Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was not genotoxic and cause neither gene mutations nor chromosomal aberrations in three in vitro guideline studies for genotoxicity. Furthermore a the substance did not induce micronuclei in an in vivo rat micronucleus test according to recent guidelines and GLP and thus showed no clastogenic or aneugenic potential in this test system.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-030-014 to 2011-05-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Followed guideline and GLP requirements. No analysis was conducted on formulations, but test item used within 4 hours of preparation and assumed stable.
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Test formulations not analyzed
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
histidine or tryptopahn locus in genome of the 5 bacterial strains

Salmonella typhimurium strains Genotype Type of mutation indicated
TA1537 his C 3076; rfa-, uvrB- frame shift mutations
TA98 his D 3052; rfa-, uvrB-; R-factor frame shift mutations
TA 1535 his G 46; rfa-; uvrB- base pair mutations
TA100 his G 46; rfa-; uvrB-; R-factor base pair substitutions

Escherichia coli trp-; uvrA- base pair substitution
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell lines (if applicable):
- Type and identity of media: obtained on culture discs or frozen
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
-Source: University of California, Berkelyor Syngenta CTL, Alderley Ledge
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal fraction from rats induced with phenobarbitone/beta-naphthoflavone at 80/100 mg/kg/day orally for 3 days
Species / strain:
E. coli WP2 uvr A
Details on mammalian cell lines (if applicable):
- Type and identity of media: obtained on nutrient agar plate and stored frozen
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
- Source: British Industrial Biological Research Association
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal fraction from rats induced with phenobarbitone/beta-naphthoflavone at 80/100 mg/kg/day orally for 3 days
Test concentrations with justification for top dose:
Range-find test: five dose levels from 50 to 5000 ug/plate (50, 150, 500, 1500, 5000 ug/plate)
Main test: 5 dose levels from 50 to 5000 ug/plate (50, 150, 500, 1500, 5000 ug/plate)
Vehicle:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: immiscible in sterile distilled water and DMSO at 50 mg/ml but fully miscible in acetone at some concentration
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Migrated to IUCLID6: WP2uvrA, TA100, TA1535 without S-9
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: for TA1537 without S-9
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: for TA1537 without S-9
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene for TA100, TA1535, TA 1537 and WP2uvrA with S-9
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: For TA 98 with S-9
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for range finding test; preincubation for main test

DURATION
- Preincubation period: 20 minutes in preincubation main test
- Exposure duration: 48 hours incubation in both preincubation and plate test

SELECTION AGENT (mutation assays): histidine or tryptophan


NUMBER OF REPLICATIONS: procedures in triplicate for each bacterial strain and each test concentration, both with and without S-9. Two complete tests: plate incorporation and preincubation tests


DETERMINATION OF CYTOTOXICITY
- Method: preliminary toxicity test using ten concentrations of test article, and acetone control used to assess toxicity in TA100 and WP2uvrA both with and without S-9. Plates assessed for revertant colonies and growth on bacterial background lawn. The test article was non-toxic to strains of bacteria used.
In the range finding test, the test article caused no reduction in growth was noted in the growth of the bacterial background lawn, either with or without activation. In the main test, the test item induced toxicity as weakened bacterial background lawns in several tester strains were noted in presence and absence of S-9 at 5000 ug/plate. No test article precipitate was noted.



METHOD OF APPLICATION: in agar (plate incorporation) and preincubation;
DURATION
- Preincubation period: 20 minutes, preincubation test
- Exposure duration: 48 hours incubation both tests

SELECTION AGENT (mutation assays): histidine or tryptophan

NUMBER OF REPLICATIONS: Each concentration tested in triplicate, with and without activation, and each positive or vehicle control. Two independent tests: plate incorporation and pre-incubation on separate days.


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (bacterial background lawn)


OTHER:

S-9 produced from Sprague Dawley male rats, 6 to 8 weeks of age, weighing approximately 250 grams. Induction was with oral phenobarbitone/beta-naphthoflavone given orally for 3 days at a dose of 80/100 mg/kg/day.
Evaluation criteria:
Acceptance criteria for valid test: all strains meet required characteristics, all tester strains exhibit characteristic number of spontaneous revertants per plate in the vehicle and untreated controls; all tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per ml; positive control chemicals must be included to demonstrate sensitivity of test strainsto mutagen exposure and the integrity of the S-9 mix. All positive controls should produce marked increases in frequency of revertant colonies, both with and without metabolic activation. There should be no evidence of excessive contamination, and there should be a minimum of 4 non-toxic dose levels.

