 276 47 1.74 201 259 40 1.68 1.53 15.1
Test substance 39.79 106 300 94 1.98 99 307 94 1.99 1.88 NC
Test substance 69.63 117 313 70 1.91 147 282 71 1.85 1.86 NC
Test substance 121.9 167 287 46 1.76 145 295 60 1.79 1.68 4.9
Experiment II: 20 h without S9 mix
Vehicle control (Ethanol) 0.5 % (v/v) 113 370 17 1.81 116 354 30 1.83 1.84  
Positive control (Demecolcin) 15.0 224 276 0 1.55 247 249 4 1.51 1.71 15.1
Test substance 79.01 97 374 29 1.86 94 366 40 1.89 1.98 NC
Test substance 118.5 108 355 37 1.86 107 355 38 1.86 1.88 NC
Test substance 177.8 178 308 14 1.67 169 316 15 1.69 1.79 4.9

P: Precipitation occurred at the end of treatment

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

NC: Not calculated as the CBPI is equal or higher than the solvent control value

Table 2: Number of micronucleated cells

  Concentration
[µg/mL]
binucleated cells with n micronuclei culture 1 sum binucleated cells with n micronuclei culture 2 sum sum in 2000 binucleated cells Micronucleated cells [%] **
    1 2 >2   1 2 >2      
Experiment I: 4 h without S9 mix
Vehicle control (Ethanol) 0.5 % (v/v) 2 0 0 2 2 0 0 2 4 0.2
Positive control (MMC) 1.0 94 10 3 107 100 8 1 109 216 10.80 S
Test substance 69.63 2 1 0 3 6 0 0 6 9 0.45
Test substance 121.9 1 0 0 1 3 0 0 3 4 0.20
Test substance 213.2 P 12 2 0 14 8 0 0 8 22 1.10 S
Experiment I: 4 h with S9 mix
Vehicle control (Ethanol) 0.5 % (v/v) 2 0 0 2 6 0 0 6 8 0.40
Positive control (CPA) 15.0 34 4 0 38 33 3 0 36 74 3.70 S
Test substance 39.79 15 0 0 15 6 0 0 6 21 1.05 S
Test substance 69.63 4 0 1 5 5 0 0 5 10 0.50
Test substance 121.9 3 0 0 3 11 0 0 11 14 0.70
Experiment II: 20 h without S9 mix
Vehicle control (Ethanol) 0.5 % (v/v) 9 0 0 9 5 1 0 6 15 0.75
Positive control (Demecolcin) 15.0 20 4 1 25 27 4 2 33 58 2.90 S
Test substance 79.01 9 1 0 10 9 0 0 9 19 0.95
Test substance 118.5 9 0 0 9 6 1 0 7 16 0.80
Test substance 177.8 # 32 2 1 35 29 1 1 311 66 1.65 S

P: Precipitation occurred at the end of treatment

S: The number of micronucleated cells is statistically significantly higher than corresponding control values

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

# The number of micronucleated cells was determined in a sample of 4000 binucleated cells

Table 3: Historical Control Data

Solvent Control without S9
Micronucleated cells in %
  Pulse treatment (4/40) Continuous treatment (20/40)
No. of experiments 50* 54**
Mean 0.61 0.55
95 % Ctrl limit 0.02 – 1.15 0.05 – 1.05
     
1x SD 0.27 0.25
2x SD 0.54 0.50
Min 0.15 0.05
Max 1.25 1.43
Solvent Control with S9
Micronucleated cells in %
  Pulse treatment (4/40)
No. of experiments 67***
Mean 0.64
95 % Ctrl limit 0.08 – 1.20
   
1x SD 0.28
2x SD 0.56
Min 0.15
Max 1.35

* Aqueous solvents – 23 Experiments; Organic solvents – 27 Experiments
** Aqueous solvents – 24 Experiments; Organic solvents – 30 Experiments

*** Aqueous solvents – 24 Experiments; Organic solvents – 43 Experiments

Conclusions:
In the in vitro micronucleus test conducted with human lymphocytes according to OECD 487, clastogenicity could be evidenced in the absence of metabolic activation after a 20-h exposure period.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Feb - 02 Mar 2016
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 (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
"his operon" (for S. typhimurium strains) and "trp operon" (for E. coli strains)
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
Test concentrations with justification for top dose:
First experiment: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate with and without metabolic activation
Second experiment:
- Strain TA 100: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate with and without metabolic activation
- All other strains: 33; 100; 333; 1000; 2500; and 5000 μg/plate with and without metabolic activation
Vehicle / solvent:
Ethanol (purity > 99 %)
The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: -S9: 4-nitro-o-phenylene-diamine, 4-NOPD for TA98 (10 µg/plate) and TA 1537 (50 µg/plate); +S9: 2-aminoanthracene, 2-AA (2.5 µg/plate) for all strains (10 µg/plate in WP2 uvrA)
Details on test system and experimental conditions:
ORIGIN OF BACTERIAL STRAINS:
The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA were obtained from Trinova Biochem GmbH (Gießen, Germany)

