Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro

In an in vitro reverse gene mutation assay in bacteria (AMES test) according to OECD guideline 471 (Toxi-Coop, 2018), the test substance showed no genotoxic potential.

In an in vitro DNA repair study (UDS assay) similar to OECD guideline 482 (Hazleton Laboratories, 1988), the test substance showed no genotoxic potential.

In an in vitro chromosome aberration assay (CA) similar to OECD guideline 473 (Hazleton Laboratories, 1988), the test substance showed clastogenic properties.

In vivo

In an in vivo mouse micronucleus test similar to EPA OPP 84 -2, the test substance showed no clastogenic potential.

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:
1988-06-27 to 1988-09-15
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
other: Chromosomal Aberration Assay
Specific details on test material used for the study:
OTHER SPECIFICS: black liquid
RT#975297, FCL#1409 6/3/88
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: laboratory of Dr. S. Wolff, University of California, San Francisco, USA
- Cell cycle length: 12 - 14 h
- Modal number of chromosomes: 21


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: McCoy's 5a culture medium which was supplemented with 10 % fetal calf serum (FCS), 1 % L-glutamine, and 1 % penicillin and streptomycin, at ~ 37 °C, in an atmosphere of ~5 % CO2 in air.
Cytokinesis block (if used):
Colcemid
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, derived from the liver of male Sprague-Dawley rats which had been previously treated with Aroclor 1254.
Test concentrations with justification for top dose:
- S9 mix: 49.9, 99.2, 149.0, and 198 µg/mL
+ S9 mix: 384.0, 765.0, 1150.0, and 1540.0 µg/mL
Based on the results of the range finding assay
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: homogenous distribution of the test substance.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding:
20 h test: 1.2E6 / 75 cm² flask
10 h test: 1.5E6 / 75 cm² flask

DURATION
- Exposure duration:
17.25 h without S9 mix
2 h with S9 mix
- Expression time: 7.75 h with S9 mix

SPINDLE INHIBITOR: 2.5 h colcemid (0.1 µg/mL)

STAIN: Giemsa staining (5 %)

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: air dried slides, the slides were stained in pH 6.8 buffered 5% Giemsa solution

NUMBER OF CELLS EVALUATED: 100 from each duplicate

OTHER EXAMINATIONS:
Cells with aberrations, but not gaps, were recorded on the data sheets by the microscope stage location. Chromatid and isochromatid gaps, if observed, were noted in the raw data and were tabulated. They were not, however, considered in the evaluation of the ability of the test article to induce chromosomal aberrations since they may not represent true chromosomal breaks.

Evaluation criteria:
The following factors were taken into account in the evaluation of the chromosomal aberrations data:
1. The overall chromosomal aberration frequencies.
2. The percentage of cells with any aberrations.
3. The percentage of cells with more than one aberration.
4. Any evidence for increasing amounts of damage with increasing dose, i.e., a positive dose response.
5. The estimated number of breaks involved in the production of the different types of aberrations which were observed, i.e. complex aberrations may have more significance than simple breaks.
Statistics:
Statistical analysis employed the Fisher's Exact Test with an adjustment for multiple comparisons (Sokal and Rohlf, 1981) to compare the percentage of cells with aberrations in each treatment group with the results from the pooled solvent and negative controls (the solvent and negative controls were statistically evaluated for similarity prior to the pooled evaluation using Fisher's Exact Test). Test article significance was established where p < 0.01.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Solubility of the test article was evaluated in McCoy's 5a culture medium and dimethyl sulfoxide (DMSO). Uneven dark emulsions were obtained in McCoy's 5a culture medium at concentrations of 500, 300, 100, and 50 mg/mL. Dark even suspensions were obtained in DMSO at 500, 300, 200, and 100 mg/mL.
- Precipitation: Subsequent 1:100 dilutions (see above) were made from these stocks in McCoy's 5a culture medium to evaluate precipitation. Small black globules precipitated and adhered to the dilution tube at final concentrations of 5000 and 3000 µg/mL. A slight clouding was observed immediately upon dosing at 2000 µg/mL, which disappeared as the compound went into solution with no precipitate visible. DMSO was the solvent of choice at a stock concentration of 200 mg/mL. A slight clouding was observed immediately upon dosing at 2000 µg/mL, which disappeared as the compound went into solution with no precipitate visible. DMSO was the solvent of choice at a stock concentration of 200 mg/mL.