Evaluation criteria: Any one or all of the following can be used to determine overall result: dose-related increase in mutant frequency over dose range tested; reproducible increase at one or more concentrations; biological relevance against in house historical control ranges; statistical analysis of data as determined by UKEMS; fold increase greater than two times the concurrent solvent control for any tester strain particularly if outside historical range.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: Weakened bacterial background lawns in preincubation test in presence and absence of S-9 at high dose.
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: Weakened bacterial background lawns in preincubation test in presence and absence of S-9 at high dose.
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Ames Test Results: Range Finding (Plate Incorporation) and Main Test (Preincubation)

Range Finding Test, Without Metabolic Activation

Test article ug/plate

TA100

TA1535

WP2uvrA

TA 98

TA 1537

0

112 (20.0)

21 (1.7)

28 (8.7)

24 (2.1)

12 (2.3)

50

102 (8.6)

21 (2.0)

24 (3.0)

22 (1.0)

10 (1.7)

150

110 (8.4)

22 (2.5)

27 (2.0)

17 (3.1)

12 (1.0)

500

99 (6.6)

22 (0.0)

25 (4.0)

21 (1.0)

9 (1.0)

1500

102 (6.7)

23 (2.3)

24 (7.0)

22 (0.6)

10 (3.8)

5000

99 (16.6)

22 (0.0)

23 (1.5)

23 (2.9)

12 (1.2)

Positve control, concentration, colonies per plate (SD)

ENNG

3 ug/plate

573 (65.4)

ENNG

5 ug/plate

383 (72.7)

ENNG

2 ug/plate

943 (107.5

4NQO

0.2 ug/plate

177 (21.2)

9AA

80 ug/plate

757 (122.2)

 (Mean Number of Revertant Colonies per Plate and Standard Deviation)

Range Finding Test, with Metabolic Activation

 (Mean Number of Revertant Colonies per Plate and Standard Deviation)

Test article ug/plate

TA100

TA1535

WP2uvrA

TA 98

TA 1537

0

97 (2/1)

14 (1.7)

31 (7.8)

25 (2.6)

12 (3.5)

50

92 (2.6)

14 (1.0)

31 (1.5)

21 (2.5)

11 (1.0)

150

82 (5.5)

14 (1.5)

30 (3.5)

23 (1.5)

12 (4.0)

500

88 (2.1)

13 (0.6)

27 (4.0)

23 (3.6)

10 (0.6)

1500

90 (4.6)

13 (0.6)

28 (2.9)

26 (4.6)

9 (1.0)

5000

91 (5.6)

14 (1.2)

28 (3.1)

21 (1.5)

13 (3.1)

Positive control, concentration, colonies per plate (SD)

2AA

1 ug/plate

1421 (218.7)

2AA

2 ug/plate

260 (23.6)

2AA

10 ug/plate

268 (31.5)

BP

5 ug/plate

284 (31.5)

2AA

2 ug/plate

231 (17.7)

Main Test, Without Activation

Test article ug/plate

TA100

TA1535

WP2uvrA

TA 98

TA 1537

0

116 (24.1)

23 (5.6)

37 (5.5)

17 (4.6)

9 (0.6)

50

95 (1.2)

21 (3.6)

38 (5.8)

15 (2.3)

4 (2.1)

150

110 (12.3)

23 (7.4)

33 (2.3)

14 (3.5)

6 (3.0)

500

112 (4.0)

21 (7.6)

33 (5.9)

13 (0.0)

7 (0.6)

1500

106 (106)

27 (6.4)

33 (0.6)

22 (3.0)

7 (1.5)

5000

122 (13.3)

23 (5.0)

25 (5.3)

8 (3.2)

4 (1.7)

Positive control, concentration, colonies per plate (SD)

ENNG

3 ug/plate

614 (60.1)

ENNG

5 ug/plate

258 (6.0)

ENNG

2 ug/plate

909 (80.5)

4NQO

0.2 ug/plate

134 (16.4)

9AA

80 ug/plate

836 (10.7)

 (Mean Number of Revertant Colonies per Plate and Standard Deviation)

Main Test, With Activation

Test article ug/plate

TA100

TA1535

WP2uvrA

TA 98

TA 1537

0

108 (1.7)

18 (3.6)

35 (34)

24 (4.4)

8 (1.2)

50

114 (14.6)

17 (3.2)

27 (1.5)

22 (4.4)

9 (0.6)

150

97 (17.9)

13 (0.6)

28 (2.0)

17 (3.5)

7 (2.9)

500

101 (2.1)

15 (4.9)

22 (4.4)

21 (1.0)

8 (3.2)

1500

93 (1.5)

13 (4.0)

31 (2.1)

19 (1.0)

8 (3.6)

5000

94 (4.4)

12 (4.0)

22 (2.1)

18 (7.8)

6 (0.0)

Positive control, concentration, colonies per plate (SD)

2AA

1 ug/plate

1249 (106.2)

2AA

2 ug/plate

440 (44.8)

2AA

10 ug/plate

345 (24.4)

BP

5 ug/plate

510 (7.5)

2AA

2 ug/plate

194 (38.9)

 (Mean Number of Revertant Colonies per Plate and Standard Deviation)

Conclusions:
The test article was considered non-mutagenic under the conditions of the test.
Executive summary:

Test article was tested in a reverse mutation assay (Ames test) using Salmonella typhimurium and Escherichia coli following OECD Guideline 471 and complying with GLP requirements. Salmonella typhimurium (strains TA 1535, 1537, TA 98 and Ta 100) and Escherichia coli (WP2uvrA) were treated with the test article using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard cofactors). Dose range for the range finder test based on a preliminary toxicity test was 50 to 5000 ug/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, fresh cultures of bacteria and fresh test article fomulations. The vehicle (acetone) gave counts of revertant colonies within normal range. All positive control chemicals induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus validity was assured. The test item caused no significant reduction in growth of the bacterial background lawn in a toxicity test so it was tested at maximum recommended dose level of 5000 ug/plate.

