Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay/Ames test (similar to OECD 471): S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA: negative with and without metabolic activation

Chromosome aberration (similar to OECD 473): numerical aberration: negative for the short-term and 24 h continuous treatment with CHL/IU cells with and without metabolic activation;

structural aberration: negative for short-term treatment with CHL/IU cells with and without metabolic activation; positive for the 24 h continuous treatment with CHL/IU cells without metabolic activation

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:
08 Jan - 05 Mar 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
All experiments for the test substance and for the positive controls were performed only in duplicates. All experiments for the solvent control were performed in triplicates, but no standard deviations were given. It should be noted that the study report is a translation from Japanese to English.
Reference:
Composition 0
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Experiments for the test substance and for the positive controls were performed only in duplicates, for the solvent control in triplicates, but no standard deviations were given. Only 2-Aminoanthracene was used as positive control with S9-mix.
Principles of method if other than guideline:
This study was conducted in accordance with "Standards for Toxicity Investiagations" (Ministry of Labor, Notification No. 77, September 1, 1988 and Notification No. 67, June 2, 1997) "Procedures of Mutagenicity Test Using Microorganisms and Evaluation of Test Results" (Ministry of Labor, Offical Notification, February 8, 1999), "III Mutagenicity test" of "Reverse-Mutation Assay in Bacteria" prescribed in "Testing Methods Relating to the New Chemical Substances" (Notification No. 1121002 of the Pharmaceutical and Food Safety Bureau, MHLW, No.2 (2003.11.13) of the Manufacturing Industries Bureau, METI & No. 031121002 of the Environmental Health Department, MOE (November 21, 2003)).
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
his operon for the S. typhimurium strains
trp operon for the E. coli strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavon
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavon
Test concentrations with justification for top dose:
Main test:
313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation

Vehicle:
- Vehicle(s)/solvent(s) used: DMSO (Lot No. WF032, 100.0% in purity, spectrophotometric grade, DOJINDO Laboratories) was used.
- Justification for choice of solvent/vehicle:
The test substance is insoluble in distilled water (information provided by the sponsor), but soluble in DMSO (solubility ≥ 500 mg/mL).
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (0.5 µg/plate in water, TA98; 1 µg/plate in water, TA100: 2 µg/plate in water, TA1535, TA1537; 20 µg/plate in water, WP2uvrA)
Remarks:
with S9 mix
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 µg/plate in DMSO, TA100, WP2uvrA; 0.1 µg/plate in DMSO, TA98); sodium azide (0.5 µg/plate in water,TA1535); 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine*2HCl (0.5 µg/plate in DMSO,TA1537)
Remarks:
without S9 mix
Details on test system and conditions:
METHOD OF APPLICATION: preincubation

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

NUMBER OF REPLICATIONS: All experiments with the negative control were performed in triplicates. All experiments with the positive controls and with the test substance were performed in duplicates.

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of reduction in the number of revertant colonies
Evaluation criteria:
The test substance may be considered positive in this test system if the following criteria are met: The test substance is considered mutagenic, if two-fold (or more) increases in mean revertant numbers are observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
for TA1537 with S9 mix at concentrations ≥ 2500 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance is insoluble in distilled water (information provided by the sponsor), but soluble in DMSO (solubility ≥ 500 mg/mL).
- Precipitation: Precipitation of the test substance was observed at test concentrations ≥ 1250 µg/plate without metabolic activation for all tester strains (see Table 2).

RANGE-FINDING/SCREENING STUDIES: The test results of the dose finding study are shown in Table 1. No increases in the number of the revertant colonies of any tester strain were observed with or without metabolic activation. Cytotoxicity was detected by a reduction in the number of revertant colonies for the tester strain TA1537 at a test concentrations of 5000 µg/plate with S9 mix. Precipitation of the test substance was observed at a test concentrations ≥ 1250 µg/plate without metabolic activation for all tester strains.