METHOD OF APPLICATION:
- Experiment I: in agar (plate incorporation)
- Experiment II: pre-incubation

DURATION
- Preincubation period in case of Experiment II: 1 h at 37 °C
- Exposure duration: 48 h at 37 °C

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn. Cytotoxicity was detected by a reduction in the number of revertants below the indication factor of 0.5.
Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
- A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. II: starting at 1000 µg/plate without S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. II: at 5000 µg/plate without S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. II: starting at 1000 µg/plate without S9 mix and starting at 2500 µg/plate with S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. II: starting at 1000 µg/plate without S9 mix and at 5000 µg/plate with S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. II: starting at 1000 µg/plate without S9 mix and at 5000 µg/plate with S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Solvent: The test item was soluble in ethanol. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria. Concentrations up to the maximum recommended test concentration (5000 µg/plate) were used.
- Precipitation: Precipitation of the test item in the overlay agar on the incubated agar plates was observed in Experiment I at concentrations from 2500 to 5000 μg/plate without S9 and at concentrations from 333 to 5000 μg/plate with S9, and in Experiment II at concentrations from 2500 to 5000 μg/plate without S9 and at concentrations from 1000 to 5000 μg/plate with S9. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: Reported as experiment I. The highest concentration (5000 µg/plate) showed no relevant toxic effects. Thus, 5000 µg/plate was chosen as maximum concentration.

Table 1: Results of Cytotoxicity study

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate)

Experiment I

Experiment II

 Strain

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

/

/

2500 – 5000

/

TA 1537

5000

/

2500 – 5000

5000

TA 98

/

/

2500 – 5000

5000

TA 100

1000 – 5000

2500 – 5000

1000 – 5000

5000

WP2 uvrA

/

/

5000

/

/= no reduction in the number of revertants (below the induction facor of 0.5)

Table 2: Results of Experiment I

EXPERIMENT 1 (Plate Incorporation)

S9-Mix

Without

 

Test item (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

E. coli

NC

14 ± 5

9 ± 3

28 ± 6

192 ± 13

46 ± 10

SC

11 ± 3

10 ± 2

36 ± 3

184 ± 22

45 ± 10

3 µg

12 ± 5

11 ± 3

41 ± 4

197 ± 18

45 ± 10

10 µg

13 ± 5

8 ± 2

32 ± 4

188 ± 9

47 ± 9

33 µg

15 ± 3

9 ± 2

35 ± 6

202 ± 17

51 ± 8

100 µg

15 ± 3

11 ± 2

31 ± 6

178 ± 21

49 ± 5

333 µg

17 ± 2

13 ± 3

33 ± 4

145 ± 28

42 ± 1

1000 µg

10 ± 2

10 ± 3

45 ± 11

67 ± 11

38 ± 12

2500 µg

 15 ± 1

6 ± 2

32 ± 6

26 ± 8

48 ± 7

5000 µg

11 ± 2

4 ± 2

23 ± 1

13 ± 2

52 ± 10

NaN3 (PC), 10 µg

1218 ± 95

 

 

2204 ± 204

 

4-NOPD (PC), 10 µg

 

 

348 ± 20

 

 

4-NOPD (PC), 50 µg

 

82 ± 11

 

 

 

MMS (PC), 2 µL

 

 

 

 

992 ± 65

S9-Mix

With

 

 

 

 

 

 

 

Test item (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

E. coli

NC

14 ± 1

11 ± 3

41 ± 2

148 ± 7

46 ± 9

SC

17 ± 4

9 ± 4

47 ± 15

187 ± 10

52 ± 7

3 µg

13 ± 4

13 ± 4

59 ± 4

158 ± 4

55 ± 10

10 µg

16 ± 2

11 ± 4

55 ± 9

207 ± 18

53 ± 7

33 µg

16 ± 2

13 ± 3

65 ± 14

169 ± 7

63 ± 8

100 µg

18 ± 2

11 ± 2

59 ± 8

196 ± 13

55 ± 11

333 µg

21 ± 2

15 ± 2

48 ± 8

162 ± 11

49 ± 6

1000 µg

14 ± 4

17 ± 4

64 ± 16

95 ± 18

63 ± 1

2500 µg

21 ± 4

9 ± 2

58 ± 4

66 ± 9

57 ± 6

5000 µg

20 ± 1

6 ± 2

55 ± 8

22 ± 6

67 ± 11

2-AA (PC), 2.5 µg

446 ± 20

147 ± 14

5381 ± 328

3427 ± 21

 

2-AA (PC), 10 µg

 

 

 

 

473 ± 21

NC = Negative/Vehicle Control

SC = Solvent Control

PC = Respective positive control substances (for details see method description)

Key to Positive Controls

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

Table 3: Results of Experiment II

EXPERIMENT 2 (Pre-Incubation)

S9-Mix

Without

 

Test item (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

E. coli

NC

9 ± 3

8 ± 2

28 ± 6

196 ± 14

51 ± 8

SC

19 ± 5

10 ± 2

32 ± 5

195 ± 9

47 ± 8

3 µg

 

 

 

181 ± 9

 

10 µg

 

 

 

187 ± 25

 