In the range finding test, no visible reduction of background lawn was seen at any dose. In the main test, weakened bacterial background lawn was seen in several tester strains with and without activation at the high dose. These results were not sufficiently severe to prevent the test item being tested up to the maximum dose of 5000 ug/plate. No precipitate of test article was seen in any plates.

No significant increase in frequency of revertant colonies was recorded for any of the strains with any dose of the test article, with or without metabolic activation. The test article, SD10, was considered non-mutagenic under the conditions of the test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Follwed guideline and GLP requirements; only one change of fixative instead of two consecutive changes of fixative were used
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
One change of fixative instead of two
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Test material information:
Composition 1
Species / strain:
Chinese hamster Ovary (CHO)
Details on mammalian cell lines (if applicable):
- Type and identity of media: complete medium (McCoy's 5A medium supplemented with 10% fetal bovine serum, 100 unites penicillin/mL, 100 ug streptomycin/mL, 2 mM L-glutamine and 2.5 ug/mL amphotericin B)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain characteristics:
other: CHO-K1 cells repository number CCL 61
Metabolic activation:
with and without
Metabolic activation system:
Aroclor1254-induced rat liver S9
Test concentrations with justification for top dose:
Chromosome Aberration Assay Test – Dose levels

Treatment Condition Treatment/Recovery Time Dose level (ug/mL)
Non-activated 4 hr/16 hr 21.9, 43.8, 87.5, 175, 350, 700, 1400, 200, 2850
20 hr/0 hr 6.5, 11.7, 21.9, 43.8, 87.5, 125, 175, 280, 350, 500
S9-activated 4 hr/16 hr 6.5, 11.7, 21.9, 43.8, 87.5,175, 350, 700, 1000



Vehicle:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: best choice for solubility and compatibility with test system
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: in S9-activated systems
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: in non-activated system
Details on test system and conditions:
METHOD OF APPLICATION: test article in solution with vehicle added to cell cultures in media or S9 mixture

DURATION

- Exposure duration: 4 hours or 20 hours
- Expression time (cells in growth medium): 16 hours or 0 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

Agents used: Colcemid two hours before scheduled harvest of metaphase cells
Cell viability: determined by Trypan blue dye exclusion

NUMBER OF REPLICATIONS: duplicate flasks per concentration

NUMBER OF CELLS EVALUATED: Mitotic index (cells in mitosis/500 cells scored) determined for each treatment flask; At least 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations

DETERMINATION OF CYTOTOXICITY
- Method: cell growth inhibition by cell counts and percent viability; viability determined by Trypan blue dye exclusion

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, from each treatment flask per 100 metaphase cells evaluated
- Determination of endoreplication: yes, from each treatment flask per 100 metaphase cells evaluated
- Other: Chromatid-type aberrations and chromosome-type aberrations recorded as well as chromatid gaps and isochromatid gaps


OTHER:
Evaluation criteria:
A positive test was considered to be when the percentage of cells with aberrations is increased in a dose-responsive manner, with one or more concentrations being statistically significant (p equal or lesser than 0.05). However, values that are statistically significant, but do not exceed the range of historical solvent controls may be judged as not biologically significant. Tests substances not demonstrating a statistically significant increase in aberrations will be considered negative.
Statistics:
Statistical analysis of percent aberrant cells was perfoed using Fisher's Exact test. Fisher's Exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's Exact test, the Cochran-Armitage test was used to measure dose-responsiveness.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
top dose chosen for scoring inhibited cell growth 50%
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: CHO-K1
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Solutions had pH of the top eight concentrations measured, and adjusted to a final pH of 7.0 to 7.5 with drops of 1 N HCl
- Effects of osmolality: In the preliminary test, osmolality of the ethanol solvent was 301 mmol/kg. Osmolality of the highest concentration (2850 ug/mL was 320 mmol/kg. As the osmolality of the test concentrations did not exceed that of the solvent by more than 20%, they are considered acceptable.
- Precipitation: none observed in any test concentrations


RANGE-FINDING/SCREENING STUDIES: Substantial toxicity (at least 50% cell growth inhibition relative to solvent control (was observed in the 2850 ug/mL in the non-activated 4 hour exposure group and at dose levels equal or greater than 285 ug/mL in both S9 activated 4 hour exposure group and the non-activated 20 hour continuous exposure group.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, acceptable

Chromosome Aberration Assay – Initial Assay Results

Treatment

Time

Recovery

Time

Harvest

Time

S9

Toxicity

highest dose scored

(ug/mL)

Mitotic

Index Reduction

**

LED for Structural

Aberrations (ug/mL)

LED for Numerical

Aberrations

(ug/mL)

4 hr

16 hr

20 hr

-

54% at 2000

34%

None

None

20 hr

0 hr

20 hr

-

52% at 280

56%

None

None

Toxicity = cell growth inhibition    ** = relative to solvent control at highest dose evaluated for chromosome aberrations LED = lowest effective dose