RANGE-FINDING/SCREENING STUDIES:
Rangefinding Assay Without Metabolic Activation
A precipitate was visible at the time of dosing at 2000 µg/mL. An unhealthy cell monolayer was observed at this concentration with floating dead cells and an accurate assessment could not be made on the presence of mitotic cells. There was no visible precipitate at the subsequent concentrations of 667 and 200 µg/mL and complete cellular toxicity (total absence of metaphase cells) was observed at these dose levels with no cell monolayer remaining. An unhealthy cell monolayer with floating dead cells was observed at 66.7 µg/mL. Results were evaluated at 0.667, 2.00, 6.67, 20.0, and 66.7 µg/mL. Severe cell cycle delay was observed at 6.67, 20.0, and 66.7 µg/mL. This delay in cell cycle persisted at 2.00 µg/mL. An extended 20 hour aberrations assay was selected for testing a dose range of 4.00, 6.00, 8.00, 20.0, 40.0, 60,0, and 80.0 µg/mL.
Rangefinding Assay With Metabolic Activation
A precipitate was visible at the time of dosing at 667 and 2000 µg/mL and complete toxicity (total absence of metaphase cells) was observed at these concentrations. There was no discernible toxicity at the subsequent concentration of 200 µg/mL. Results were analyzed at 20.0, 66.7, and 200 µg/mL. There was no significant cell cycle delay at these test concentrations. A regular 10 hour harvest was selected for the aberrations assay testing a dose range of 34.9, 52.4, 69.9, 175, 349, 524, and 699 µg/mL.

CYTOKINESIS BLOCK: Colcemid

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Prior to the harvest of the cultures visual observations of toxicity were made. These observations included an assessment of the percent confluence of the cell monolayer within the culture flasks. The cultures were also evaluated for the presence of mitotic (large rounded cells) or dead cells floating in the medium.

Metabolic Activation: - S9 Mix

Number and Type of Aberration

 

 

 

 

 

 

Not computed

simple

Complex

 

 

 

 

Conc. (µg/mL)

Cells Scored

TG

SG

UC

TB

SB

IM

ID

TR

QR

CR

D

R

CI

Other GT

No. of Aberrations/Cell

% Cells with Aberrations

% Cells with >1 Aberrations

Controls, Negative and Solvent

 

200

3

1

 

1

1

 

 

1

 

 

2

 

 

 

0.03

2.5

0.0

Positive: MMC

0.08

25

2

1

 

4

2

 

1

2

3

 

 

 

2

 

0.60

36.0*

120.*

Test Substance

99.2

200

6

1

 

5

4

 

 

 

 

 

 

1

 

 

0.05

3.0

0.5

Test Substance

149

200

18

1

 

15

6

 

 

 

 

 

3

1

 

 

0.13

6.5

3.0

Test Substance

198

200

17

1

 

14

4

 

 

 

 

 

5

 

 

1

>0.16

8.5* 

2.5

* Significantly greater than the pooled negative and solvent controls, p < 0.01

Metabolic Activation: - S9 Mix

 

Number and Type of Aberration

 

 

 

 

 

 

Not computed

simple

 

Complex

 

 

 

 

Conc. (µg/mL)

Cells Scored

TG

SG

UC

TB

SB

IM

ID

TR

QR

CR

D

R

CI

DF

Other GT

No. of Aberrations/Cell

% Cells with Aberrations

% Cells with >1 Aberrations

Controls, Negative and Solvent

 

200

7

5

 

 

1

 

 

 

 

 

 

 

 

 

 

0.01

0.5

0.0

Positive:  CP

12.5

25

1

 

 

4

3

 

1

2

1

 

 

 

3

 

 

0.56

40.0*

16.0*

Test substance

384

200

8

4

 

 

 

 

 

 

 

 

1

 

 

 

 

0.01

0.5

0.0

Test Substance

769

200

15

1

 

2

1

 

 

1

 

1

 

 

 

1

 

0.03

2.5

0.5

Test Substance

1150

200

20

5

 

6

6

 

2

7

3

 

1

 

1

 

 

0.13

11.0*

2.0

Test Substance

1540

100

10

4

 

10

43

 

1

1

3

 

 

 

 

 

 

0.58

41.0*

13.0*

* Significantly greater than the pooled negative and solvent controls, p < 0.01

 

DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED CELLS:

 

NOT COMPUTED

TG Chromatid Gap: ("tidgap"). An achromatic (unstained) region in one chromatid, the size of which is equal to or smaller than the width of a chromatid. These are noted but not usually included in final totals of aberrations as they may not all be true breaks.

SG Chromosome Gap: ("isochromatidgap, IG"). Same as chromatid gap but at the same locus in both sister chromatids.

UC Uncoiled Chromosome Failure of chromatin packing. Probably not a true aberration.

PP Polyploidcell: A cell containing multiple copies of the haploid number (n) of chromosomes. Only indexed if very common.Not counted in the cells scored for aberrations.

E Endoreduplication: 4n cell in which separation of chromosome pairs has failed. Only indexed if very common. Not counted in the cells scored for aberrations.

 

SIMPLE

TB Chromatid Break: An achromatic region in one chromatid, larger than the width of a chromatid. The associated fragment may be partially or completely displaced.

SB Chromosome Break: Chromosome has a clear break, forming anabnormal (deleted) chromosome with an acentric fragment that is dislocated. This classification now includes the acentric fragment (AF). The AF was different from the SB only in that it was not apparently related to any specific chromosome.