COMPARISON WITH HISTORICAL CONTROL DATA: The values for the positive and the negative controls are in the range of the historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxicity was detected by a reduction in the number of revertant colonies for the tester strain TA1537 at test concentrations ≥ 2500 µg/plate with S9 mix (see Table 2).

Table 1. Test results of the dose finding study (preincubation method).

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 2 or 3 plates*)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvrA

TA98

TA1537

– S9 -mix

0

97

13

29

22

15

– S9 -mix

4.88

116

7

35

21

13

– S9 -mix

19.5

100

9

29

17

14

– S9 -mix

78.1

99

11

26

21

19

– S9 -mix

313

117

13

26

21

17

– S9 -mix

1250

101 P

8 P

32 P

20 P

8 P

– S9 -mix

5000

105 P

6 P

38 P

22 P

7 P

Positive controls, – S9 -mix

Name

AF-2

NaN3

AF-2

AF-2

ICR-191

Concentrations

(μg/plate)

0.01

0.5

0.01

0.1

0.5

Mean No. of colonies/plate

(average of 2 plates)

603

468

311

517

1580

+ S9 -mix

0

112

13

33

39

30

+ S9 -mix

4.88

102

11

33

23

18

+ S9 -mix

19.5

100

10

39

25

21

+ S9 -mix

78.1

104

8

33

28

20

+ S9 -mix

313

127

12

30

31

25

+ S9 -mix

1250

118

13

43

29

11

+ S9 -mix

5000

104

6

34

31

7

Positive controls, + S9 -mix

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

1

2

10

0.5

2

Mean No. of colonies/plate

(average of 2 plates)

962

246

634

278

234

 * All experiments of the solvent control were performed in triplicates. All experiments for the positive controls and for the test substance were performed in duplicates.

AF-2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3= Sodium azide

ICR-191 = 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine*2HCl

2AA = 2-Aminoanthracene

P = Precipitate

 

Table 2. Test results of the main study (preincubation method).

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 2 or 3 plates*)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvrA

TA98

TA1537

– S9 -mix

0

103

10

28

25

10

– S9 -mix

313

117

7

26

21

9

– S9 -mix

625

108

9

31

23

5

– S9 -mix

1250

104 P

8 P

32 P

26 P

6 P

– S9 -mix

2500

110 P

12 P

32 P

15 P

6 P

– S9 -mix

5000

108 P

8 P

26 P

24 P

6 P

Positive controls, –S9 -mix

Name

AF-2

NaN3

AF-2

AF-2

ICR-191

Concentrations

(μg/plate)

0.01

0.5

0.01

0.1

0.5

Mean No. of colonies/plate

(average of 2 plates)

674

344

377

516

1441

+ S9 -mix

0

114

7

33

32

24

+ S9 -mix

313

105

7

30

28

16

+ S9 -mix

625

140

7

29

22

11

+ S9 -mix

1250

113

8

28

30

14

+ S9 -mix

2500

112

6

30

37

7

+ S9 -mix

5000

117

11

35

31

7

Positive controls, +S9 -mix

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

1

2

10

0.5

2

Mean No. of colonies/plate

(average of 2 plates)

1039

202

657

371

284

 * All experiments with the solvent control were performed in triplicates. All experiments with the positive controls and with the test substance were performed in duplicates.

AF-2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3= Sodium azide

ICR-191 = 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine*2HCl

2AA = 2-Aminoanthracene

P = Precipitate

The test result of the main test are shown in Table 2. No increases in the number of the revertant colonies of any test strain were observed with or without metabolic activation. Cytotoxicity was detected by a reduction in the number of revertant colonies for the tester strain TA1537 at a test concentrations 2500 µg/plate with S9 mix. Precipitation of the test substance was observed at test concentrations ≥ 1250 µg/plate without metabolic activation for all tester strains.