Chromosome Aberration Assay – Repeat Assay Results

Treatment

Time

Recovery

Time

Harvest

Time

S9

Toxicity

highest dose scored

(ug/mL)

Mitotic

Index Reduction

**

LED for Structural

Aberrations (ug/mL)

LED for Numerical

Aberrations

(ug/mL)

4 hr

16 hr

20 hr

+

51% at 2000

39%

None

None

Toxicity = cell growth inhibition    ** = relative to solvent control at highest dose evaluated for chromosome aberrations LED = lowest effective dose

Cytogenetic Analysis of CHO Cells Treated with SD10 in Absence of Activation

4 hour treatment, 16 hour recovery (Initial Assay)

Group

(ug/ml)

Mitotic Index

(A, B)

Cells scored

(#/structural)

% Aberrant

Cells

(#/structural)

Gaps

Chromatid

Structural aberrations

(br/ex)

Chromosome

Structural

Aberrations

(br/dic/ring)

Avg. aberr.

per cell

Ethanol

15.4

13.6

100/100

100/100

4/0

4/0

0

0

0/0

0/0

0/0/0

0/0/0

0.000

0.000

175

13.4

14.0

100/100

100/100

4/0

5/0

1

0

0/0

0/0

0/0/0

0/0/0

0.000

0.000

700

10.4

10.0

100/100

100/100

5/1

6/0

0

0

1/0

0/0

0/0/0

0/0/0

0.010

0.000

2000

10.2

 9.0

100/100

100/100

3/1

4/1

0

1

0/1

0/1

0/0/0

0/0/0

0.010

0.010

MMC (0.2)

9.4

10.0

100/50

100/50

5/18

5/16

1

0

6/4

7/4

0/1/0

0/0/0

0.220

0.220

Mitotic index = (Cells in mitosis/500 cells scored) x 100

% aberrant cells: numerical cells (#) include polyploid and endoreplicated cells

                        : structural cells exclude cells with only gaps

Chromatid breaks: (br) include chromatid and isochromatid breaks and fragments

Chromatid exchanges: (ex) include quadriradials, triradials, and complex rearrangements

Chromosome breaks: (br) include breaks and accentric fragments

Dic: dicentric chromosomes

Severely damaged cells: includes cells with one or more pulverized chromosomes and cells with 10 or more aberrations: None were present in controls or treated groups scored in this treatment

Average aberrations per cell: severely damaged cells and pulverizations were counted as 10 aberrations

Cytogenetic Analysis of CHO Cells Treated with SD10 in Absence of Activation

20 hour treatment, (Initial Assay)

Group

(ug/ml)

Mitotic Index

(A, B)

Cells scored

(#/structural)

% Aberrant

Cells

(#/structural)

Gaps

Chromatid

Structural aberrations

(br/ex)

Chromosome

Structural

Aberrations

(br/dic/ring)

Avg. aberr.

per cell

Ethanol

15.4

15.8

100/100

100/100

3

2

1

0

1/0

0/0

0/0/2

0/0/1

0.030

0.010

43.8

15.6

14.2

100/100

100/100

4

3

1

1

0/1

1/0

1/1/0

0/0/0

0.030

0.010

125

11.0

10.0

100/100

100/100

2

2

1

1

0/0

0/0

1/0/0

1/1/1

0.010

0.030

280

6.6

7.0

100/100

100/100

2

2

1

2

0/1

2/0

1/1/0

2/0/1

0.030

0.050

MMC (0.1)

8.4

9.0

100/50

100/50

3

3

1

0

4/3

8/1

3/1/1

1/0/0

0.240

0.200

Cytogenetic Analysis of CHO Cells Treated with SD10 in Presence of Activation

4 hour treatment, 16 hour recovery (Repeat Assay)

Group

(ug/ml)

Mitotic Index

(A, B)

Cells scored

(#/structural)

% Aberrant

Cells

(#/structural)

Gaps

Chromatid

Structural aberrations

(br/ex)

Chromosome

Structural

Aberrations

(br/dic/ring)

Avg. aberr.

per cell

Ethanol

13.0

14.0

100/100

100/100

0/1

2/2

0

0

0/1

2/1

0/0/0

0/0/0

0.010

0.030

175

14.6

14.4

100/100

100/100

4/1

3/4

0

1

0/1

2/2

0/0/0

0/0/0

0.010

0.040

700

10.4

11.6

100/100

100/100

2/1

4/1

0

0

0/0

1/0

1/0/0

0/0/0

0.010

0.010

2000

8.4

8.2

100/100

100/100

3/2

3/4

1

0

1/1

2/2

0/0/0

0/1/0

0.020

0.050

CP (10)

2.8

2.6

100/50

100/50

1/22

4/20

0

0

4/3

8/4

7/0/0

1/0/0

0.280

0.260

Conclusions:
Interpretation of results (migrated information):
negative

The test susubstance was negative for induction of structural and numerical chromosome aberrations in CHO cells in both the non-activated and S9-activated systems.
Executive summary:

The test substance was tested in a chromosome aberration assay using Chinese hamster ovary (CHO) cells both in the presence and absence of an Arocolor-induced rat liver S9 metabolic activation system to evaluate clastogenic potential of the substance.. A preliminary toxicity test established the dose range for the assay. Cytotoxicity was determined in the preliminary and definitive assays based on the increase in cell counts relative to that of baseline cell counts.