Conclusions:
In this in vitro chromosome aberration assay (CA) similar to OECD guideline 473, the test substance showed genotoxic properties.
Executive summary:

In this study, cultured CHO cells were exposed to the test substance at concentrations of 49.6, 99.2, 149, or 198 µg/mL for 17.25 h in absence of the S9 metabolic activation. In the presence of the S9 activation, the CHO cells were exposed to the test substance at concentrations of 115, 384, 769, 1150, or 1540 µg/mL for 2 h. After exposure to the test substance, the treated cells were washed with buffered saline, and complete McCoy’s a medium containing 0.1 µg/mL colcemid was added to the washed cells. The cells were then incubated for 2.5 h (without S9) or 7.5 h (with S9). The metaphase cells were then harvested, and slides prepared for analysis. The results showed that without S9 activation, the test substance at concentrations of 149 and 198 µg/mL induced chromosomal aberrations, consisting mainly of simple chromatid breaks. In the presence of S9 activation, 1150 and 1540 µg/mL of the test substance caused a statistically significant and dose-related increase in chromosomal aberrations, consisting of simple chromatid and chromosome breaks.

The test substance was tested up to cytotoxic concentrations, based on the results of a rangefinding study. The positive controls (without S9 mix: 0.08 µg/mL mitomycin C, with S9 mix: 12.5 µg/mL cyclophosphamide) did induce the appropriate response. There was a concentration related positive response of induced chromosomal aberrations compared to untreated and solvent control.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1988-06-08 to 1988-07-29
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Version / remarks:
1986-10-23
GLP compliance:
yes
Type of assay:
other: unscheduled DNA synthesis
Specific details on test material used for the study:
OTHER SPECIFICS: thick black-brown liquid
RT #975297, FCL #1409
Species / strain / cell type:
hepatocytes: adult male Fischer 344 rat
Metabolic activation:
without
Test concentrations with justification for top dose:
0.025, 0.050, 0.101, 0.252, 0.504, 1.01, 2.52, and 5.04 µg/mL
Higher doses than the top dose were lethal to the primary hepatocytes.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was incompletely soluble in the cell culture medium.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 2 h
- Exposure duration: 18 - 20 h
- Expression time (cells in growth medium): 2 - 4 h
- Fixation time (start of exposure up to fixation or harvest of cells): 20 - 24 h

STAIN: ³H-thymidine (5µCi/mL)

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: the nuclei in the labeled cells were swollen by addition of 1 % sodium citrate to the coverslips for 8 - 10 min, and then the cells were fixed in acetic acid:ethanol (1:3) and dried for atleast 24 h. The coveslips were mounted on glass slides (cells up), dipped in an emulsion of Kodak NTB2, and dried.The coated slides were stored for 7 - 10 days at +4 °C in light-tight boxes containing packets of Drierite. The emulsions were then developed in D19, fixed, and stained with Williams´modified hematoxylin and eosin procedure.

NUMBER OF CELLS EVALUATED: 50/coverslip; 150/treatment group

DETERMINATION OF CYTOTOXICITY
- Method: trypan blue exclusion
Key result
Species / strain:
hepatocytes: adult male Fischer 344
Remarks:
Charles River Breeding Laboratories, Inc.
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test material was incompletely soluble in WMEI (Cel Culture Medium), therefore dimethylsulfoxide (DMSO) was investigated and chosen as the solvent. A solution in DMSO was serially diluted with DMSO and each stock was then diluted 1:100 (or greater) into WMEI to obtain the final desired concentrations of test material.
- Definition of acceptable cells for analysis: The hepatocytes for the UDS assay were collected at a calculated viability of 87.5 % (determined by trypan blue exclusion), and 75.0 % of the viable cells attached to the culture dishes during the 2-hour settling period. The treatments were initiated about 1.5 hours later with cell monolayers that were 88.2 % viable. After an additional 20.5 hours in culture (which encompassed the nearly 18.5-hour treatment period), the average viable cell count in the control cultures was 85.5 % of the viable count at the beginning of the treatments. This stability in cell number and the normal morphological appearance of the cells indicated that the hepatocyte cultures were in good metabolic condition for the UDS assay.

RANGE-FINDING/SCREENING STUDIES: The dose selection procedure was an integral part of the UDS assay in order to select appropriate doses for a particular, fresh primary culture of hepatocytes. A range of fifteen concentrations was applied initially to the cells. A viable cell count (trypan blue exclusion) was then obtained about 20.5 hours after initiation of the treatments. If possible at least six concentrations were chosen for analysis of nuclear labeling, starting with the highest dose that resulted in a sufficient number of survivors with intact morphologies and proceeding to successively lower doses. If the test material was freely soluble and relatively nontoxic, a maximum concentration of 5 mg/ml (or 5 microliters/ml) or another limit specified by the sponsor was tested.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: trypan blue exclusion

Summary of Data from Rat Hepatocyte UDS Assay

Test condition

Conc.