Conclusions:
Under the conditions of the Ames test the test substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvr A) tested with and without metabolic activation.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Nov 2008 - 15 Apr 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Limited documentation, only one experiment. It should be noted that the study report is a translation from Japanese to English.
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
: limited documentation; only one experiment; evaluation criteria not according to OECD 473
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Principles of method if other than guideline:
This study was conducted in accordance with "III Mutagenicity Test: Chromosomal Aberration Test Using Cultured Mammalian Cells" prescribed in "Concerning Testing Methods Relating to the New Chemical Substances" on Japanese Test Guideline (Notification No. 1121002 of the Pharmaceutical and Food Safety Bureau, MHLW, No.2 (November 13, 2003) of the Manufacturing Industries Bureau, METI & No. 031121002 of the Environmental Health Department, MOE (November 21, 2003)).
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration test
Test material information:
Composition 1
Target gene:
Not applicable.
Species / strain:
other: Chinese hamster lung fibroblasts (CHL/IU) cells)
Details on mammalian cell lines (if applicable):
- Type and identity of media: Eagle's minimum essential medium (Nissui Pharmaceutical Co., Ltd, Lot No. 588803) supplemented with L-glutamine (final concentration: 0.292 g/L), sodium hydrogen carbonate (final concentration: approximately 1.85 g/L) and 10 vol% heat-inactivated newborn calf serum (NBCS, Sanko Junyaku Co., Ltd.)
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Short-term treatment
Without S9-mix: 6.91, 9.77, 13.8, 19.5, 27.6, 39.1 and 55.2 µg/mL
With S9-mix: 78.1, 110, 156, 221, 313 and 442 µg/mL

24 h continuous treatment:
Without S9 mix: 4.88, 6.91, 9.77, 13.8, 19.5 and 27.6 µg/mL
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance is insoluble in distilled water (information provided by the sponsor), but soluble in DMSO (solubility ≥ 500 mg/mL).
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
with S9 mix
Positive control substance:
other: cyclophosphamide monohydrate
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
without S9 mix
Positive control substance:
mitomycin C
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 6 h treatment: 24 h; 24 h treatment: 24 h

SPINDLE INHIBITOR (cytogenetic assays): colcemid (10 µg/mL)
STAIN (for cytogenetic assays): Giemsa 2% (v/v) in phosphate buffer solution

NUMBER OF REPLICATIONS: one experiment for the short-term treatment and one experiment for the continuous treatment; each experiment in duplicates

NUMBER OF CELLS EVALUATED: 100 per culture (200 metaphases per concentration)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
A test substance is considered positive (clastogenic) in the chromosome aberration test if a) the number of cells with structural or numerical aberrations is ≥ 10% and shows a dose-related increase; b) the number of cells with structural or numerical aberrations is ≥ 5% both in the chromosomal aberration test and the confirmation test.
Key result
Species / strain:
other: Chinese hamster lung fibroblasts (CHL/IU cells)
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Remarks:
numerical aberration: negative for the short-term and 24 h continuous treatment with and without S9-mix; structural aberration: negative for short-term treatment with and without S9-mix; positive for the 24 h continuous treatment without S9-mix
Cytotoxicity:
yes
Remarks:
Short-term treatment: cytotoxicity was observed at concentrations ≥ 39.1 µg/mL without S9 mix and at concentrations ≥ 313 µg/mL with S9 mix. 24 h continuous treatment: cytotoxicity was observed at concentrations ≥ 19.5 µg/mL.
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance is insoluble in distilled water (information provided by the sponsor), but soluble in DMSO (solubility ≥ 500 mg/mL).
- Precipitation: In presence of S9 mix, precipitation of the test substance was observed at all concentrations.

RANGE-FINDING STUDIES: The test concentrations were set based on the outcome of a preliminary cell growth inhibition test. The IC50 values were calculated at 27 µg/mL in the short-term treatment without S9 mix, at 270 µg/mL in the short-term treatment with S9 mix and at 14 µg/mL in the continuous treatment.