           Ethanol was used as the solvent based on test substance solubility and compatibility with the target cells. Solubility of the test substance in ethanol was about 500 mg/ml. The maximum test substance dose in the preliminary toxicity assay was 2850 ug/mL (10mM). No precipitates were seen in the ethanol and treatment medium at all dose levels tested.

           Substantial toxicity (at least 50% cell growth inhibition relative to solvent control) was observed at 2850 ug/mL in the non-activated 4-hour exposure group, and at dose levels equal or greater than 285 ug/mL in both S9-activated 4-hour and the non-activated 20 hour continuous exposure group. Based on these findings, doses chosen for the chromosome aberration assay ranged from 21.9 to 2850 ug/mL for the non-activated 4-hour exposure group, from 6.5 to 1000 ug/mL for the S9-activated 4-hour exposure group, and from 6.5 to 500 ug/mL for the non-activated 20-hour continuous exposure group.

           In the initial chromosome aberration assay, the cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. No precipitates of test article were seen. Selection of doses for microscopic analysis was based on toxicity (the lowest dose with at least 50% reduction in cell growth and two lower doses) in the non-activated 4 and 20 hour exposure groups. In the S9-activated 4-hour exposure group, due to inconsistent cytotoxicity, the chromosome aberration assay was repeated.

           The percentage of cells with structural or numerical aberrations in the non-activated 4- and 20-hour exposure groups was not significantly increased relative to solvent control at any dose level (p>0.05, Fisher’s Exact test).

In the repeat assay, dose levels tested ranged from 175 to 2850 ug/mL. Cells were treated for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. No precipitates were seen. Selection of doses for microscopic analysis was based on toxicity (the lowest dose with at least 50% reduction in cell growth and two lower doses). The percentage of cells with structural or numerical aberrations in the S9-activated 4 hour exposure group was not significantly increased relative to solvent control at any dose level. (P > 0.05, Fisher’s Exact test).

           Based on findings in this study, the test article was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells in borth the non-activated and the S-9activated test systems.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Followed guideline and GLP requirements; Analyses of formulations not conducted, but test performed at maximum feasible concentration as to precipitation. Noble agar in cloning medium was 0.24% instead of between 0.22 and 0.23%, dilution scheme for the mutation assay was performed as intended, but not according to the written dilution scheme.
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
Formulations were not analyzed, but tested at highest concentration possible; noble agar concentration in cloning medium 0.01% higher than protocol; dilution scheme conducted as intended, but not according to written scheme
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Test material information:
Composition 1
Target gene:
Thymidine kinase locus of L5178Y mouse lymphoma cells
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
- Type and identity of media: Restrictive media as per Clive and Spector, 1975
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain characteristics:
other: clone 3.7.2C obtained from Glaxo Wellcome
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-1254 induced rat liver S9
Test concentrations with justification for top dose:
Initial mutagenicity assay, with and without activation, 4 hour exposure: Chosen for cloning: 25, 75, 150, 250 and 300 ug/mL without activation, and 25, 50, 100, 250 ug/mL with S9
Extended mutagenicity assay, non-activated, 24 hour exposure: Chosen for cloning: 5.0, 10, 15, and 25 ug/mL.
Vehicle:
- Vehicle(s)/solvent(s) used: ethanol;
- Justification for choice of solvent/vehicle: bested for solubility and lack of vehicle effect on system
Solvent controls:
yes
Remarks:
ethanol
Positive controls:
yes
Remarks:
DMSO diluent, in activated system
Positive control substance:
7,12-dimethylbenzanthracene
Solvent controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: water diluent, for non activated system
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): For expression of mutant phenotype: 24 and 48 hours , and 48 and 72 hours for extended exposure
For expression of TK+/- cells, plated for 10 to 14 days

NUMBER OF REPLICATIONS: Duplicate cultures per concentration, 3 plates per concentration culture for VC colonies (total 6 plates per concentration) and TFT Colonies (total 6 plates per concentration)


DETERMINATION OF CYTOTOXICITY
- Method: Preliminary test evaluated cell populaton density and measured suspension growth of treated cultures relative to solvent control cultures after 48 hours. In mutagenicity studies, cytotoxic effects of each treatment condition were expressed relative to solvent-treated control for suspension growth over the two days post-treatment and for total growth (suspension growth corrected for plating efficiency).