UDS*grains/nucleus

Avg.# % of nuclei with ≥ 6 grains

Mean***
Cytoplasmic Grain Count

Survival ## at 20.5 h

Solvent Control (DMSO)

1        %

-0.66**

2.0**

0.0**

100.0

Positive Control
(2-AAF)

0.1 µg/mL

13.45

91.3

33.0

98.6

Test substance

5.04 µg/mL

0.03

4.7

0.0

95.3

Test substance

2.52 µg/mL

-0.12

5.3

0.0

95.3

Test substance

1.01 µg/mL

-0.75**

4.7**

0.0**

95.5

Test substance

0.504 µg/mL

-1.08

0.7

0.0

96.4

Test substance

0.0252 µg/mL

0.3**

0.7**

0.0**

97.0

Test substance

0.101 µg/mL

-0.27

0.0

0.0

ND

Test substance

0.050 µg/mL

-0.37

0.7

0.0

ND

Test substance

0.025 µg/mL

-1-11

4.0

0.0

ND

*             UDS = average of net nuclear grain counts on triplicate coverslips (150 total cells).

**          One slide unreadable. UDS = Average of net nuclear grain counts on triplicate slides.

***        Average of three areas per cell, 50 cells per coverslip for total of 150 cells

#             Average value for triplicate coverslips

##          Survival = Number of viable cells per unit area relative to the solvent control

2-AAF   2-Acetylaminofluorene

ND         Not determined

 

 

Conclusions:
In this in vitro DNA repair study (UDS assay) similar to OECD guideline 482, the test substance showed no genotoxic potential.
Executive summary:

In this study, primary rat hepatocyte cultures isolated from adult male Fischer 344 rats were exposed to the test substance in DMSO at concentrations of 0, 0.025, 0.050, 0.101, 0.252, 0.504, 1.01, 2.52, and 5.04 µg/mL for 18 – 20 h. The test substance was tested up to max. non-cytotoxic concentration, determined by cell-viability via trypan blue staining. The positive control (0.1 µg/mL 2-acetylaminofluorene) induced the appropriate response.

There was no evidence that unscheduled DNA synthesis, as determined by radioactive tracer procedure (³H-thymidine (5 µCi/mL) was induced.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-23 to 2018-02-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
his/trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver.
Test concentrations with justification for top dose:
160, 100, 50, 16, 5, 1.6 and 0.5 μg/plate.
Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test (informatory toxicity test).
Vehicle / solvent:
- Solvent used: Based on the results of preliminary tests, the test item was dissolved in dimethyl sulfoxide (DMSO).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylenediamine, NPD
Remarks:
TA98, without S9 mix, 4 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100, TA1535, without S9 mix, 2 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537, without S9 mix, 50 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
E. coli, without S9 mix, 2 µL/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
all S. typhimurium strains tested, with S9 mix, 2 µg/plate; E.coli, with S9 mix, 50 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS:
3

DETERMINATION OF CYTOTOXICITY
- Method: growth of background lawn and number of revertant colonies

Rationale for test conditions:
According to OECD guideline 471.
Evaluation criteria:
The colony numbers on the controls (untreated, vehicle, positive) and the test item plates were determined (counted manually, evaluated by unaided eye), the mean values and appropriate standard deviations and mutation rates were calculated.

Mutation rate = Mean revertants at the test item (or control*) treatments / mean revertants of vehicle control


* : untreated, vehicle or positive control

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results. A statistical evaluation of the results was not regarded as necessary.
Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
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
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
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
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Not observed

Validity of the test

Valid tests were performed since the tester strains (used in this study) demonstrated the specific phenotype characteristics, agreed with the corresponding historical control data ranges, and showed the adequate strain culture titer. Each batch of the S9 fraction used in this test had the appropriate biological activity (according to the provided Certificates) and was active in the applied system (2AA treatments).

Each of the investigated reference mutagens showed the expected, biological relevant increase (at least a 3.0-fold increase respectively a 2.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control in all experimental phases. The spontaneous revertant colony numbers of dimethyl sulfoxide (DMSO) vehicle control plates showed the characteristic mean numbers agreed with the actual historical control data ranges in the main experiments.

Table 1: Summary Table of the Results of the Intial Mutation Test

Concentration (µg/plate)

Salmonella typhimuriumtester strains

 

