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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial mutagenicity

Key; 175116; Ames (OECD 471); GLP; S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538; 1 – 1000 µg/plate (+ S9), 1 - 100 (-S9); negative for TA 98, TA 1537, TA 1538 ± S9 and positive for TA 100 and TA 1535 ±S9

Gene mutations in mammalian cells

Key; 0174-EV1; HPRT (OECD 476); GLP, V79 cells; 10 - 56 µg/mL (+ S9) and 1 – 10 µg/mL (–S9); negative ± S9

Cytogenetic assay in mammalian cells

Key; T5558.337; Chromosome aberration in vitro (OECD 473); GLP; CHO cells; 0.2 - 3 µg/mL (+ S9) and 0.003 – 0.05 µg/mL (–S9); negative ± S9

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:
19 Jan - 26 Feb 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 2020
Deviations:
yes
Remarks:
Missing strain for AT reversion site (S. thyphimurium TA 102 or E.coli WP2)
Qualifier:
according to guideline
Guideline:
EPA OPP 84-2
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
His operon
Species / strain / cell type:
bacteria, other: TA 98, 100, 1535, 1538 and 1537
Additional strain / cell type characteristics:
other: His auxotrophy, defective DNA repair activity (delta uvrB) and defective LPS barrier (rfa) Ampicillin resistance (TA 98 and TA 100 only)
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Wistar rats which received a single injection of 500 mg/kg bw Aroclor 1254 in olive oil 5 days before sacrifice.

The protein concentration of the S9 preparation was usually between 20 and 45 mg/mL.

The S9 fraction was mixed with cofactors to final concentrations of: 8 mM MgCI2, 33 mM KCI, 5 mM Glucose-6-phosphate, 5 mM NADP in 100 mM sodium-ortho-phosphate buffer (pH 7.4).

S9 fraction was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 5.6 % v/v in the S9 mix. Each batch of S9 was routinely tested for its capability to activate the known mutagen 2-aminoanthracene in the Ames test.
Test concentrations with justification for top dose:
The standard plate incorporation assay was performed using 1, 10, 33.3, 100, 333.3, 666.6 and 1000 µg/plate of the test item with S9 mix using Salmonella strains TA 98, TA 100, TA 1535, TA 1538 and TA 1537.

Without S9 mix Salmonella strains TA 100, TA 1535,TA 1538 and TA 1537 were used in the presence of 1, 3.3, 10, 33.3, 66.6 and 100 µg test substance/plate.

Strain TA 98 was tested with 10, 33.3, 100, 333.3, 666.6 and 1000 µg test substance/plate, without S9 mix.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine, 4-NOPD, TA 1537, TA 1538, TA 98, 50 μg/plate in DMSO; - S9 mix, 2-aminoanthracene, 2-AA, TA 1535, TA 1537, TA 1538, TA 98, TA 100, 10 µg/plate in DMSO, + S9 mix
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicates
- Number of independent experiments: 2 (with and without metabolic activation, plus a pre-test for strains TA 98 and TA 100)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 72 h


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition
Rationale for test conditions:
A pre-experiment for testing the toxicity of the test article was performed with strains TA 98 and TA 100. 8 concentrations were tested for toxicity and mutagenicity with each 3 plates.
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 100) or thrice (strains TA 1535, TA 1537, TA 1538, TA 98) the colony count of the corresponding solvent control is observed. Also, a significant dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential.

Acceptability:
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the historical data
Statistics:
No statisitcal analysis was performed.
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxic effects occurred with and without metabolic activation at 1000 µg/plate (exp.II)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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:
toxic effects occurred with and without metabolic activation at 1000 µg/plate (exp.II).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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:
toxic effects occurred at 333.3 and 1000µg/plate (exp.I) with and without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
dose-dependent and significant increase in revertant colony numbers with and without metabolic activation
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
normal background growth up to 1000 µg/plate with S9 mix and up to 100 µg/plate in the absence of metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
dose-dependent and significant increase in revertant colony numbers with and without metabolic activation.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
normal background growth up to 1000 µg/plate with S9 mix and up to 100 µg/plate in the absence of metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment the concentration range of the test item was 1 – 5000 μg/plate and only in strains TA 98 and 100. Genotoxicity was not observed up to the highest concentration tested. At 5000 µg/mL, no spontanous revertants occurred. Data can be found in Attachment 1 in the attached background material.

STUDY RESULTS
The test material produced a dose-dependent and significant increase in revertant colony numbers observed in strains TA1535 and TA100 in the two experiments performed. Signs of toxicity with and without metabolic activation. With and without S9 mix, toxic effects occurred in strains TA98 at 333.3 and 1000µg/plate (exp.I) and in the strains TA1537 and TA1538 at 1000 µg/plate (exp.II). With metabolic activation toxic effects occurred in strains TA1537 (exp.I) and TA100 (exp.I and II) at 1000 µg/plate. Strains TA1535, TA1537, TA1538 and TA100 showed normal background growth up to 1000 µg/plate with S9 mix and up to 100 µg/plate in the absence of metabolic activation.

For results of both experiment I and experiment II as well as the pre-experiment, please refer to the attached background material
(Attachment 2) under "Attached background material"



HISTORICAL CONTROL DATA
- Positive historical control data: no
- Negative (solvent/vehicle) historical control data: yes, can be found in Attachment 3 in the attached background material.