Evaluation criteria:
Positive result: if concentration-related increase in induced mutation frequency observed in treated cultures and one or more treatment conditions with 10% or greater total growth exhibited mutant frequencies of 90 or greater mutants per 10^6 clonable cells (based on the average mutant frequency of duplicate cultures). If average of solvent control mutant frequencies was greater than 90 per 10^6 clonable cells, a doubling of mutant frequency over the background would be required. Some other situations for which a chemical would be considered when there was no culture showing between 10-20% survival: 1) no evidence of mutagenicity (no dose response or increase in induced mutant frequency between 45 and 89 mutants per 10^6 clonable cells) in a series of data points within 100% and 20% survival and there was at least one negative data point between 20 and 25% survival. 2) There was no evidence of mutagenicity (no dose response or increase in induced mutant frequency between 45 and 89 mutants per 10^6 clonable cells) in a series of data points between 100% and 25% survival and there was also a negative data point between 10% and 1% survival. In this case it would be acceptable to count the TFT colonies of cultures exhibiting < 10% growth.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: Test requres a high test concentration causing 80 to 90% cytotoxicity
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Initial Assay: 4 hour exposure, without activation

Dose level ug/mL

Total

Susp. Growth

% Susp. Growth

VC Colonies

Mean Plate Count

TFT Colonies

Mean Plate Count

Total Mutant Frequency

(per 10^6 cells)

Induced Mutant Frequency (per 10^6 cells)

% Total Growth

Solvent A

24.6

100

177

57

64

NA

100

Solvent B

21.5

100

203

54

53

NA

100

25 A

15.9

69

196

46

47

-12

71

25 B

13.9

60

180

48

54

-5

57

75 A

13.1

57

186

46

50

-9

56

75 B

9.8

43

234

54

46

-13

52

150 A

7.2

31

235

76

65

6

39

150 B

6.7

29

233

73

63

4

35

250 A

5.0

22

181

68

75

16

21

250 B

3.6

16

220

53

48

-11

18

300 A

4.3

18

210

57

55

-4

20

300 B

4.1

18

184

60

65

6

17

400 A

1.3

8

-

-

-

-

-

400 B

1.2

8

-

-

-

-

-

Positive control: methyl methanesulfonate (ug/mL)

20

8.9

39

39

134

681

623

8

15

10.9

47

78

207

530

471

19

Mean Solvent Total Suspension Growth: 23.0

Mean Solvent Cloning Efficiency: 95%

Mean Solvent Mutant Frequency: 59 per 10^6 cells

- means no data due to toxicity to great to clone

Initial Assay: 4 hour exposure, with activation

Dose level ug/mL

Total

Susp. Growth

% Susp. Growth

VC Colonies

Mean Plate Count

TFT Colonies

Mean Plate Count

Total Mutant Frequency

(per 10^6 cells)

Induced Mutant Frequency (per 10^6 cells)

% Total Growth

Solvent A

15.4

100

166

76

92

NA

100

Solvent B

13.2

100

209

73

70

NA

100

25 A

11.9

83

166

93

112

31

74

25 B

14.9

104

180

77

85

4

100

50 A

9.9

69

182

89

98

17

68

50 B

11.0

77

204

75

74

-7

83

75 A

6.4

44

186

87

93

12

44

75 B

4.6

32

186

82

88

7

32

150 A

2.3

19

164

91

111

30

17

150 B

4.0

28

*

136

250 A

1.8

16

151

84

111

30

13

250 B

1.6

19

173

109

126

45

17

300 A

0.3

0

-

-

-

-

-

300 B

0.2

0

-

-

-

-

-

Positive control: 7,12-dimethylbenz(a)anthracene (ug/mL)

1.25

3.3

23

136

279

681

328

17

1

5.6

39

172

253

530

214

36

Mean Solvent Total Suspension Growth: 14.3

Mean Solvent Cloning Efficiency: 94%

Mean Solvent Mutant Frequency: 81 per 10^6 cells

- means no data due to toxicity to great to clone * means culture lost to error

Extended Treatment Assay: 24 hour exposure, without activation

Dose level ug/mL

Total

Susp. Growth

% Susp. Growth

VC Colonies

Mean Plate Count

TFT Colonies

Mean Plate Count

Total Mutant Frequency

(per 10^6 cells)

Induced Mutant Frequency (per 10^6 cells)

% Total Growth

Solvent A

37.9

100

155

36

46

NA

100

Solvent B

33.2

100

180

39

43

NA

100

5 A

31.4

89

141

36

51

6

74

5 B

22.3

63

167

47

57

12

63

10 A

18.7

53

173

57

66

22

54

10 B

19.8

56

144

35

48

4

48

15 A

15.7

44

147

41

56

11

39

15 B

13.3

38

152

57

75

30

34

25 A

4.0

8

114

56

98

53

6

25 B

2.8

7

113

59

105

60

5

40 A

2.8

7

674

-

-

-

-

40 B

1.5

5

418

-

-

-

-

Positive control: methyl methanesulfonate (ug/mL)

7.5

16.5

47

70

253

718

674

20

5

22.2

63

109

252

462

418

41

Mean Solvent Total Suspension Growth: 35.5

Mean Solvent Cloning Efficiency: 84%

Mean Solvent Mutant Frequency: 44 per 10^6 cells

- means no data due to toxicity to great to clone 

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of the study, the test article was negative in th eL5178Y/TK+/- Mouse Lymphoma Assay.
Executive summary:

The test article was tested in the L5178Y/TK+/- Mouse Lymphoma Assay (OECD Guideline 476 In Vitro Mammalian Cell Gene Mutation Assay) in the presence and absence of Aroclor-induced rat liver S9. Ethanol was the solvent based on solubility of test article and compatibility with target cells. Test article was soluble in ethanol at about 500 mg/mL, the maximum concentration tested. In a preliminary toxicity test, the maximum test article concentration was 2850 ug/mL (10 mM). No precipitate was noted in the test media at any concentration. Suspension growth relative to solvent control was 0% at 1500 ug/mL or higher without activation and a 4 hour exposure, at 500 ug/mL and higher with S9 activation and a 4 hour exposure, and at 150 ug/mL and higher without activation and a 24 hour exposure.