E.coli WP2 uvrA

TA98

TA100

TA1535

TA1537

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

19.3

1.09

21.3

0.89

82.0

1.21

91.3

1.13

9.3

1.12

9.0

1.04

4.0

1.09

5.3

1.14

33.0

1.27

42.7

1.29

DMSO Control

17.7

1.00

24.0

1.00

68.0

1.00

80.7

1.00

8.3

1.00

8.7

1.00

3.7

1.00

4.7

1.00

26.0

1.00

33.0

1.00

Ultrapure Water Control

79.7

1.00

9.7

1.00

26.0

1.00

160

3.3

0.19

4.3

0.18

36.7

0.54

31.0

0.38

3.0

0.36

0.7

0.08

1.0

0.27

1.3

0.29

10.0

0.38

9.3

0.28

100

12.0

0.68

14.0

0.58

55.7

0.82

72.3

0.90

5.3

0.64

7.0

0.81

3.0

0.82

3.3

0.71

18.3

0.71

20.3

0.62

50

13.3

0.75

14.3

0.60

59.7

0.88

78.3

0.97

9.3

1.12

11.0

1.27

4.7

1.27

4.3

0.93

22.3

0.86

30.0

0.91

16

15.0

0.85

18.7

0.78

65.3

0.96

77.3

0.96

10.7

1.28

9.7

1.12

2.3

0.64

4.3

0.93

35.7

1.37

38.3

1.16

5

14.3

0.81

24.0

1.00

71.0

1.04

74.7

0.93

7.7

0.92

10.7

1.23

4.7

1.27

4.7

1.00

27.7

1.06

33.3

1.01

1.6

14.0

0.79

15.0

0.63

73.3

1.08

82.7

1.02

9.0

1.08

8.3

0.96

3.7

1.00

4.0

0.86

27.7

1.06

40.0

1.21

0.5

16.0

0.91

20.0

0.83

74.3

1.09

90.3

1.12

10.0

1.20

9.3

1.08

4.3

1.18

5.7

1.21

26.3

1.01

37.7

1.14

NPD

(4 μg/plate)

481.3

27.25

SAZ

(2 μg/plate)

796.7

10.00

1093.3

113.10

9AA

(50 μg/plate)

581.3

158.55

MMS

(2 μL/plate)

562.7

21.64

2AA

(2 μg/plate)

1290.7

53.78

1629.3

20.20

194.3

22.42

123.0

26.36

2AA

(50 μg/plate)

215.3

6.53

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine;SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Table 2: Summary Table of the Results of the Confirmatory Mutation Test

Concentration (µg/plate)

Salmonella typhimuriumtester strains

 

E.coli WP2 uvrA

TA98

TA100

TA1535

TA1537

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

23.3

1.35

28.7

1.19

87.7

0.97

108.7

1.20

11.3

1.10

12.0

1.24

8.7

1.18

5.7

0.89

32.7

1.18

35.3

0.88

DMSO Control

17.3

1.00

24.0

1.00

90.7

1.00

90.3

1.00

10.3

1.00

9.7

1.00

7.3

1.00

6.3

1.00

27.7

1.00

40.3

1.00

Ultrapure Water Control

80.0

1.00

12.0

1.00

35.7

1.00

160

6.7

0.38

7.3

0.31

39.7

0.44

40.3

0.45

2.3

0.23

3.0

0.31

5.3

0.73

4.7

0.74

8.7

0.31

8.3

0.21

100

17.3

1.00

16.7

0.69

56.3

0.62

70.0

0.77

10.7

1.03

6.0

0.62

6.3

0.86

7.7

1.21

22.3

0.81

27.7

0.69

50

20.0

1.15

30.0

1.25

81.0

0.89

78.3

0.87

11.0

1.06

9.3

0.97

7.0

0.95

8.3

1.32

32.3

1.17

32.3

0.80

16

22.0

1.27

24.3

1.01

78.7

0.87

91.3

1.01

10.0

0.97

13.3

1.38

7.0

0.95

7.0

1.11

37.7

1.36

50.0

1.24

5

23.3

1.35

32.0

1.33

80.7

0.89

102.7

1.14

9.7

0.94

9.0

0.93

8.7

1.18

4.7

0.74

30.7

1.11

44.0

1.09

1.6

24.3

1.40

31.3

1.31

82.3

0.91

96.0

1.06

9.3

0.90

12.7

1.31

7.0

0.95

5.3

0.84

29.0

1.05

44.3

1.10

0.5

18.0

1.04

33.0

1.38

82.3

0.91

97.7

1.08

9.7

0.94

9.7

1.00

6.3

0.86

6.0

0.95

28.7

1.04

48.7

1.21

NPD (4 μg/plate)

566.7

32.69

SAZ (2 μg/plate)

976.0

12.20

682.3

56.86

9AA

(50 μg/plate)

472.7

64.45

MMS

(2 μL/plate)

1040.0

29.16

2AA (2 μg/plate)

1442.7

60.11

1088.0

12.04

133.0

13.76

109.3

17.26

2AA

(50 μg/plate)

251.3

6.23

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Conclusions:
In an in vitro reverse gene mutation assay in bacteria according to OECD guideline 471, the test item did not show a genetoxic potential.
Executive summary:

In a bacterial in vitro reverse mutation assay (Ames) according to OECD guideline 471, five bacterial strains were used to investigate the mutagenic potential of the test item in two independent experiments, in a plate incorporation test (experiment I, initial mutation test) and in a pre-incubation test (experiment II, confirmatory mutation test). Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. In the performed experiments all of the validity criteria regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix). Sporadic increases in revertant colony numbers compared to the vehicle control values were observed in both independently performed main experiments. They were within the actual historical control data ranges. Furthermore, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

The concentrations examined in the main experiments were chosen in regard to the OECD 471 guideline recommendations, taking into account the solubility and unequivocal, strong cytotoxic effects of the test item. In the performed experiments (I and II) inhibitory effect of the test item, indicated by affected background lawn development: reduced or slightly reduced background lawn and/or decreased revertant colony counts (mostly below the corresponding historical control data ranges) was observed in all examined strains at the concentration of 160 μg/plate in the absence and presence of exogenous metabolic activation (±S9 Mix). Furthermore, 100 μg/plate was found to be the lowest cytotoxic concentration, observed in initial and confirmatory mutation tests in the case of Salmonella typhimurium TA100 strain, in the absence of exogenous metabolic activation (-S9 Mix). No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