Table 1. Test results of the short-term treatment (6 h) and the 24 h continuous treatment

Test item

Concentration

Cell growth rate

Aberrant cells in %*

 

in µg/mL

in %

Structural aberration

Numerical aberration

Exposure period 6h, fixation time 24h, without S9 mix

DMSO

-

100

1.5

0

MMC

0.1

n.d.

45

1.0

Test substance

6.91

78.9

n.o.

n.o.

9.77

72.4

n.o.

n.o.

13.8

66.2

n.o.

n.o.

19.5

58.3

2.5

0.5

27.6

50.3

2.0

0

39.1

42.6

4.0

0

55.2

31.8

n.o.

n.o.

Exposure period 6h, fixation time 24h, with S9 mix

DMSO

-

100

1.0

0

CP

6.0

n.d.

36.0

0.5

Test substance

78.1P

82.3

n.o.

n.o.

110 P

81.3

n.o.

n.o.

156 P

70.4

3.5

0.5

221 P

57.4

1.5

1.0

313 P

42.6

3.0

0

442 P

21.3

n.o.

n.o.

Exposure period 24h, fixation time 24h, without S9 mix

DMSO

100

2.5

1.0

MMC

0.05

n.d.

59.5

0

Test substance

4.88

84.6

4.5

0

6.91

73.2

11.5

0

9.77

65.3

8.5

0.5

13.8

58.2

6.5

2.5

19.5

48.2

3.0

1.0

27.6

34.4

n.o.

n.o.

MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)

n.o.: not observed

P: Precipitation of the test substance

*The frequency of cells with chromosomal aberrations was calculated by observation of 200 metaphases per concentration.

 

Conclusions:
Under conditions of the in vitro chromosome aberration test in CHL/IU cells the test substance did not induce numerical aberrations after short-term or 24 h continuous treatment with or without metabolic activation but induced structural aberrations only after 24 h continous treatment without metabolic activation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

micronucleus assay (according to OECD 474): negative (3 doses (150 mg/kg bw, 300 mg/kg bw, 600 mg/kg bw) administered by gavage to mice)

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
No analytical purity is given.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
THE DEPARTEMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM.
Type of assay:
other: micronucleus assay
Test material information:
Composition 1
Species:
mouse
Strain:
other: Hsd: ICR (CD-1®)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK
- Age at study initiation: 5-8 weeks old
- Weight at study initiation: 23-30 g
- Assigned to test groups randomly: yes; selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card
- Housing: in groups of up to 7, by sex, in solid-floor polypropylene cages with wood-flake bedding
- Diet: Harlan Teklad 2014 Rodent Pelleted Diet; ad libitum
- Water: drinking water; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Arachis Oil BP
- Amount of vehicle: 10 mL/kg
- Lot/batch no.: W72
Duration of treatment / exposure:
24 h for all dose groups
48 h for the high dose group
Frequency of treatment:
single treatment
Post exposure period:
Animals were scarificed by cervical dislocation 24 or 48 h following treatment.
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
7 (exception: in the positive control group only 5 animals were used)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral (via gavage)
- Doses: 50 mg/kg
Tissues and cell types examined:
Femur; bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on the outcome of the range-finding study (see table 1), the maximum tolerated dose (MTD) of the test substance is 600 mg/kg. In addition, 150 and 300 mg/kg of the test substance were selected as lower dose levels for the use in the main test.

DETAILS OF SLIDE PREPARATION: Immediately following termination (24 h or 48 h following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone morrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald/Giemsa, allowed to air-dry and a cover slip applied using mounting medium.
Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