Table 1: Revertant colonies mean from three plates with and without metabolic activation

Treatment

Conc.

[µg/plate]

S9 mix

TA 1535

Exp I/ ExpII

TA 1537

Exp I/ ExpII

TA 1538

Exp I/ ExpII

TA 98

Exp I/ ExpII

TA 100

Exp I/ ExpII

Negative control

 

-

22 ; 9

10 ; 9

18 ; 12

19 ; 19

95 ; 77

Solvent controlA

 

-

18 ;16

6 ; 7

12 ; 12

18 ; 20

89 ; 75

test substance

1.0

-

27 ; 18

9 ; 15

10 ; 14

- ; -

107 ; 104

 

3.3

-

17; 22

11 ; 8

15 ; 15

- ; -

132 ; 109

 

10.0

-

33; 23

16 ; 17

11 ; 17

25 ; 32

205 ; 147

 

33.3

-

54 ;41

12 ; 17

12 ; 15

33 ; 31

277 ; 220

 

66.6

-

56; 53

12 ; 16

16 ; 14

- ; -

271 ; 274

 

100.0

-

44; 51

12 ; 12

14 ; 21

34 ; 31

368 ; 256

 

333.3

-

 

 

 

8 ; 33

 

 

666.6

-

 

 

 

7 ; 16

 

 

1000.0

-

 

 

 

1 ; 12

 

Positive controlB

 

-

133 ; 1100

305 ; 220

2594 ; 2421

2031 ; 170

1299 ; 1069

Negative control

 

+

11 ; 11

10 ; 13

16 ; 13

33 ; 31

104 ; 87

Solvent controlA

 

+

11 ; 12

11 ; 13

18 ; 13

32 ; 29

89 ; 92

 test substance

1.0

+

20 ; 14

9 ; 12

17 ; 13

31 ; 30

95 ; 98

 

10.0

+

23 ; 27

11 ; 13

20 ; 15

38 ; 28

145 ; 187

 

33.3

+

52 ; 44

14 ; 18

12 ; 16

35 ; 40

220 ; 269

 

100.0

+

70 ; 54

19 ; 11

16 ; 19

40 ;42

298 ; 293

 

333.3

+

45 ; 34

11  ; 11

10 ; 17

33 ; 27

247 ; 304

 

666.6

+

53 ; 39

12 ; 11

15 ; 9

47 ;45

247 ; 266

 

1000.0

+

37 ; 26

0 ; 4

11 ; 3

31 ; 43

0 ; 0

Positive controlB

 

+

507 ; 109

414 ; 89

2243 ; 152

2604 ; 1672

2564 ; 837

ADMSO,B2-aminoanthracene,SD - Standard deviation

 

 

Conclusions:
The study was performed according to OECD guideline 471 and compliant with GLP. Under the conditions of this study, the test substance induced point mutations by base pair changes in the genome of the strains TA1535 and TA100 with and without metabolic activation.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Aug 1985 - 1 May 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopten in 2016
Deviations:
yes
Remarks:
For details on guideline deviations, please refer to "Principles of method if other than guideline".
Principles of method if other than guideline:
Deviations from current OECD guideline 476 (adopted 2016)
- No information on chromosome number and mycoplasma contamination checks
- No information on cell cycle length
- Chosen concentrations were too low as the relative cell survival did not reach 20%
- No Historical Control Data provided
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine guanine phosporibosyl transferase (HPRT) locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Department Toxicology, Agricultural University of Wageningen, The Netherlands
- Suitability of cells: standard cells for the respective assay

For cell lines:
- Methods for maintenance in cell culture: cell density was kept below 4x10^6 cells per 75 cm² flask

- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media: Ham's F-10 medium (without thymidine and hypoxanthine) supplemented with 10% newborn calf serum, L-glutamine (2 mM), and penicillin/streptomycin/cefoxtamin (50 U/mL, 50 µg/mL and 2 µg/mL, respectively)
- Exposure medium: F-10 culture medium buffered with 20 mM Hepes in the absence of serum
- CO2 concentration: 5% CO2
- humidity level: humid atmosphere (95%)
- Temperature: 37 °C
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver homogenate (S9) was prepared previously. The S9/cofactor mixture consisted of

1.02 mg MgCl2, 2.46 mg KCl, 1.7 mg glucose-6-phosphate, 3.4 mg NADP, 4 µmol HEPES and 0.5 mL S9.