33 µg

15 ± 5

10 ± 2

30 ± 9

171 ± 18

44 ± 5

100 µg

18 ± 8

10 ± 3

27 ± 3

173 ± 27

46 ± 8

333 µg

16 ± 3

12 ± 4

35 ± 8

135 ± 30

50 ± 3

1000 µg

15 ± 1

8 ± 1

31 ± 3

56 ± 6

37 ± 11

2500 µg

2 ± 1

1 ± 1

1 ± 1

17 ± 8

39 ± 3

5000 µg

1 ± 1

0 ± 0

0 ± 1

1 ± 1

8 ± 4

NaN3 (PC), 10 µg

1394 ± 96

 

 

2314 ± 75

 

4-NOPD (PC), 10 µg

 

 

397 ± 18

 

 

4-NOPD (PC), 50 µg

 

97 ± 13

 

 

 

MMS (PC), 2 µL

 

 

 

 

798 ± 28

S9-Mix

With

 

 

 

 

 

 

 

Test item (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

E. coli

NC

12 ± 5

15 ± 6

41 ± 2

202 ± 7

62 ± 4

SC

16 ± 2

16 ± 1

45 ± 10

195 ± 21

67 ± 14

3 µg

 

 

 

206 ± 19

 

10 µg

 

 

 

185 ± 7

 

33 µg

18 ± 3

16 ± 3

43 ± 8

191 ± 9

56 ± 3

100 µg

23 ± 5

17 ± 3

53 ± 9

185 ± 11

62 ± 12

333 µg

19 ± 4

16 ± 1

46 ± 7

188 ± 20

63 ± 2

1000 µg

23 ± 2

16 ± 2

50 ± 1

161 ± 23

69 ± 12

2500 µg

18 ± 3

13 ± 3

49 ± 6

113 ± 26

71 ± 12

5000 µg

12 ± 2

6 ± 2

17 ± 3

53 ± 3

55 ± 3

2-AA (PC), 2.5 µg

485 ± 19

112 ± 14

5361 ± 195

3413 ± 74

 

2-AA (PC), 10 µg

 

 

 

 

453 ± 19

NC = Negative/Vehicle Control

SC = Solvent Control

PC = Respective positive control substances (for details see method description)

Key to Positive Controls

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

Conclusions:
Based on the results of the conducted study, the test substance did not show mutagenic properties in TA 98, TA 100, WP2 uvrA, TA 1535 and TA 1537 with and without metabolic activation, respectively.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo alkaline comet assay (OECD 489): equivocal in duodenum and glandular stomach but negative in the liver of male Crl:Wistar Han rats

An mammalian erythrocyte micronucleus test according to OECD guideline 474 is proposed.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 Oct 2019 - 26 May 2020
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:
adopted 29 Jul 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Method B.62 (In Vivo Mammalian Alkaline Comet Assay)
Version / remarks:
EC Commission Regulation No. 2017/735
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Wistar
Remarks:
Crl: Wistar Han
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at administration: ca. 54 - 60 days
- Weight at study initiation: 219 - 262 g
- Assigned to test groups randomly: yes
- Housing: in groups; animals were given access to small soft white untreated wood (Aspen) chew blocks and a plastic shelter for environmental enrichment
- Diet: pelleted Envigo Teklad 2014C, ad libitum
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 (minor deviation on one day with 19 °C reported)
- Humidity (%): 40 - 70 (minor deviation noticed on 2 distinct days, with 38% humidity measured for ca. 1 h)
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: corn oil
- Concentration of test material in vehicle: 18.75, 37.5 and 75 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Supplier: KTC Edibles Ltd, United Kingdom
- Lot/batch no.: 9154K
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
An accurately weighed amount of the test item was ground with a mortar and pestle, the vehicle was gradually added during continuous rubbing. The formulations were homogenised for 5 min and sonicated for 5 min until a homogenous suspension was achieved. Formulations were magnetically stirred until and during administration. The test item formulations were prepared and analysed for achieved concentration on each day of dosing.

Duration of treatment / exposure:
2 days
Frequency of treatment:
daily (the second dose being administered approximately 24 hours after the first dose)
Post exposure period:
sampling time point: 3 h after the second dose administration
Dose / conc.:
187.5 mg/kg bw/day (actual dose received)
Dose / conc.:
375 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6 males (test item and vehicle control) and 3 males (positive control)
Control animals:
yes, concurrent vehicle
Positive control(s):
ethylmethanesulphonate (EMS)
- Route of administration: oral gavage (single treatment approximately 3 h prior to termination)
- Doses / concentrations: 200 mg/kg bw / 20 mg/mL (dose volume 10 mL/kg bw)
Tissues and cell types examined:
liver, duodenum and glandular stomach
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Dose levels were selected based on the results of a preliminary toxicity in male and female rats (please refer to "Additional information on results"). The highest dose level for the main comet assay was chosen to represent the maximum tolerated dose (MTD), defined as the highest dose that will be tolerated without evidence of study-limiting toxicity, relative to the duration of the study period.