The reported data of this mutagenicity assay show, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In an in vivo mouse micronucleus test similar to EPA OPP 84 -2 (Hazleton Laboratories, 1989), the test substance showed no genotoxic potential.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-06-13 to 1989-08-15
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPP 84-2
GLP compliance:
yes
Type of assay:
other: mouse micronucleus assay
Species:
mouse
Strain:
ICR
Details on species / strain selection:
This healthy, random bred strain was selected to maximize genetic heterogeneity and at the same time assure access to a common source.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Frederick, MD., USA
- Age at study initiation: adult, ~8.5 weeks
- Weight at study initiation: ♂: 29.0 - 38.5 g; ♀: 21.8 - 28.3 g
- Assigned to test groups randomly: yes

- Housing: group-housed up to five per cage in sanitary polycarbonate cages.
- Diet: ad libitum, Purina Certified Laboratory Chow #5002
- Water: ad libitum
- Acclimation period: at least 7 days
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
- Justification for choice of solvent/vehicle: solubility of the test item
- Amount of vehicle: 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing solutions for the assay were prepared by making a 80 mg/mL stock for the high dose (800 mg/kg). Dilutions of this stock were prepared for the 400 and 80 mg/kg dose levels.
Frequency of treatment:
single exposure
Post exposure period:
treatment group: 24, 48, and 72 h
positive and vehicle control group: 24 h
Dose / conc.:
80 mg/kg bw/day
Dose / conc.:
400 mg/kg bw/day
Dose / conc.:
800 mg/kg bw/day
No. of animals per sex per dose:
5
Remark: A second group of animals (designated Secondary Dose Group, 10 animals/sex/dose) was also assigned to the study and was dosed with the high dose of the test article. These animals were only used in the assay as replacements for any which died in the primary dose group.
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral gavage
- Dose: 80 mg/kg bw
Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose levels used in this assay were based upon the results of a previously conducted dose range finding assay.

TREATMENT AND SAMPLING TIMES: The doses selected for this assay were 80, 400, and 800 mg/kg body weight and were administered by a single oral gavage.

DETAILS OF SLIDE PREPARATION: At the appropriate harvest time, the animals were euthanatized with CO2 and the adhering soft tissue and epiphyses of both tibiae were removed. The marrow was flushed from the bone and transferred to centrifuge tubes containing 3 mL fetal calf serum (one tube for each animal). Following centrifugation to pellet the tissue, most of the supernatant was drawn off, the cells were resuspended, and the suspension spread on slides and air-dried. The slides were then fixed in methanol, stained in May-Gruenwald solution followed by Giemsa, and rinsed in deionized water (Schmid, 1975). After being air-dried, the slides were coverslipped using Depex® mounting medium.

METHOD OF ANALYSIS: The coded slides were scored for micronuclei and the polychromatic (PCE) to normochromatic (NCE) cell ratio. Standard forms were used to record these data. Where possible, one thousand PCEs were scored from each animal. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this mouse strain is about 0.0 - 0.42. The frequency of PCEs versus NCEs was determined by scoring the number of NCEs observed in the optic fields while scoring the 1000 PCEs for micronuclei.
Evaluation criteria:
The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ring-shaped micronuclei occasionally occur. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size of the PCE. The unit of scoring was the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus was counted as one micro-nucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color of PCEs and NCEs (bluish-grey and red, respectively).
The criteria for determining a positive response involved a statistically significant dose-related increase in micronucleated PCEs, or the detection of a reproducible and statistically significant positive response for at least one dose level. A test article that induced neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level was considered negative. In either case, the final decision was based on scientific judgment.
Statistics:
Data were summarized to include tables indicating the individual animal results and in tables with animal results summarized by sex and dose groups at the different time points. The analysis of the data was performed using an Analysis of Variance on the square root arcsine transformation which was performed on the proportion of cells with micronuclei per animal (square root arcsine proportion, Sokal and Rohlf, 1981). Once the Analysis of Variance had been performed, Tukey's Studentized range test (HSD) with adjustment for multiple comparisons was used at each harvest time to determine which dose groups, if any, were significantly different from the negative control. Analyses were performed separately for each harvest time and sex combination, and also at each harvest time for the sexes combined.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
mortality, within 24 h after administration, 11 animals died in the 800 mg/kg dose group and 1 animal in the 400 mg/kg dose group.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: No
- Ratio of PCE/NCE: see: Any other information on results incl. tables
- Statistical evaluation: The analysis of the data was performed using an Analysis of Variance on the square root arcsine transformation which was performed on the proportion of cells with micronuclei per animal (square root arcsine proportion, Sokal and Rohlf, 1981). Once the Analysis of Variance had been performed, Tukey's Studentized range test (HSD) with adjustment for multiple comparisons was used at each harvest time to determine which dose groups, if any, were significantly different from the negative control.