0.2 mL of the S9/cofactor mixture was added to 1 mL of medium for cytotoxicity or mutagenicity testing.
Test concentrations with justification for top dose:
1, 3.3, 5.6 or 10 µg /mL without S9 mix
10, 18, 33 or 56 µg/mL in the presence of S9 mix for 2 h
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9-mix: Ethylmethane sulfonate (EMS); 6 mM With S9-mix: Dimethylnitrosamine (DMN); 4 and 8 mM
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 1 culture per dose
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 6 x 10^6 (exposure), 10^6 (expression)
- Test substance added in cell culture medium as described above (serum-free, buffered with Hepes)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2 h (with and without metabolic activation)
- Harvest time after the end of treatment (sampling/recovery times): 14 - 17 days

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days, during this time, the cells were subcultured every 2 or 3 days to maintain exponential growth
After the expression period, in total 10^6 cells were plated into 10 petri dishes with selective medium.
- Selective agent: 10 µM 6-thioguanine, 7 - 10 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 10^6 cells in total for mutant selection after the expression period, 2 x 200 cells of each dose were plated in cloning medium directly after exposure to assess cell survival (cloning efficiency)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency (CE)
CE: (Average No. of colonies on cloning plates)/200 *100%

Mutant Frequency per 10^5 survivors: (100/CE)*(Total number of mutant colonies on selective plates/number of seeded cells)
Rationale for test conditions:
A pre-test was performed assessing the cytotoxicity of the cells. 3x10^6 cells were treated in the presence (0.001 - 10 µg/mL) or absence (0.01 - 100 µg/mL) of S9 mix.
Evaluation criteria:
A mutant assay was considered acceptable if the following criteria were met:
- The absolute colony forming efficiency of the solvent control is 60%
- At least 3/4 doses of the test substance have an acceptable number of surviving cells (10^6) analysed for HPRT mutations.
- The spontanous mutant frequency in the untreated or solvent treated control is <3 per 10^5 survivors.
- The positive controls induced significant (at least 3-fold) increases in mutant frequencies.

A test substance is considered mutagenic if
- It induces a mutant frequency that is at least 3 x times higher than the spontaneous mutant frequency of the untreated control.
- The results show a dose response relationship and are reproducible in an independently repeated test.

A test substance is considered negative (nonmutagenic) if
- None of the test concentrations induce a mutant frequency that is at least 3 x times higher than the spontaneous mutant frequency of the untreated control.
- The results are reproducible in an independently repeated test.
Statistics:
No statistical evaluation was performed.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Chosen concentrations were too low as the cell survival did not reach 20%
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: A concentration of 10 µg/mL,without metabolic activation, reduced cloning efficiency by approximatively 50%. In the presence of S9 mix, cytotoxicity occurred only from 30 µg/mL. Summarized results can be found in Attachment 1 in the attached background material.

STUDY RESULTS
- Genotoxicity: No statistically significant increases in mutant frequency or dose– response were observed.
- Concurrent vehicle negative and positive control data: The solvent and positive control induced satisfactory mutagenicicty rates, indicating the adequacy of the experimental conditions.
- Results from cytotoxicity measurements: the test item was observed to be significantly cytotoxic at the level of 10 µg/mL in the absence of S9 mix (CE was reduced by approcx. 55 - 75%). In the presence of S9 mix, the cloning efficiency was only reduced by 25 - 50%, even though the test item concentrations were higher (up to 56 µg/mL).

For summarized results, please refer to Attachment 1 in the attached background material.

HISTORICAL CONTROL DATA: not provided
Conclusions:
The present study was conducted in compliance with GLP and EPA OPP 84-2, and similar to OECD test guideline 476. Under the conditions of the study, no statistically significant increase in mutant frequency was observed for any dose level in the absence or presence of metabolic activation. No significant dose-response was observed either. Therefore, the test substance is concluded not to be mutagenic in CHO cells in vitro.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Jun - 4 Aug 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted in 2016
Deviations:
yes
Remarks:
methodological limitations (2x 50 metaphases analysed instead of 2 x 100, no historical control data available, missing information on cell system)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: American Type Culture Collection, Rockville, MD, USA

MEDIA USED
- Type and composition of media: McCoy's 5A medium supplemented with 10% or 2.5% FBS, 100 U/mL peniciliin, 100 µg/mL streptomycin and 2mM L-glutamine (complete medium)
- CO2 concentration: 4 - 6%
- Temperature: 36 - 38 °C
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from livers of male Sprague-Dawley rats treated with Aroclor 1254 (500 mg/kg bw i.p.) 5 days prior to sacrifice. Immediatly prior to use, the S9 fraction was mixed with the cofactor pool to contain 15 µl S9 fraction, 1.4 mg NADP and 2.7 mg isocitric acid per mL growth medium.
Test concentrations with justification for top dose:
- S9 mix: 0.003, 0.006, 0.012, 0.023 and 0.05 µg /mL
+ S9 mix: 0.2, 0.4, 0.8, 1.5 and 3 µg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: –S9 mix: Triethylenemelamine (TEM, 1 µg/mL) +S9 mix: Cyclophosphamide (CP, 10 µg/mL)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicates
- Number of independent experiments: 1 pre-test and 1 main experiment

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 x 10^5 cells/25 cm² flask
- Test substance added in complete medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: after seeding, the cells were incubated in complete medium for 16 - 24 h before treatment
- Exposure duration/duration of treatment: 14 h (-S9 mix), 2 h (+S9 mix)
- Harvest time after the end of treatment: 2 h (-S9 mix), 8 h (+S9 mix)