DETAILS OF SINGLE CELL SUSPENSION PREPARATION:
The animals were sacrificed about 3 hours after administration of the second dose. Positive control animals were sacrificed approximately 3 h after a single dosing. Sections of the liver,duodenum and glandular stomach were prepared and placed into ice cold mincing solution; all samples were stored on ice before processing for Comet analysis. Single cell suspensions were prepared using the following tissue specific methods:

Liver
Approximately 0.5 cm³ of liver tissue was cut and washed in fresh complete mincing solution until as much blood was removed as possible. 2 mL of fresh complete mincing solution was added to the liver and then cut into several small pieces. The pieces were transferred to 150 µm bolting cloth held over a falcon tube and the liver was pushed through the cloth. An additional 2 mL of complete mincing solution was added, and any remaining liver pushed through the cloth. The sample was stored on ice until slide preparation.

Duodenum
The duodenum was removed, washed free of waste using complete mincing solution and placed into a falcon tube and covered with approximately 2 mL of mincing solution. The samples were incubated on ice for approximately 30 min (± 10 min). After incubation the tissue was removed and was washed free of waste using complete mincing solution for a second time. The tissue was transferred to a clean Petri dish, 1.5 mL of complete mincing solution was added, and the tissue was minced using a scalpel blade to release the cells. The cells were poured into a clean falcon tube and stored on ice until slide preparation.

Glandular Stomach
The stomach was removed, cut open, washed free from food using mincing solution and incubated in a falcon tube on ice for 30 min (± 10 min). Following incubation, the stomach was removed and surface epithelial was gently scraped using a cell scraper. The layer was discarded, and gastric mucosa was rinsed with fresh mincing solution until all food was removed. 1.5 mL of fresh mincing solution was added to a clean petri dish and the stomach was scraped 4 - 5 times to release the cells. The cells were stored on ice until slide preparation.

DETAILS OF SLIDE PREPARATION:
Comet slides were prepared from all cell suspensions.
Frosted end glass slides were dipped in 1% normal melting point agarose and left to air dry prior to addition of the cell suspension layer.
For each tissue type, an appropriate dilution of the cell suspensions were made and mixed with the appropriate volume of 0.5% low melting point agarose. A 75 µL aliquot of the cell/agar mix was dispensed onto the appropriate pre-dipped slides and cover-slipped. Once the agar had set the cover slips were removed and the slides immersed in chilled lysis solution in a light proof box. These were stored at 2 - 8 ºC overnight prior to electrophoresis.
Electrophoresis
The slides were randomly placed onto a dry, level platform of a horizontal electrophoresis unit containing chilled electrophoresis buffer. The slides from each treatment were spread across the platform to avoid any positional effects. The buffer reservoir of the unit was topped up with electrophoresis buffer until the surfaces of the slides are covered. The slides were left for 20 min to allow the DNA to unwind. After alkali unwinding, the slides were electrophoresed at 18 V with a starting current of approx. 300 mA (between 0.7 to 1.0 V/cm) for 30 min.
The temperature of the electrophoresis solution during unwinding and electrophoresis was maintained at a low temperature, usually 2 – 10 °C. When electrophoresis was completed, the slides were washed on three occasions (separated by 5 min intervals) with neutralisation buffer and stored, refrigerated, in lightproof boxes with moistened tissues (to prevent agar dehydration).

METHOD OF ANALYSIS:
Microscopic Examination
Coded slides were stained with SYBR GOLD® (Life Technologies) and visualised under a fluorescence microscope. The comet images from the microscope were captured via a CCD camera and measured using Perceptive Instruments COMET IVTM image analysis system. Initially the slides were examined for any overt toxicity, e.g. an increase in background debris and/or an increase in the incidence of excessively damaged cells (i.e. hedgehog cells). These cells were excluded from the analysis, along with any cells that had unusual staining artefacts.
50 cells were scored per slide to give a total number of 150 cells per tissue per animal. The extent of DNA migration and hence damage is reflected by the percentage of tail intensity (% TI) defined as the fluorescence detected by image analysis in the tail, which is proportional to the amount of DNA that has moved from the head region into the comet tail.

OTHER:
Histopathology
Sections of the liver, duodenum and glandular stomach were stored in 10% buffered formalin. Sections of the duodenum and glandular stomach from the vehicle control animals and animals administered test item doses of 187.5, 375 and 750 mg/kg bw/day were embedded in paraffin wax, sectioned and stained with haematoxylin and eosin and assessed for signs of cytotoxicity, necrosis and apoptosis.

Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
a) At least one of the test doses exhibits a statistically significant increase in % tail intensity compared with the concurrent vehicle control.
b) The increase in % tail intensity is dose-related when evaluated with an appropriate trend test.
c) Any of the results are outside the distribution of the historical vehicle control data.

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if:
a) None of the test concentrations exhibits a statistically significant increase in % tail intensity compared with the concurrent vehicle control.
b) There is no concentration-related increase in % tail intensity when evaluated with an appropriate trend test.
c) All results are inside the distribution of the historical vehicle control data.

There is no requirement for verification of a clearly positive or negative response.