METHOD OF ANALYSIS:
Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.
Evaluation criteria:
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.
A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group.
If these criteria were not fulfilled, then the test material was considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity was demonstrated when the dose groupan polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.
Statistics:
All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a  √(X + 1) transformation using Student's t-test (two tailed) and any significant results were confird using the one way analysis of variance.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Clinical signs were observed in animals dosed with the test material at 600 mg/kg in both the 24 and 48‑hour groups, these were as follows: Hunched posture, lethargy, ataxia and ptosis.
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 600, 800, 1000 and 2000 mg/kg bw
- Clinical signs of toxicity in test animals: In animals dosed with the test material via the oral route premature deaths occurred at 800 and 2000 mg/kg (see table 1). Clinical signs were observed at and above 600 mg/kg as follows: Hunched posture, ptosis, ataxia, lethargy, pilo-erection, splayed gait, decreased respiratory rate, laboured respiration, elevated tail and tonic convulsions. The test material showed no marked difference in its toxicity to male or female mice, it was therefore considered to be acceptable to use males only for the main test. 
- Other: Adequate evidence of test material toxicity was demonstrated via the oral route of administration, therefore, this route was selected for use in the main test. The maximum tolerated dose (MTD) of the test material, 600 mg/kg, was selected for use in the main test, with 300 and 150 mg/kg as the lower dose levels.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test substance when compared to the concurrent vehicle control groups (see table 2).
- Ratio of PCE/NCE: There were no statistically significant decreases in the PCE/NCE ratio in the 24- or 48-hour test material groups when compared to their concurrent vehicle control groups (see table 3). However, the observation of clinical signs was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.
- Other: There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at 600 mg/kg in both the 24 and 48‑hour groups, these were as follows: Hunched posture, lethargy, ataxia and ptosis. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the test system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Table 1: Range-finding toxicity test

Dose Level

(mg/kg)

Sex

Number of Animals Treated

Route

Deaths on Day

Total Deaths

0

1

2

2000

Male

1

oral

1*

-

-

2/2

Female

1

1

-

-

1000

Male

1

oral

0

0

0

0/2

Female

1

0

0

0

800

Male

1

oral

0

0

0

1/2

Female

1

1

-

-

600

Male

1

oral

0

0

0

0/2

Female

1

0

0

0

600

Male

2

oral

0

0

0

0/2

Female

0

-

-

-

*= Killed in extremis

Table 2: Results of the in-vivo micronucleus assay in male animals.

 

Total PCE + MN

at sampling time

Total NCE + MN at sampling time

Treatment group

Number

of animals

Dose [mg/kg]

24 h

48 h

24 h

48 h

Number scored

Vehicle control

(Arachis oil BP, 10 mL/kg)

7

0

5

3

3

0

566 ± 46 (24 h)

500 ± 44 (48 h)

Test substance

7

600

4

5

0

1

510 ± 70 (24 h)

504 ± 31 (48 h)

Test substance

7

300

11

n.d.

0

n.d.

555 ± 45

Test substance

7

150

7

n.d.

0

n.d.

570 ± 87

Positive control

(cyclophosphamide)

5

50

150

n.d.

0

n.d.

615 ± 55

n.d.= not determined; *The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE) per animal was scored; **The number of normochromatic erythrocytes (NCE) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.

 

Table 3: Summary of the results of the in-vivo micronucleus assay.

Treatment group

Dose

[mg/kg]

Sampling time [h]

Mean frequency of PCE with MN per 2000 PCE

 

PCE/NCE ratio

Vehicle control (Arachis oil BP, 10 mL/kg)

0

48

0.4 ± 0.5

1.02 ± 0.18

 

0

24

0.7 ± 0.8

0.78 ± 0.16

Test substance

600

48

0.7 ± 0.8

0.99 ± 0.12

 

600

24

0.6 ± 0.8

1.0 ± 0.32

 

300

24

1.6 ± 1.5

0.81 ± 0.15

 

150

24

1.0 ± 1.0

0.79 ± 0.30

Positive control (Cyclophosphamide)

50

24

30.0 ± 4.7***

0.64 ± 0.14

***=P<0.001

 

Conclusions:
Under the experimental conditions of the in vivo micronucleus test the test substance did not induce micronuclei in erythrocytes of mice treated with the test substance.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

not applicable

Additional information

Genetic toxicity, in-vitro:

The in-vitro genetic toxicity of the test substance was investigated in a bacterial reverse mutation assay (Ames test) according to Japanese guidelines for screening mutagenicity testing of chemicals (Ogura, 2009). The preincubation method was conducted with S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA at concentrations up to 5000 µg/plate in the presence and absence of a metabolic activation system. The test substance did not induce reversions in any of the S. typhimurium strains or in E. coli WP2 uvrA with or without metabolic activation. No cytotoxic effects were observed except for TA1537 at test concentrations ≥ 2500 µg/plate in the presence of S9 mix. Precipitation of the test substance was noted for all tester strains at concentrations ≥ 1250 µg/plate in the absence of metabolic activation. Under the test conditions, the test substance showed no evidence of a mutagenic potential in bacteria.

The genotoxic potential of the test substance in-vitro was assessed in a chromosomal aberration test in mammalian chinese hamster lung fibroblasts (CHL/IU) cells according to Japanese guidelines for screening mutagenicity testing of chemicals (Ogura, 2009).

Based on the findings of a cell growth inhibition test, the following concentrations were chosen for the chromosomal aberration test: 6.91, 9.77, 13.8, 19.5, 27.6, 39.1 and 55.2 µg/mL in short-term treatment in the absence of metabolic activation, 78.1, 110, 156, 221, 313 and 442 µg/mL in short-term treatment in the presence of metabolic activation and 4.88, 6.91, 9.77, 13.8, 19.5 and 27.6 µg/mL in 24 h continuous treatment without metabolic activation.

As a result of the chromosomal aberration test, the frequencies of numerical aberration cells were below 5% at all test concentrations in all treatment methods. The frequencies of cells with structural aberrations were below 5% only for the short-term treatment. In the 24 h continuous treatment group, the frequencies of cells with structural aberrations were 4.5%, 11.5%, 8.5%, 6.5% and 3.0% at 4.88, 6.91, 9.77, 13.8 and 19.5 µg/mL, respectively. For the highest concentration of 27.6 µg/mL, no structural aberrations were observed. As the cell growth rates were about 50% at 13.8 and 19.5 µg/mL and the frequency of metaphases was low at 19.5 µg/mL, it was considered that the appearance of chromosomal aberration was inhibited by cytotoxicity at ≥ 9.77 µg/mL. Since the frequencies of cells with structural aberrations at 4.88 µg/mL were 4.5%, it was evaluated that a concentration related increase in structural aberrations was observed between 4.88 and 6.91 µg/mL in the 24 h continuous treatment. All controls were valid. Under the test conditions, the test substance did not induce numerical aberration but induced structural aberration.

Based on the results of the chromosomal aberration test, it is ambiguous if the test substance induces clastogenicity in-vitro. Therefore, a micronucleus assay in mice was performed.

Genetic toxicity, in-vivo

The genetic toxicity of the test substance in-vivo was tested in the micronucleus assay in mice, performed according to OECD 474 (Flanders, 2009). Seven males per dose group received 150, 300 or 600 mg/kg bw of the test substance by gavage. 600 mg/kg bw was the maximum tolerated dose (MTD) based on the outcome of a range-finding test. The animals were sacrificed 24 or 48 h following treatment and bone marrow smears from the femurs were prepared. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were microscopically scored for the presence of micronuclei. There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test substance at 600 mg/kg in both the 24 and 48 h groups: Hunched posture, lethargy, ataxia and ptosis. There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test substance compared the control groups.

No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48 h dose groups when compared to their concurrent control groups. However, the observation of clinical signs indicated that systemic absorption had occurred and exposure to the target tissue had been achieved. The positive control (cyclophosphamide) showed a clear clastogenic effect. Under the test conditions, there was no indication for a clastogenic effect in male mice after oral administration of up to 600 mg/kg of the test substance. In conclusion, the test substance did not induce clastogenicity in-vivo.

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 but not sufficient for classification.