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colcemid was added 2 h before harvest time (-S9 mix: t = 14 h, +S9 mix: t = 8 h), the final concentration was 0.1 µg/mL
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): To prepare slides, the harvested cells were centrifuged, the supernatant discarded and the cells were resuspended in Carnoy's fixative. 1 - 2 drops of cell suspension were dropped onto the center of a moist glass slide and allowed to air dry overnight. Dried slides were stained with 5% Giemsa, air dried and permanently mounted.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 50 metaphases/slide, 2 slides per dose, in total 100 metaphases per dose
- Scored: Cells were evaluated for chromatid fragments, acentric fragments, dicentrics, rings, triradicals, quadriradicals, complex rearrangements, pulverized chromosome(s) and cells and severly damaged cells (> 10 aberrations).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative cell growth and cell cycle delay
Rationale for test conditions:
A pre-test for cytotoxicity was conducted assessing the influence of the test substance on cell growth potential and cell cycle delay. CHO cells were seeded for each treatment condition at approx. 5x10^5 cells/25 cm² flask and after 16 - 24 h were incubated with the test substance for 6 h (-S9 mix) or 2 h (+S9 mix). The concentrations ranged from 0.03 - 330 µg/mL. After the incubation period, cells were washed with PBS, and incubated with normal growth medium containing 0.01 mM BrdU for 24 h. 2 h before the end of the treatment, Colcemid was added to each flask (final conc. 0.1 µg/mL). Cells were harvested by trypsinization and counted for estimation of relative cell growth. Metaphase preparations were prepared and stained for sister chromatid differentiation using a modified fluorescence plus giemsa technique.
Evaluation criteria:
The number of cells with chromosome aberrations in the negative control group must be no greater than 6%. The number of cells with chromosome aberrations in the positive control must be statistically increased (p<0.05, Student's t test) relative to the solvent control or to the untreated control.
Statistics:
Statistical analysis of the frequency of structural aberrations per cell was performed using the student's t-test (all comparisons were made to the control group). Statistical analysis of the percentage of aberrant cells was performed using the Fisher's exact test (all comparisons were made to the control group).
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
38 % (-S9 mix)/ 53 % (+S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: At the highest concentration in the pre-test tested (330 µg/mL), the test article was soluble in the solvent, but slight precipitation occurred when added to the medium. In the main study, no preciupitation was observed.

RANGE-FINDING/SCREENING STUDIES: The test substance induced cell cycle delay at all concentrations tested in the nonactivated study; therefore, the harvest time was set at 16 h in order to ensure that all cells evaluated were in first division metaphase. No cell cycle delay was observed in the S9 activated study and the harvest time was left at 10 h.

Summarized results can be found in Attachment 1 in the attached background material.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Negative, solvent and positive controls induced expected results. The test system is therefore proven to be sensitive for the assay.
- Results from cytotoxicity measurements: At the time of harvest, the high dose of 0.05 µg/ml (in the absence of S9 mix) was observed to be slightly toxic upon microscopic examination of the cell monolayer. This dose was not evaluated for chromosome aberrations due to insufficient number of metaphase cells. In the presence of S9 mix, at the time of harvest, dose levels of 0.8, 1.5 and 3 µg/ml were observed to be slightly to moderately toxic upon microscopic examination of the cell monolayer. The highest dose was not evaluated for chromosome aberrations due to insufficient number of metaphase cells.
Relative cell growth without S9: 0.03 µg/ml: 53%; with S9: 3 µg/ml: 38%
- Genotoxicity results: The frequency of cells with aberrations or the mean aberrations per cell in the test article treatment groups was not statistically increased above that of the study control (p>0.05, Fishers exact test or student’s t-test).

Summarized results can be found in Attachment 1 in the attached background material.

HISTORICAL CONTROL DATA: No data
Conclusions:
The present study was in principles conducted similarly to OECD guideline 473, under GLP conditions. Under the conditions of the assay, the test item was not clastogenic in CHO cells when tested in the presence and absence of metabolic activation system.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Gene mutations in mice

Key; CCR200902, Mouse spot test, (OECD 484); GLP, mouse, 75 – 750 mg/kg bw, negative

Mouse germ-cell cytogenetic assay

Key; CCR175127; Chromosome aberration in vivo (similar to OECD 483); GLP, mouse sperm; 75 - 750 mg/kg bw; negative

Cytogenetic assays in bone marrow cells

Key; T5558.122; MNT (similar to OECD 474); GLP, mouse bone marrow; 38 - 377 mg/kg bw; negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Oct 1990 - 29 Jan 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 484 (Genetic Toxicology: Mouse Spot Test)
Version / remarks:
adopted in 1986
Qualifier:
according to guideline
Guideline:
EU Method B.24 (Mouse Spot Test)
Deviations:
yes
Remarks:
consecutively to negative results, statsitical analysis was not performed
GLP compliance:
yes
Type of assay:
mouse spot test
Species:
mouse
Strain:
other: NMRI (female), DBA/2 (male)
Details on species / strain selection:
The mouse is the most suitable species with strains expressing coat colour mutations which are well characterized with regard to the genetic background.
In addition, the mouse is an experimental animal in many physiological , pharmacological, and toxicological studies. Data from such experiments also may be useful for the design and the performance of the mouse spot test.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Laboratory for Mutagenicity Testing, Darmstadt, Germany (females), BRL- Tierfarm Füllinsdorf, Germany (males)
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: approx. 30 g
- Housing: individually after treatment in Makrolon Type II cages with wire mesh top
- Diet: pelleted standard diet (ALTROMIN, Lage/Lippe, Germany)
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 20 -80
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 4 Oct 1990 To: 29 Jan 1991
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose); 1%
- Justification for choice of solvent/vehicle: toxicity profile in animals
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dose was adjusted to the most recent body weight.
Duration of treatment / exposure:
single oral dose at Day 9 of pregnancy
Frequency of treatment:
single oral dose
Post exposure period:
3 weeks. Offspring was examined between day 39-43 after birth. For a detailed study schedule, please refer to "Any other information on materials and methods incl. tables".
Dose / conc.:
75 mg/kg bw/day
Dose / conc.:
750 mg/kg bw/day
No. of animals per sex per dose:
Treated animals were:
Solvent control: 21 pregnant females
Positive control: 30 pregnant females
75 mg/kg bw: 29 pregnant females
750 mg/kg bw: 79 pregnant females
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethylnitrosurea (ENU)
- Route of administration: orally, once
- Doses / concentrations: 20 mg/kg bw
- Volume: 10 mL/kg bw
- Solvent: physiological saline, pH 4.5
- The solution was prepared on the day of administration.
Tissues and cell types examined:
The offspring were coded and scored for the occurrence of pigmented spots.
Three classes of spots were distinguished:

1. WMVS (white mid-ventral spots);
White spots within 5 mm of the mid-ventral line which are presumed to result from cell killing.

2. MDS (misdifferentiation spots):
Yellow, agouti-like spots associated with mammaet genital ia, throat, axillary and inguinal areas and mid-forehead, which are presumed to result from misdifferentiation.

3. RS (recessive spots) :
pigmented and white spots randomly distributed on the coat which are presumed to result from somatic mutation.

All three classes of spots were scored but only the last, RS, is of genetic relevance. Microscopic analysis of mounted coloured hairs was performed only if there were doubts in classification of a spot.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A pre-experiment with 750 mg/kg bw was conducted.


Evaluation criteria:
The test article is classified as mutagenic if it induces a statistically significant dose-related increase in the frequency of genetically relevant spots or a statistically significant positive response for at least one of the test points compared with the negative control.

The test article is classified as non-mutagenic if it induces no statistically significant dose-related increase in the frequency of genetical ly relevant spots and no statistically significant positive response for at least one of the test points compared with the negative control.

Both biological and statistical significance should be considered together in the evaluation.
Statistics:
A statistical analysis of the results was not necessary to perform as after treatment with the test article the frequency of recessive spots which are presumed to result from somatic mutation were in the range of the negative control value.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
750 mg/kg bw = MTD
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 750 mg/kg bw
- Results: A pre-experiment was realised in order to evaluate the toxicity of the test susbtance. After a single oral dose of 750 mg/kg bw, the treated animals expressed toxic reactions (reduction of spontaneous activity, ptosis, apathy); this dose was estimated to be the MTD.

RESULTS OF DEFINITIVE STUDY
In the mutation assay the mean litter size was not reduced after treatment with the test article indicating no embryotoxic effects. Experimental conditions as well as criteria for the determination of test responses are well defined and appropriate. Controls gave the expected response. In the vehicle test group one recessive spot (RS) was observed. After treatment with 75 or 750 mg/kg bw thiram, the frequency of RS was 0.75 and 0.62%, respectively, which are in the range of the historical control data (0.00%- 1.00%). The positive control showed a distinct increase in induced RS frequency.

Summarized results can be found in Attachment 1 in the attached background material.
Conclusions:
The study was conducted according to the OECD guideline 484 and under GLP conditions. During the mutagenicity assay described and under the experimental conditions reported, the test article did not induce mutations in somatic cells of the mouse.
Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Mar - 26 Oct 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 483 (Mammalian Spermatogonial Chromosome Aberration Test)
Version / remarks:
adopted in 2016
Deviations:
yes
Remarks:
100 instead of 200 metaphases were analyzed per animal.
GLP compliance:
yes
Type of assay:
mammalian germ cell cytogenetic assay
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf, Germany
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: approx. 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: 5 h (food)
- Housing: individually in Makrolon Type I cages with wire mesh top
- Diet: pelleted standard diet (ALTROMIN, Lage/Lippe, Germany), ad libitum (except for fasting period before study)
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 Mar 1990 To: 26 Sep 1990
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose) 1%
- Justification for choice of solvent/vehicle: toxicity profile in animals
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test article was formulated in 1% carboxymethylcellulose suspension. All animals received a single standard dose volume adjusted to the body weight.
Duration of treatment / exposure:
single dose
Frequency of treatment:
single dose
Post exposure period:
6 h: high dose
24 h: negative and positive control; low, mid and high dose
48 h: high dose
Dose / conc.:
75 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
750 mg/kg bw/day
No. of animals per sex per dose:
6 males (Evaluation of five)
Control animals:
yes, concurrent vehicle
Positive control(s):
Doxorubicin-sulfathydrochloride
- Route of administration: intraperitoneally, single application
- Doses / concentrations: 7.5 mg/kg bw
- Volume administered: 10 mL/kg bw
Tissues and cell types examined:
spermatogonia; 100 metaphases per animal
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A pre-test was conducted, showing that 750 mg/kg bw was a dose in which suffcient metaphases for evaluation were available.