In case the response is neither clearly negative nor clearly positive as described above and in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions (e.g. dose spacing, other routes of administration, other sampling times or other tissues) could be useful.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and will therefore be concluded as equivocal.
To assess the biological relevance of a positive or equivocal result, cytotoxicity at the target tissue should also be discussed. Histopathological information can help in the interpretation of a positive result in the comet assay. Careful interpretation of increased or decreased % tail DNA in the presence of severe cytotoxicity is therefore essential.
Statistics:
Please refer to pdf document "Statistical analysis" under "attached background material"
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Clinical signs were observed at all dose levels and comprised piloerection at ≥ 187.5 mg/kg bw/day and fast breathing at 750 mg/kg bw/day.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: liver tissue
Key result
Sex:
male
Genotoxicity:
positive
Remarks:
statistically significant increase in % tail intensity at 187.5, 375 and 750 mg/kg bw/day
Toxicity:
yes
Remarks:
Clinical signs were observed at all dose levels and comprised piloerection at ≥ 187.5 mg/kg bw/day and fast breathing at 750 mg/kg bw/day.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: duodenum tissue
Key result
Sex:
male
Genotoxicity:
positive
Remarks:
statistically significant increase in % tail intensity at 750 mg/kg bw/day
Toxicity:
yes
Remarks:
Clinical signs were observed at all dose levels and comprised piloerection at ≥ 187.5 mg/kg bw/day and fast breathing at 750 mg/kg bw/day.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: glandular stomach tissue
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In the preliminary toxicity study, the animals were initially treated at 1000 mg/kg bw/day with 2 doses administered 24 h apart. At 1000 mg/kg bw/day, clinical signs of toxicity in male and female animals included fast breathing, salivation, piloerection, elevated gait and prominent eyes (both). The clinical signs observed were mainly classified as moderate and persisted until the end of the working day. On the basis of this result the maximum tolerated dose had been exceeded, therefore, an additional group of two male and two female animals was administered 500 mg/kg bw/day. At 500 mg/kg bw/day, clinical signs of toxicity in male and female animals included fast breathing, salivation, piloerection, elevated gait and prominent eyes (both). Clinical signs were mainly classified as mild and any moderate signs transitioned to mild after around 15 minutes post-gavage. On the basis of this result the maximum tolerated dose had not been achieved, therefore, an additional group of two male and two female animals was administered 750 mg/kg bw/day. At 750 mg/kg bw/day, clinical signs of toxicity in male and female animals included fast breathing, salivation, pilo erection, elevated gait and prominent eyes (both). Clinical signs were mainly classified as mild and moderate signs were transient in nature.
On the basis of these results the maximum tolerated dose was considered to be 750 mg/kg bw/day. There was not considered to be a substantial difference in toxicity between the sexes, therefore, in line with current guidelines, the comet test was conducted using male animals only.

RESULTS OF DEFINITIVE STUDY
Hedgehog cells were excluded from the analysis, along with any cells that had unusual staining artefects.

- Results on tail intensity (% TI):

Vehicle control group:
The vehicle control group mean and median % tail intensity values for the liver, duodenum and glandular stomach of male Crl: Wistar Han rats were within the current vehicle historical control range for the individual tissue (95% confidence limits). For details on historical control data please refer to Tables 5 and 6 under "Any other information on results incl. tables".

Positive control group:
The positive control compound, EMS, produced a significant increase (p < 0.001) in the median % TI when compared to vehicle control values in all tissues analysed and all % TI values were comparable to the positive historical control range. For details on historical control data please refer to Tables 5 and 6 under "Any other information on results incl. tables".

Test item group:
Liver:
There were no statistically significant increases observed in the median % TI in the liver of male rats administered 187.5, 375 or 750 mg/kg bw/day. The group (mean and median) % TI values from all treatment groups were within the current vehicle historical control range (95% confidence limits). For details please refer to Table 2 under "Additional information on results incl. tables".

Duodenum:
There were statistically significant increases observed in the median % TI in the duodenum of male rats administered 187.5 mg/kg bw/day (p<0.05), 375 and 750 mg/kg bw/day (p < 0.001). The group (mean and median) % TI values were outside of the current vehicle historical control range (95% confidence limits). For details please refer to Table 3 under "Additional information on results incl. tables".

Glandular stomach:
There were statistically significant increases observed in the median % TI in the glandular stomach of male rats administered 750 mg/kg bw/day (p < 0.001). The group (mean and median) % TI values were outside of the current vehicle historical control range (95% confidence limits). For the 187.5 and 375 mg/kg bw/day dose groups, no statistically significant increases in the median % TI in the glandular stomach were observed and the group (mean and median) %TI values were within the current vehicle historical control range (95% confidence limits). For details please refer to Table 4 under "Additional information on results incl. tables".

- Results on Hedgehog cells:
In the liver, hedgehog cells were observed after administration of 750 mg/kg bw/day only.
In the duodenum, there was a dose-related increase in the number of hedgehog cells observed at 187.5, 375 or 750 mg/kg bw/day, compared to the concurrent vehicle control (group mean: vehicle control, 187.5, 375 or 750 mg/kg bw/day: 0.5, 14.2, 23.7 and 33.2 respectively).
In the glandular stomach, there was a dose-related increase in the number of hedgehog cells observed after administration of 187.5, 375 or 750 mg/kg bw/day, compared to the concurrent vehicle control (group mean: vehicle control, 187.5, 375 or 750 mg/kg bw/day: 0.0, 0.0, 2.8 and 29.3 respectively).

