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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 / chromosome aberration study in mammalian cells
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:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study planned
Study period:
2018
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out:
Zinc, bis(O,O-bis(1-methylethyl) phosphorodithioato-.kappa.S)bis(cyclohexanamine)-, (T-4)- (CAS 52585-16-7)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies:
A reliable in vitro gene mutation study in bacteria according to OECD TG 471 and GLP is available (Envigo CSR, 2016). The test substance (CAS 52585-16-7) was tested at concentrations up to 5000 µg/plate in 2 independent experiments, including the plate incorporation and preincubation method. Precipitates were visible at the two highest doses tested. Vehicle and positive controls were valid and lay within or marginally above the range of historical control data. Based on the results, the test substance (CAS 52585-16-7) did not induce an increase in reversions in the S. typhimurium strains TA 98, 100, 1535 and 1537, and in the E.coli strain WP2 uvrA with or without metabolic activation. Therefore, Zinc, bis(O,O-bis(1-methylethyl) phosphorodithioato-.kappa.S)bis(cyclohexanamine)-, (T-4)- (CAS 52585-16-7) can be considered as non-mutagenic in bacterial cells.
The clastogenic activity of Zinc, bis(O,O-bis(1-methylethyl) phosphorodithioato-.kappa.S)bis(cyclohexanamine)-, (T-4)- (CAS 52585-16-7) 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). In the first experiment, the test substance was tested in the absence and in the presence of metabolic activation for a 4-h exposure time. In the second experiment, the test substance was tested for a 20-h treatment period in the absence of S9 mix. 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 test 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, when tested up to precipitating or the highest evaluable concentrations.
- Available non-GLP studies: not available
- Historical human data: not available
- (Q)SAR: not applicable
- In vitro methods: Both in vitro studies are described under “available GLP-studies”.
- Weight of evidence: not available
- Grouping and read-across: not available

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- According to Reach Regulation (EC) 2006/1907, Annex VIII, 8.4.3, Column 2, appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII.
FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: An in vitro Mammalian Cell Micronucleus Test conducted according to OECD 487 with peripheral cultured human lymphocytes revealed a clastogenic potential for the registered substance. Therefore, additional in vivo data are needed, and the alkaline single-cell gel electrophoresis assay (Comet Assay according to OECD TG 489) is considered the most appropriate follow up test, to confirm or not the positive results of the in vitro test. In fact, the detection of DNA strand breaks is the principle of this method. These DNA strand breaks may result from direct interactions with DNA, alkali labile sites or as a consequence of incomplete excision repair. Therefore, the alkaline comet assay recognizes primary DNA damage that would lead to gene mutations and/or chromosome aberrations, but also detects DNA damage that may be effectively repaired or lead to cell death. The comet assay can be applied to almost every tissue of an animal from which single cell or nuclei suspensions can be made, including specific site of contact tissues.
Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

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, when tested up to precipitating or the highest evaluable concentrations.

Conclusion for genetic toxicity

The available data show the registered substance showed clastogenic properties without metabolic activation in cultured peripheral human lymphocytes.

Justification for classification or non-classification

The available experimental test data on genetic toxicity are insufficient for the purpose of classification under Regulation (EC) No.1272/2008. Therefore, an testing proposal for the conduct of an in vivo alkaline single-cell gel electrophoresis assay (Comet Assay according to OECD TG 489) has been submitted to the ECHA. Based on the data of this study a decision on the classification on the classification under Regulation (EC) No.1272/2008 will be made.