TREATMENT AND SAMPLING TIMES: All animals were dosed once, the low and middle dose as well as the positive and solvent control were only evaluated after 24 h, the high dose after 6, 24 and 48 h.

DETAILS OF SLIDE PREPARATION: Animals were sacrificed by cervical dislocation. Both testes were collected, the tunicas removed, the tubules were separated mechanically in physiological saline and dissociated enzymatically with collagenase resulting in a single cell suspension. After removal of the enzyme solution, the cells were incubated hypotonic trisodium citrat solution (1% w/v) for 20 min. The suspension was centrifuged, the supernatant discarded and the cells were fixed in 3:1 absolute methanol : glacial acetic acid fixative for 30 min, resuspended, the fixative was changed and enough fixative was added to make a relatively thin cell suspension. The fixative-cell suspension was spread by flame-drying and stained with Giemsa solution. At least 1 slide was made from each animal.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Gaps, breaks, fragments, deletions , exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. At least 100 well spread metaphases per animal were scored for cytogenetic damage on coded slides. Only metaphases with the characteristic chromosome number of 40 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis; 1000 cells are scored) was determined.
Five animals per test group were evaluated as described. The remaining animal of each test group was evaluated in case an animal died in its test group or was used to replace unscorable preparations.
Evaluation criteria:
A test article is classified as mutagenic if it induces a statistically or biologically significant increase in the number of structural chromosomal aberrations.
A test article producing no increase in the number of structural chromosomal aberrations is considered as non-mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test
However, both biological and statistical significance should be considered together.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric-Mann-Whitney test.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
2 pre-experiments were realized in which 8 mice/dose received orally a single dose of 1000 or 2000 mg/kg bw. In the second pre-experiment, 4 additional animals received a single dose of 750 mg/kg bw.
- Dose range: 750 - 2000 mg/kg bw
- Clinical signs of toxicity in test animals:
- 2000 mg/kg bw: reduction of spontaneous activity, eyelid closure, apathy in both pre-tests.
- 1000 mg/kg bw: reduction of spontaneous activity (first pre-experiment), in both pre-experiments, 1 animals died 24 h after treatment.
- 750 mg/kg bw: No clinical signs and no mortalities were observed.
- Harvest times: The animals receiving 2000 mg/kg bw (both pre-experiments) were sacrificed 48 h after dosing. The animals receiving 1000 mg/kg bw (both pre-experiments) and 750 mg/kg bw were sacrificed 24 h after dosing. Only in the 750 mg/kg bw group a sufficient number of scorable metaphases was available.


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels: At no preparation interval and with no dose of the test article the chromosome aberration frequency was significantly increased as compared to the negative control value.
- Cytotoxicity: Determination of the mitotic indices demonstrated that the test item induced cytotoxic effects (preparation interval 24 hours: 250 and 750 mg/kg bw) as compared to the negative control.
- Solvent and positive control induced the expected results.

Summarized results can be found in Attachment 1 in the attached background material.
Conclusions:
The study was conducted according to the OECD guideline 483 and under GLP conditions. Under the experimental conditions reported, the test article did not induce chromosome aberrations in spermatogonia of the mouse.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Jul - 9 Nov 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted in 2014
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 84-2
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory, Kingston, NY, USA
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: 27.1 - 35.9 g (males), 23.8 - 30.7 g (females)
- Assigned to test groups randomly: yes
- Housing: up to 5 per cage in plastic autoclavable cages with wire lids
- Diet:certified laboratory rodent chow, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26.67
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 Jul 1987 1987 To: 15 Oct 1987
Route of administration:
intraperitoneal
Vehicle:
CMC (carboxymethyl cellulose), 1%
- Justification for choice of solvent/vehicle: toxicity profile in animals
- Concentration of test material in vehicle: 3.8, 18.9 and 37.7 for the low, mid and high dose, respectively
- Volume administered: 10 mL/kg bw
- Lot/batch no.: 7-2-87
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing solutions were prepared freshly and based on the most recently measured body weight (directly before injection).
Duration of treatment / exposure:
single dose
Frequency of treatment:
single dose
Post exposure period:
24, 48 and 72 h
Dose / conc.:
38 mg/kg bw/day
Dose / conc.:
189 mg/kg bw/day
Dose / conc.:
377 mg/kg bw/day
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine
- Route of administration: IP injection
- Doses / concentrations: 0.25 mg/kg bw
- Vehicle: water
Tissues and cell types examined:
Bone marrow from femur was prepared.
Details of tissue and slide preparation:
Toxicits test: A toxicity test was conducted with 6 groups of five male and five female rats each. Animals were observed after dose administration and daily thereafter for 7 days for clinical signs of chemical effect. Body weights were recorded prior to dose administration and 1 and 3 days after dose administration

TREATMENT AND SAMPLING TIMES: Animals were observed after dosing for clinical signs. At the scheduled time of sacrifice, 5 mice/sex were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just about the knee and the bone marrow was aspired into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL FBS. The bone marrow cells were pelleted by centrifugation.