- Results on histopathology:
There were no test item-related changes in the glandular stomach and the duodenum. The liver was not examined for histopathological changes.






Mortality and clinical signs of toxicity:

No mortalities were observed throughout the duration of the comet assay. Clinical signs of toxicity comprised piloerection in 5/6 animals at 187.5 mg/kg bw/day, in 2/6 animals at 375 mg/kg bw/day and in 6/6 animals at 750 mg/kg bw/day. In addition, fast breathing was observed in 1/6 animals at 750 mg/kg bw/day.

Body weight development:

Marginal body weight loss was noted in animals of the 375 mg/kg bw/day group (-0.4%) and in animals of the 750 mg/kg bw/day group (-1.5%) when compared to the vehicle control animals.

Result tables

Table 2: Summary of results in liver tissue

Male animal data - Liver

Treatment

Dose
(mg/kg bw/day)

Number of cells scored

Group mean tail intensity% (SD) #

Group mean of median tail intensity% (SD) #

Vehicle

-

900

2.57 (0.5)

0.58 (0.5)

Test item

187.5

900

2.69 (0.6)

0.47 (0.3)

Test item

375

900

3.03 (0.6)

0.50 (0.3)

 Test item

750

900

3.01 (0.8)

0.47 (0.4)

EMS

200

450

54.14 (8.4)

55.16 L+***(9.5)

SD: Standard deviation

EMS: Ethylmethane sulfonate

# Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):

p values for comparisons with control using Williams’ test, unless indicated otherwise (+ t-test)

*** p < 0.001 (statistically significant)

L: Analysis performed upon logarithmically transformed data

Table 3: Summary of results in duodenum tissue

Male animal data - Duodenum

Treatment

Dose
(mg/kg bw/day)

Number of cells scored

Group mean tail intensity% (SD) #

Group mean of median tail intensity% (SD) #

Vehicle

-

900

5.83 (1.4)

2.06 (1.0)

Test item

187.5

900

7.65 (1.9)

3.87 *(0.9)

Test item

375

900

10.35 (1.9)

5.38 ***(1.3)

Test item

750

900

13.81 (2.0)

7.26 ***(1.3)

EMS

200

450

61.96 (6.0)

63.66 L+***(8.1)

SD: Standard deviation

EMS: Ethylmethane sulfonate

# Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):

p values for comparisons with control using Williams’ test, unless indicated otherwise (+ t-test)

*** p < 0.001, * p < 0.05 (statistically significant)

L: Analysis performed upon logarithmically transformed data

Table 4: Summary of results in glandular stomach tissue

Male animal data - Glandular stomach

Treatment

Dose
(mg/kg bw/day)

Number of cells scored

Group mean tail intensity% (SD) #

Group mean of median tail intensity% (SD) #

Vehicle

-

900

4.74 (1.1)

1.69 (0.9)

Test item

187.5

900

3.39 (0.5)

0.93 L(0.3)

Test item

375

900

4.98 (2.4)

2.00 L(1.3)

Test item

750

900

17.36 (2.7)

11.42 L***(2.7)

EMS

200

450

58.11 (8.0)

59.84 L+***(8.2)

SD: Standard deviation

EMS: Ethylmethane sulfonate

# Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):

p values for comparisons with control using Williams’ test, unless indicated otherwise (+ t-test)

*** p < 0.001 (statistically significant)

L: Analysis performed upon logarithmically transformed data

Historical control data

Table 5: Mean tail intensity historical control data generated in the testing facility in Mar 2018 - Mar 2020 (liver and duodenum tissue) and in Dec 2011 - Mar 2020 (glandular stomach tissue), combined data generated in Sprague-Dawley and Wistar Han rats

Mean % Tail Intensity

Historical control data: liver tissue

Historical control data: duodenum tissue Historical control data: glandular stomach tissue
Vehicle Control Values Positive Control Values Vehicle Control Values Positive Control Values Vehicle Control Values Positive Control Values
172 Animals  30 Studies  87 Animals  29 Studies  124 Animals  22 Studies  66 Animals  22 Studies  71 Animals  12 Studies  54 Animals  12 Studies 
Individual Group Individual Group Individual Group Individual Group Individual Group Individual Group
Minimum 0.9 1.5 15.9 18.4 1 1.3 27 29.4 1.5 2.4 28 34.4
Maximum 5.2 3.7 51.4 41.3 7.7 6.7 63 52.7 14.3 12.6 70.3 64.8
Mean 2.3 2.3 29.8 29.8 4 4 39.3 39.3 4.7 4.8 54.5 53.4
Standard Deviation 0.8 0.5 7.2 6.3 1.5 1.3 8.1 7.1 2.5 2.6 9.5 9.2
Lower 95% CL 0.7 1.3 15.3 17.1 1 1.3 23.1 25.1 0 0 35.5 35.1
Upper 95% CL 4 3.3 44.2 42.4 7.1 6.7 55.5 53.5 9.7 10.1 73.5 71.8