 

Treatment

Dose

Harvest Time (h)

% Micronucleated PCEs
Mean of 1000/Animal ± S.E. (1)

Ratio PCE:NCE
Mean ± S.E.

Total

Neg. Control

10.0 mL/kg

24

0.12 ± 0.04

0.06 ± 0.02

0.09 ±0.02

0.36 ± 0.03

0.45 ± 0.04

Pos. Control

80 mg/kg

24

1.54 ± 0.68*

1.38 ± 0.41*

1.46 ± 0.38*

0.55 ± 0.01

0.36 ±0.04

Test Substance

80 mg/kg

24

0 06 ± 0.02

0.08 ± 0.05

0.07±0.03

0.34 ± 0.04

0.54 ± 0.05

48

0.06 ± 0.04

0.02 ± 0.02

0.04 ± 0.02

0.48 ± 0.05

0.56 ± 0.08

72

0.14 ± 0.07

0.04 ± 0.04

0.09 ± 0.04

0.35 ± 0.03

0.49 ± 0.06

400 mg/kg

24

0.02 ± 0.02

0.04 ± 0.02

0.03 ± 0.02

0.50 ± 0.10

0.37 ± 0.06

48

0.06 ± 0.04

0.08 ± 0.06

0.07 ± 0.03

0.46 ± 0.03

0.62 ± 0.09

72

0.00 ± 0.00

0.05 ± 0.04

0.03 ± 0.02

0.38 ± 0.02

0.35 ± 0.03

800 mg/kg

24

0.10 ± 0.03

0.02 ± 0.02

0.06 ± 0.02

0.52 ± 0.15

039 ± 0.05

48

0.01 ± 0.05

0.16 ± 0.05

0.13 ± 0.04

0.55 ± 0.04

0.47 ± 0.06

72

0.02 ± 0.02

0.10 ± 0.04

0.06 ± 0.03

0.43 ± 0.06

0.31 ± 0.03

(1) One male in the 24 h 800 mg/kg group had only 491 PCEs analyzed for micronuclei.

* Significanty greater than the corresponding negative control, p < 0.05.

Conclusions:
In an in vivo mouse micronucleus test similar to EPA OPP 84 -2, the test substance showed no clastogenic potential.
Executive summary:

The objective of this in vivo assay was to evaluate the ability of the test substance to induce micronuclei in bone marrow polychromatic erythrocytes of mice (strain ICR). The study was conducted similar to the EPA OPP 84-2 guideline. The test article was suspended in corn oil and dosed by oral gavage at 80, 400, and 800 mg/kg based upon the results of a previously conducted dose range finding assay. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. The animals were dosed with the test article and were euthanatized 24, 48 and 72 hours after dosing for extraction of the bone marrow. A second group of animals (designated Secondary Dose Group, 10 animals/sex/dose) was also assigned to the study and was dosed with the high dose of the test article. These animals were only used in the assay as replacements for any which died in the primary dose group. Vehicle (corn oil) and positive control (cyclophosphamide, 80 mg/kg bw) groups euthanatized 24 hours after dosing were included in the assay.

All animals were observed immediately after dosing and periodically throughout the duration of the assay for toxic symptoms and/or mortalities. Within ten minutes of dosing nine in the 800 mg/kg dose groups and one male in the 400 mg/kg dose groups had expired. Approximately 1.5 hours after dosing one additional male in the high dose group was found dead. The following morning, approximately 19 hours after dosing, one in the 800 mg/kg dose group was found dead. All other animals appeared normal and remained healthy until the appropriate harvest times. Apparent test article induced bone marrow toxicity was observed in one male in the 24 hour high dose group, since only 491 PCE could be analyzed for micronuclei. The data from this animal were included in the statisitical analysis although no difference in the evaluation of the test article would occur by its exclusion. The criteria for the identification of micronuclei were those of Schmid (1976). No statistically significant reduction in the PCE:NCE ratio relative to the negative control was detected at any dose level. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 1.54 ± 0.68 and 1.38 ± 0.41 for the males and females, respectively.

The test substance did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test.

 

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

Additional information

In vitro

In an bacterial in vitro reverse mutation assay (Ames) according to OECD guideline 471 (Toxi-Coop, 2018), five bacterial strains were used to investigate the mutagenic potential of the test item in two independent experiments, in a plate incorporation test (experiment I, initial mutation test) and in a pre-incubation test (experiment II, confirmatory mutation test). Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. In the performed experiments all of the validity criteria regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analysable concentration levels were fulfilled. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix). Sporadic increases in revertant colony numbers compared to the vehicle control values were observed in both independently performed main experiments. They were within the actual historical control data ranges. Furthermore, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

The concentrations examined in the main experiments were chosen in regard to the OECD 471 guideline recommendations, taking into account the solubility and unequivocal, strong cytotoxic effects of the test item. In the performed experiments (I and II) inhibitory effect of the test item, indicated by affected background lawn development: reduced or slightly reduced background lawn and/or decreased revertant colony counts (mostly below the corresponding historical control data ranges) was observed in all examined strains at the concentration of 160 μg/plate in the absence and presence of exogenous metabolic activation (±S9 Mix). Furthermore, 100 μg/plate was found to be the lowest cytotoxic concentration, observed in initial and confirmatory mutation tests in the case of Salmonella typhimurium TA100 strain, in the absence of exogenous metabolic activation (-S9 Mix). No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

The reported data of this mutagenicity assay show, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

To assess the genotoxic/clastogenic potential of the test substance, in vitro studies in mammalian cells were performed.