DETAILS OF SLIDE PREPARATION: After centrifugation, the pellet was were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each animal. The slides were fixed in methanol stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS: Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 1000 polychromatic erythrocytes were scored for the presence of micronuclei. The proportion of polychromatic erythrocytes to total erythrocytes and the number of micronucleated normocytes in the field of 1000 polychromatic erythrocytes were also enumerated.
Evaluation criteria:
The test article is considered to induce a positive response if a treatment—related increase in micronucleated polychromatic erythrocytes is observed relative to the vehicle control (P<0.05, Kastenbaum—Bowman Tables) . The positive response must be dose—dependent or must be observed at a single dose level at adjacent sacrifice times. If a single treatment group is significantly elevated at one sacrifice time, the assay is considered a suspect or unconfirmed positive and a repeat assay will be recommended.

The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes in the negative (vehicle) control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be statistically significantly increased relative to the negative control (P<0.05, Kastenbaum—Bowman Tables) .
Statistics:
The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was presented for each animal and treatment group.

Statistical significance was determined using the Kastenbaum—Bowman tables which were based on the binomial distribution.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF TOXICITY STUDY
- Dose range: 200 - 1000 mg/kg bw
- Results: Deaths were observed in all test article—treated groups, with 1, 6, 9, 7 and 9 deaths occurring at dose groups of 200, 400, 600, 800 and 1000 mg/kg bw, respectively. Clinical observations for the above groups included lethargy, paralysis, and piloerection. The LD50 was calculated by probit analysis to be approximately 471 mg/kg bw. 377 mg/kg bw was estimated to be 80% of the LD50 and was selected as the high dose for the micronucleus test.

RESULTS OF DEFINITIVE STUDY
At the highest dose level, mortality (15/38 animals), clinical signs and bone marrow toxicity (reduced ratio PCE/Total E) were observed. These effects were also apparent in some animals in the mid-dose group. No increase in the number of micronucleated polychromatic erythrocytes was detected at any of the sampling times. Experimental conditions as well as the criteria for the determination of test responses are well defined and appropriate. Positive controls gave the expected response.

Summarized results can be found in Attachment 1 in the attached background material.
Conclusions:
The study was conducted similar to OECD guideline 474 and under GLP conditions.

Under the conditions of the test, the test substance did not increase the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female CD-1 mice.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Please refer to the respective endpoint summary. 

Additional information

Genotoxicity in vitro

An Ames test, a HPRT and a Chromosomal aberration assay were conducted with the test substance in vitro. All tests were performed according to or similar to OECD guidelines and are therefore considered valid and can serve as key studies. Additionally, an in vitro UDS assay is available as a supporting study.

An Ames test (Rep. No. 175116) was conducted with Salmonella thypimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 with and without metabolic activation. The concentrations tested were 1 - 100 µg/mL without metabolic activation and 1 - 1000 µg/mL with metabolic activation. The test substance produced a dose-dependent and significant increase in revertant colony numbers observed in strains TA1535 and TA100 with and without metabolic activation, in the two experiments performed.

With and without S9 mix, toxic effects occurred in strains TA98 at 333.3 - 666.6 and 1000µg/plate (Experiment I) and in the strains TA1537 and TA1538 at 1000 µg/plate (Experiment II). With metabolic activation toxic effects occurred in strains TA1537 (Experiment I) and TA100 (Experiment I and II) at 1000 µg/plate. Strains TA1535, TA1537, TA1538 and TA100 showed normal background growth up to 1000 µg/plate with S9 mix and up to 100 µg/plate in the absence of metabolic activation.

 

A HPRT test was conducted with V79 cells (0174-EV1) with and without metabolic activation. The concentrations tested were 1 – 10 µg/mL without metabolic activation and 10 – 56 µg/mL with metabolic activation. Positive controls were ethyl methane sulfonate (-S9) and dimethyl nitrosamine (+S9) and gave the expected response. The experiment was repeated once.

A concentration of 10 µg/mL, without metabolic activation, reduced the cloning efficiency by approximatively 50%. In the presence of S9 mix, cytotoxicity occurred only from 30 µg/ mL. In both experiments, none of the tested doses induced a significant increase in mutant frequency at the HPRT-locus. There was only a doubling of the incidence, and in addition, the effect was not replicated in one independent experiment). The addition of S9 mix did not influence these results.

Under the conditions of the study, no statistically significant increase in mutant frequency was observed for any dose level in the absence or presence of metabolic activation. No significant dose-response was observed either. Therefore, the test substance is concluded not to be mutagenic in CHO cells in vitro.

In a chromosome aberration test in vitro (Rep. No. T5558.337), CHO cells were incubated with the test substance in the absence and presence of metabolic activation. The concentrations tested were 0.003 – 0.05 µg/mL in the absence of S9 mix and 0.2 – 3 µg/mL in the presence of S9 mix.

At the time of harvest, the high dose of 0.05µg/mL (in the absence of S9 mix) was observed to be slightly toxic upon microscopic examination of the cell monolayer. This dose was not evaluated for chromosome aberrations due to insufficient number of metaphase cells. In the presence of S9 mix, at the time of harvest, dose levels of 0.8, 1.5 and 3 µg/mL were observed to be slightly to moderately toxic upon microscopic examination of the cell monolayer. The highest dose was not evaluated for chromosome aberrations due to insufficient number of metaphase cells. The frequency of cells with aberrations or the mean aberrations per cell in the test group was not statistically increased above that of the study control.