CL: Confidence limits

Table 6: Median tail intensity historical control data generated in the testing facility in Mar 2018 - Mar 2020 (liver and duodenum tissue) and in Oct 2017 - Mar 2020 (glandular stomach tissue), combined data generated in Sprague-Dawley and Wistar Han rats

Median % Tail Intensity

Historical control data: liver tissue Historical control data: duodenum tissue Historical control data: glandular stomach tissue
Vehicle Control Values Positive Control Values Vehicle Control Values Positive Control Values Vehicle Control Values Positive Control Values
172 Animals  30 Studies  87 Animals  29 Studies  124 Animals  22 Studies  66 Animals  22 Studies  71 Animals  12 Studies  54 Animals  12 Studies 
Individual Group Individual Group Individual Group Individual Group Individual Group Individual Group
Minimum 0 0.1 11.9 14.9 0.1 0.3 23.2 26 0.2 0.5 25 32.1
Maximum 1.4 0.7 52.6 40.9 4.1 3.2 65.4 53 3.9 2.6 63.4 56.8
Mean 0.4 0.4 27.4 27.4 1.4 1.3 37.5 37.5 1.2 1.2 43.6 43.6
Standard Deviation 0.2 0.2 8.4 7.4 1.1 0.9 9.5 8.2 1.2 1.2 14.4 12.5
Lower 95% CL 0 0.1 10.5 12.7 0 0 18.4 21 0 0 14.8 18.7
Upper 95% CL 0.8 0.7 44.3 42.1 3.5 3.1 56.5 54 3.6 3.6 72.5 68.5

CL: Confidence limits

Conclusions:
Oral administration of the test substance within the present comet assay resulted in an increase in DNA strand breaks in the duodenum and glandular stomach of male Crl:Wistar Han rats. However, due to the confounding presence of hedgehog cells, these increases are considered to be linked with cytotoxicity. There were no treatment-related findings at histopathological examination. Based on the experimental findings, the test item is considered to be equivocal for DNA damage in the duodenum and glandular stomach, whereas it is negative for DNA damage in the liver.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Genetic toxicity in bacteria

The in vitro genetic toxicity of Zinc, bis[O,O-bis(1-methylethyl) phosphorodithioato-.kappa.S]bis(cyclohexanamine)-, (T-4)- (CAS 52585-16-7) was assessed in a bacterial reverse mutation assay (Ames test) according to GLP criteria and OECD 471 (Envigo CSR, 2016). The mutagenic potential of the test substance was assessed in S. typhimurium tester strains TA 98, 100, 1535 and 1537 and in the Escherichia coli tester strain WP2 uvrA at concentrations up to 5000 µg/plate in 2 independent experiments, including the plate incorporation and preincubation method, with and without metabolic activation, respectively. Precipitates were visible at the two highest doses tested. The test substance did not induce an increase in reversions in any of the S. typhimurium strains or the E.coli strain with or without metabolic activation. In the plate incorporation test cytotoxicity was observed at 5000 µg/plate in TA 1537, in TA 100 from 1000 to 5000 µg/plate and from 2500 to 5000 µg/plate without and with metabolic activation, respectively. In the pre-incubation test and in the absence of metabolic activation cytotoxicity was observed at a concentration of 1000 µg/plate and higher in TA1537, in TA 1535, TA 98 and TA 100. In WP2uvrA, cytotoxic properties were detected at 5000 µg/plate without metabolic activation. With metabolic activation, cytotoxicity was observed in TA 98 and TA 100 at 5000 µg/plate, and in TA 1537 at 2500 µg/plate and higher. The vehicle and positive controls were valid and lay within or marginally above the range of historical control data.

 

In vitro Cytogenicity in mammalian cells

The clastogenic activity of the registered substance was investigated in an in vitro mammalian cell micronucleus test in cultured peripheral human lymphocytes according to OECD guideline 487 and GLP (Envigo CSR, 2016). The test substance was dissolved in ethanol and two independent experiments were performed. In the first experiment, the test substance was tested at 69.63, 121.9 and 213.2 µg/mL in the absence of metabolic activation and at 39.79, 69.63 and 121.9 µg/L in the presence of metabolic activation for a 4-h exposure time following a recovery period of 16 h and a cytochalasin B exposure of 20 h, respectively. In the second experiment, the test substance was tested 79.01, 118.5 and 177.8 µg/mL for a 20-h treatment period in the absence of S9 mix following a cytochalasin B exposure of 20 h. In each experimental group two parallel cultures were analyzed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage. In Experiment I, a statistical significant increase (1.05%) in micronucleated cells at the lowest evaluated concentration (39.79 μg/mL) in the presence of S9 mix and a statistical significant increase (1.10%) in micronucleated cells at the highest evaluated concentration (213.2 μg/mL) in the absence of S9 mix was observed. However, both values can be declared as biologically irrelevant, because they are well within the range of the historical laboratory control data (0.02 – 1.15% in the absence and 0.08 – 1.20% in the presence of S9 mix). In Experiment II in the absence of S9 mix a statistical significant increase (1.65%) in micronucleated cells was observed at the highest evaluated concentration (177.8 μg/mL). The value is outside of the 95% control limit of the laboratory historical control data (0.05 – 1.05%). Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the registered substance induced micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, Zinc, bis(O,O-bis(1-methylethyl) phosphorodithioato-.kappa.S)bis (cyclohexanamine)-,(T-4)- is considered to be clastogenic in the in vitro micronucleus test in the absence of metabolic activation, when tested up to precipitating or the highest evaluable concentrations.