In an in vitro gentoxicity study in mammalian cells according to OECD Guideline 482 (Hazleton Laboratories, 1988), primary rat hepatocyte cultures isolated from adult male Fischer 344 rats were exposed to the test substance in DMSO at concentrations of 0, 0.025, 0.050, 0.101, 0.252, 0.504, 1.01, 2.52, and 5.04 µg/mL for 18 – 20 h. The test substance was tested up to max. non-cytotoxic concentration, determined by cell-viability via trypan blue staining. The positive control (0.1 µg/mL 2-acetylaminofluorene) induced the appropriate response. There was no evidence that unscheduled DNA synthesis, as determined by radioactive tracer procedure (³H-thymidine (5µCi/mL) was induced.

The clastogenic potential of the test item was assessed in an in vitro chromosome aberration assay according to OECD guideline 473 (Hazleton Laboratories, 1988). In this study, cultured CHO cells were exposed to the test substance at concentrations of 49.6, 99.2, 149, or 198 µg/mL for 17.25 h in absence of the S9 metabolic activation. In the presence of the S9 activation, the CHO cells were exposed to the test substance at concentrations of 115, 384, 769, 1150, or 1540 µg/mL for 2 h. After exposure to the test substance, the treated cells were washed with buffered saline, and complete McCoy’s a medium containing 0.1 µg/mL Colcemid was added to the washed cells. The cells were then incubated for 2.5 h (without S9) or 7.5 h (with S9). The metaphase cells were then harvested, and slides prepared for analysis. The results showed that without S9 activation, the test item at concentrations of 149 and 198 µg/mL induced chromosomal aberrations, consisting mainly of simple chromatid breaks. In the presence of S9 activation, 1150 and 1540 µg/mL of the test substance caused a statistically significant and dose-related increase in chromosomal aberrations, consisting of simple chromatid and chromosome breaks. The test substance was tested up to cytotoxic concentrations, based on the results of a range finding study. The positive controls (without S9 mix: 0.08 µg/mL mitomycin C, with S9 mix: 12.5 µg/mL cyclophosphamide) did induce the appropriate response.

There was a concentration related positive response of induced chromosomal aberrations compared to untreated and solvent control.

The positive outcome of the OECD 473 guideline study made a confirmatory in vivo study necessary.

In vivo

The objective of this in vivo assay was to evaluate the ability of the test substance to induce micronuclei in bone marrow polychromatic erythrocytes of mice (strain ICR). The study was conducted similar to the EPA OPP 84-2 guideline. The test article was suspended in corn oil and dosed by oral gavage at 80, 400, and 800 mg/kg based upon the results of a previously conducted dose range finding assay. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. The animals were dosed with the test article and were euthanatized 24, 48 and 72 hours after dosing for extraction of the bone marrow. A second group of animals (designated Secondary Dose Group, 10 animals/sex/dose) was also assigned to the study and was dosed with the high dose of the test article. These animals were only used in the assay as replacements for any which died in the primary dose group. Vehicle (corn oil) and positive control (cyclophosphamide, 80 mg/kg bw) groups euthanatized 24 hours after dosing were included in the assay.

All animals were observed immediately after dosing and periodically throughout the duration of the assay for toxic symptoms and/or mortalities. Within ten minutes of dosing nine in the 800 mg/kg dose groups and one male in the 400 mg/kg dose groups had expired. Approximately 1.5 hours after dosing one additional male in the high dose group was found dead. The following morning, approximately 19 hours after dosing, one in the 800 mg/kg dose group was found dead. All other animals appeared normal and remained healthy until the appropriate harvest times. Apparent test article induced bone marrow toxicity was observed in one male in the 24 hour high dose group, since only 491 PCE could be analysed for micronuclei. The data from this animal were included in the statistical analysis although no difference in the evaluation of the test article would occur by its exclusion. The criteria for the identification of micronuclei were those of Schmid (1976). No statistically significant reduction in the PCE:NCE ratio relative to the negative control was detected at any dose level. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 1.54 ± 0.68 and 1.38 ± 0.41 for the males and females, respectively.

The test substance did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test.

Conclusion: The test substance did not show genotoxic properties in a bacterial reverse mutation assay (Ames) and in an unscheduled DNA synthesis assay in primary rat hepatocytes. In contrast, a clastogenic potential of the test substance was assessed in a chromosome aberration assay in CHO cells. However, this result was not confirmed in an in vivo mouse micronucleus assay. Therefore, the test substance is considered as non-genotoxic.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the results of the conducted studies, the test substance is considered not to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.