Under the conditions of the assay, the test item was not clastogenic with and without enzymatic activation. Cytotoxicity was observed in the presence and absence of the S9 mix but was more pronounced without metabolic activation.

Further, an in vitro UDS assay (Rep. No. 0174-ER156) was performed to assess DNA damage in freshly isolated primary rat hepatocytes. Cytotoxicity was evaluated with test substance concentrations ranging from 0.05 to 250 μg/mL in trypan blue exclusion after exposure to the test substance. The concentration which produced a 50% decrease in viability comparing with control (EC50) was determined. The highest dose level used in the main assay was the EC50.

In the main assay approximately 10^5 cells/well were exposed during 18 h in concentrations at 0, 0.03, 0.1, 0.3, 1.0, 3.0 and 10 μg/mL in two independent experiments. In both experiments the EC50 value for cytotoxicity appeared to be 1 μg/mL. Therefore, the compound was tested to a cytotoxic level. In one replicate of cells treated with 10 μg/mL, a rather large amount of grains was found over nuclei from cells devoid of cytoplasm. This finding, in the highest concentration tested, is probably irrelevant to the conclusion since it occurred above the EC50 cytotoxicity value. The positive control, DMBA, produced a significant increase in the number of grains per nucleus

Under the conditions of this study, it is concluded that there are no evidences of DNA-damaging activity in primary rat hepatocytes. 

Genotoxicity in vivo

To assess the genotoxicity of the test substance in vivo, three assays were conducted, according to or similar to OECD guidelines and under GLP conditions. All three studies are valid and can serve as key studies.

In the mouse spot assay (Rep. No. CCR200902), NMRI females (nonagouti a/a; albino c/c) were mated with DBA/2 males (nonagouti a/a; brown b/b; dilute d/d) on Day 0. Pregnant female mice were treated with the test substance (75 – 750 mg/kg bw) once on Day 9 of pregnancy and the offspring was observed for the occurrence of pigmented spots. No difference was found between the offspring of the treatment groups when compared to the control group. The mean litter size was not reduced after treatment with the test article indicating no embryotoxic effects. Experimental conditions as well as criteria for the determination of test responses were well defined and appropriate. Controls gave the expected response. In the vehicle test group one recessive spot (RS) was observed. After treatment with 75 or 750 mg/kg bw, the frequency of RS was 0.75 and 0.62%, respectively, which are in the range of the historical control data (0.00% - 1.00%). The positive control showed a distinct increase in induced RS frequency, thereby proving the sensibility and suitability of the test system. Under the experimental conditions of the test, the test article did not induce mutations the mouse.

In a chromosomal aberration study in mouse sperm cells (Rep. No. CCR175127) male mice were administered the test substance in carboxymethyl cellulose (1%) in doses of 75, 250 and 750 mg/kg bw/day. Animals receiving the vehicle only served as a solvent control. The doses were based on a preliminary dose range finding study that showed 750 mg/kg bw to be the maximum tolerated dose (MTD). Groups of 6 animals were sacrificed after 6 h (high dose), 24 h (solvent control, positive control, all treatment groups) or 48 h (high dose) and their sperm cells were analysed for chromosomal aberration. Determination of the mitotic indices demonstrated that the test item induced cytotoxic effects (preparation interval 24 hours: 250 and 750 mg/kg bw) as compared to the negative control. At no preparation interval and with no dose of the test article the chromosome aberration frequency was significantly increased as compared to the negative control value. The positive control induced the expected result and demonstrated the suitability and sensitivity of the test item. Under the experimental conditions reported, the test article did not induce chromosome aberrations in spermatogonia of the mouse.

A micronucleus assay (Rep. No. T5558.122) was conducted similar to OECD guideline 474 and under GLP conditions. Groups of 15 mice per sex and dose received the test substance in 1% carboxymethylcellulose in doses of 38, 189 or 377 mg/kg bw/day. Animals receiving the vehicle only served as a solvent control. 5 animals per sex and dose each were sacrificed 24, 48 and 72 h after administration of the test substance and their bone marrow cells were examined for the occurrence of micronuclei. At the highest dose level, mortality (15/38 animals), clinical signs and bone marrow toxicity (reduced ratio PCE/Total E) were observed. These effects were also apparent in some animals in the mid-dose group. No increase in the number of micronucleated polychromatic erythrocytes was detected at any of the sampling times. The positive control (triethylenemelamine) induced the expected result and demonstrated the suitability and sensitivity of the test item. Under the conditions of the test, the test substance did not increase the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female CD-1 mice.

 

Conclusion:

The test substance was mutagenic in bacteria, but negative in mammalian test systems, including in-vivo assays. No indication for mutagenic properties was obtained in in vitro and in vivo indicator tests for gene mutation, including the in vitro UDS and in vivo mouse spot test. The overall available data indicate that the positive response observed with the test substance in the bacterial mutagenicity test is not relevant in vivo. Several reliable in vitro mammalian (CA, HPRT and UDS) and in vivo studies (MNT, CA, Mouse spot test assays) are available and neither mutagenicity nor genotoxicity could be evidenced with respect to the present test substance. Thus, the test item does not pose a significant genotoxic risk to humans.

Justification for classification or non-classification

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

This is in line with the harmonised classification of the test substance (Annex VI of Regulation (EC) 1272/2008, index number 006-005-00-4).