 

In vivo alkaline Comet assay

The registered substance was assessed for its potential to induce DNA strand breaks in a Comet assay conducted in male Crl : Wistar Han rats. The study was performed according to OECD guideline 489 and in compliance with GLP (Covance Laboratories, 2020). The test substance was formulated in corn oil and administered to the animals by gavage for two consecutive days. Dose levels were chosen based on the results of a preliminary toxicity study in male and female rats, in which 750 mg/kg bw/day were identified as the maximum tolerated dose (MTD) causing mild to moderate clinical signs of toxicity of transient nature. As no substantial difference in toxicity was observed between the sexes, the comet assay was conducted in male animals only. In the main study, groups of 6 animals received two test item doses of 187.5, 375 or 750 mg/kg bw/day 24 h apart. A similar constituted group of 6 males received the vehicle corn oil and served as control. In addition, a group of 3 positive control animals was included and treated with a single oral dose of 200 mg/kg bw/day ethylmethanesulphonate (EMS). Clinical signs of toxicity and individual body weights were monitored. The animals were sacrificed 3 h after the last dosing and cell suspensions were obtained from the liver, duodenum and glandular stomach. Following slide preparation and gel electrophoresis, a total of 150 cells/animal was scored for DNA damage, which was reflected as percentage of tail intensity (% TI). In addition, histopathology was performed on the duodenum and glandular stomach tissues.

Following treatment with the test substance, piloerection was noted in the animals of all test item dose groups and fast breathing was observed at 750 mg/kg bw/day. A statistically significant and dose-dependent increase in the % TI value in the duodenum of all test item-treated male rats, as compared to the vehicle control group was noted. Further, the median % TI value was statistically significantly increased in the glandular stomach of the high dose group (750 mg/kg bw/day). There was no increase in median % TI observed for the liver at any dose level. The increase in DNA damage observed for the duodenum and the glandular stomach was accompanied by an increase in hedgehog cells. Hedgehog cells were observed in the liver at 750 mg/kg bw/day, whereas they were reported in the duodenum and glandular stomach at all dose levels, with a dose-dependent increase in number. Although the exact cause of the hedgehog cells remained unclear, it is clear that they, or their cause, have in this study compromised the comet response and it is considered, that they are in this case linked with cytotoxicity. Therefore, it could not be excluded that the DNA damaging effects observed with the test item were related to cytotoxicity. Histopathological examination of the duodenum and the glandular stomach revealed no treatment-related findings.

The tail intensities observed for the vehicle and positive controls remained within the range of the laboratories historical control data, thus demonstrating the sensitivity and validity of the test.

In conclusion, based on the experimental findings, Zinc, bis(O,O-bis(1-methylethyl) phosphorodithioato-.kappa.S)bis (cyclohexanamine)-,(T-4)- has not shown any evidence of causing an increase in DNA strand breaks in the liver, whereas evidence of causing an increase in DNA strand breaks in the duodenum and glandular stomach of male rats was noted. However, due to the confounding presence of hedgehog cells, these increases are considered to be linked with cytotoxicity. Thus, the results of the Comet assay are considered equivocal with respect to the induction of DNA strand breaks in both, the duodenum and the glandular stomach of male Wistar rats treated with the registered substance.

 

Conclusion for genetic toxicity

Based on the available data, the registered substance was negative for gene mutation in bacteria (Ames test) but showed clastogenic properties without metabolic activation in cultured peripheral human lymphocytes in vitro. An equivocal result on induction of DNA damage in duodenum and glandular stomach by the test substance was obtained in an in vivo Comet assay. Considering all available data, the test item is currently considered to be inconclusive for genetic toxicity. Thus, a mammalian erythrocyte micronucleus test according to OECD guideline 474 is proposed.

Justification for classification or non-classification

The data as available for genotoxicity/mutagenicity consists of an in vitro Ames test, which was negative with and without metabolic activation, an in vitro Micronucleus test, which was positive in the absence but negative in the presence of metabolic activation, and a newly conducted in vivo Comet assay with male rats. The findings of the Comet assay are considered equivocal with respect to both, the duodenum and glandular stomach of rat, whereas the result was negative for the liver. Hereby it needs to be mentioned that the result of the glandular stomach is of no particular relevance for man.

Thus, the whole set of data as yet available is not considered sufficient to support any classification and labelling of the test item for genotoxicity/mutagenicity according to the CLP Regulation (EC) No. 1272/2008.