Based on the preliminary toxicity assay, the concentrations in the initial mutagenicity assay ranged from 25 to 750 ug/mL for the non-activated and S9-activated cultures with a 4 hour exposure. No precipitate was noted. Concentrations chosen for cloning were 25, 75, 150, 250 and 300 ug/mL without activation, and 25, 50, 100, 250 ug/mL with S9 activation. No cloned cultures exhibited induced mutation frequencies of 90 or higher mutants per 10^6 clonable cells. There was no concentration-dependent increase in mutant frequency.

The concentrations in the extended treatment assay ranged from 5.0 to 150 ug/mL for non-activated cultures with a 24 hour exposure. No precipitates were noted in the treatment medium. Concentrations chosen for cloning were 5.0, 10, 15, and 25 ug/mL. No cloned cultures exhibited induced mutation frequencies of 90 or higher mutants per 10^6 clonable cells. There was no concentration-dependent increase in mutant frequency.

The trifluorothymidine-resistant colonies for the positive and solvent control cultures from both assays were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS and DMBA positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods to detect small colony mutants) and large colonies.

Under the conditions of the study, the test article was considered to be negative in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay.

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

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle:
- Amount of vehicle (if gavage or dermal):
- Type and concentration of dispersant aid (if powder):
- Lot/batch no. (if required):
- Purity:
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

Duration of treatment / exposure:
A single gavage dose was given.
Frequency of treatment:
A single gavage dose was given.
Post exposure period:
Animals sacrificed at either 24 or 48 hours
Remarks:
Doses / Concentrations:
0, 100, 200, or 400 mg/kg
Basis:
nominal conc.
400 mg/kg MTD based on preliminary test
No. of animals per sex per dose:
5 animals per sex per treated dose group, vehicle control group, positive control group, and reserve group dosed at high dose.
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: same as treated animals - oral gavage
- Doses / concentrations: 50 mg/kg
Tissues and cell types examined:
Femoral bone marrow prepared as slides; polychromatic erythrocytes, micronucleated PCEs, total erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: MTD was determined to be 400 mg/kg based on mortality and clinical signs seen at 600 mg/kg in a preliminary study, and LD50 estimate of 550 mg/kg in previous toxicity study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Single gavage, with sacrifice at 24 or 48 hours. Femurs exposed, cut above the knee, and bone marrow aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a labelled centrifug tube containing about 1 ml fetal bovine serum. The cells were pelleted by centrifugation at about 100 g for five minutes and supernatent drawn off. the cells were resuspended.

DETAILS OF SLIDE PREPARATION: A drop of bone marrow suspension was spread on a clean glass slide, affixed with a computer generated label with experiment and animal number. Two slides were prepared per animal. Slides were air dried and fixed in methanol. One set of sldes was stained with a nucleic acid-specific stain (acridine orange) and used for microscopic evaluation.

METHOD OF ANALYSIS: Slides were coded by random number table by person not involved in scoring. Using a fluorescent microscope and medium magnification (400x; blue excitation filter in range of 440 to 490 nm and barrier filter combination at 520 nm0, an area of acceptable quality was selected so that the cells were well spread and stained. Using oil immersion (1000x), the following cellular components were evaluated and enummerated: polychromatic erythrocytes (PCEs), normochromatic erythrocytes (NCEs), micronuclei (M), and the PCEs/ECs ratio.

OTHER:
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
tested at MTD
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes

Summary of Bone Marrow Micronucleus Analysis

Treatment

(mg/kg)

Sex

Hours

Group

Size

PCE/Total erythrocytes (Mean +/- SD)

Change from control (%)

MPCE/1000 PCE (Mean +/- SD)

MPCE/ PCE scored

Vehicle

M

F

24

24

5

5

0.591 (0.06)

0.573 (0.05)

- - -

- - -

0.4 (0.42)

0.2 (0.27)

4/10000

2/10000

 SD10

100

M

F

24

24

5

5

0.538 (0.06)

0.539 (0.06)

-9

-6

0.2 (0.27)

0.2 (0.27)

2/10000

2/10000

SD10

200

M

F

24

24

5

5

0.562 (0.08)

0.486 (0.06)

-5

-15

0.2 (0.27)

0.0 (0.0)

2/10000

0/10000

SD10

400

M

F

24

24

5

5

0.533 (0.06)

0.563 (0.05)

-10

-2

 

 

0/10000

2/10000

CP

50

M

F

24

24

5

5

0.443 (0.05)

0.422 (0.05)

-25

-26

9.3 (0.57)

11.4 (0.82)

*93/10000

*114/10000

Vehicle

M

F

48

48

5

5

0.530 (0.02)

0.530 (0.05)

- - -

- - -

 

0.0 (0.0)

0/10000

0/10000

SD10

400

M

F

48

48

5

5

0.486 0.06)

0.584 (0.06)

-8

10

0.1 (0.22)

0.1 (0.22)

1/10000

1/10000

  • Statistically significant increase compared to vehicle control p +/< 0.05 (binomial distribution, Kastenbaum-Bowman tables)
  • PCE: Polychromatic Erythrocytes      MPCEs: micronucleated polychromatic erythrocytes
Conclusions:
Interpretation of results (migrated information): negative
Test article was negative in the rat micronucleus assay because it did not induce a significant increase in micronucleated polychromatic erythrocytes in bone marrow of male and female Hsd:SD rats given a single oral gavage dose at maximum tolerated dose and lower.
Executive summary:

The test article was evaluated for genotoxicity potential (clastogenicity/aneugenicity) as measured by its ability to increase the incidence of micronucleated polychromatic erythrocytes (MPCEs) in bone marrow of male and female Hsd:SD rats. Methods complied with OECD Guideline 474 (Genetic Toxicology: Mammalian Erythrocyte Micronucleus Test). Test article was administered orally by single oral dose gavage of a fomulation in arachis (peanut) oil, at a dose volume of 10 mL/kg. Vehicle control was the arachis oil, and positive control substance was cyclophosphamide monohydrate (50 mg/kg).

           The test was conducted in a dose range finding phase (toxicity) and a definitive phase (micronucleus test). The dose range find study consisted of exposing 5 rats per sex per group to 300, 400, or 600 mg/kg test article. Based on mortality and clinical signs, 400 mg/kg was determined to be the maximum tolerated dose. Mortality was one of five males and two of five females dosed at 600 mg/kg. Animals dosed at 600 mg/kg showed piloerection and lethargy, as well as crusty noses, diarrhea, prostration and hunched posture. Pilorection only was noted in the 300 mg/kg group animals, while the animals in the 400 mg/kg group had piloerection and lethargy. No appreciable reduction in mean group body weights was noted in the study period.

           The definitive study consisted of 7 groups with 5 rats per sex in each. Five of the groups were dosed with control (vehicle or positive) substances or with the test article at 100, 200, or 400 mg/kg, and were euthanized at 24 hours post dose. Rats in the two remaining groups were dosed with either vehicle or the high dose test article formulation and euthanized at 48 hours. An additional 5 rats/sex were dosed at the high dose to be used as replacement animals if needed, but mortality did not occur, and bone marrow was not collected from that group. At the scheduled time point, femoral bone marrow was collected, and marrow smears (slides) were prepared and stained with a nucleic acid specific stain (acridine orange stain). Bone marrow cells (polychromatic erythrocytes (2000 PCEs/animal)] were examined for presence of micronuclei (micronucleated PCEs, MPCEs). Statistical analysis of the data was performed using the Kastenbaum-Bowman Tables (binomial distribution, p </= 0.05).

           No mortality was noted in the definitive study. Piloerection was noted in the 400 mg/kg group animals, and lethargy was noted in the 400 mg/kg group sacrificed at 48 hours. Reductions of up to 15% in the PCEs/ECs ratio of polychromatic erythrocytes to total erythrocytes relative to the vehicle control groups was observed in some of the test groups without a dose response, suggesting that the substance did not markedly inhibit erythropoiesis. No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in test substance groups relative to the respective vehicle control groups was observed in male or female rats at 24 or 48 hours post dose administration. (p > 0.05, binomial distribution, Kastenbaum-Bowman Tables). The positive control substance (cyclophosphamide monohydrate) induced a statistically significant increase in the incidence of micronucleated PCEs in male and female rats (p < 0.05, binomial distribution, Kastenbaum-Bowman Tables). The number of MPCEs in the vehicle control groups did not exceed historical control vehicle ranges. All criteria were met for a valid test.

           Under the conditions of the study, a single oral dose of test article at doses up to and including a dose of 400 mg/kg (MTD) did not induce a significant increase in micronucleated polychromatic erythrocytes in bone marrow of male and female Hsd:SD rats. Therefore, the test article was concluded to be negative in the rat micronucleus assay.

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

Additional information

The substance was tested in three in vitro assays following recent guidelines and GLP. No genotoxic or clastogenic response was observed.

Test article was tested in a reverse mutation assay (Ames test) using Salmonella typhimurium and Escherichia coli following OECD Guideline 471 and complying with GLP requirements. Salmonella typhimurium (strains TA 1535, 1537, TA 98 and Ta 100) and Escherichia coli (WP2uvrA) were treated with the test article using both the Ames plate incorporation and pre-incubation methods at five concentration levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard cofactors). The test item caused no significant reduction in growth of the bacterial background lawn in a toxicity test so it was tested at maximum recommended concentration level of 5000 ug/plate.

In the main test, weakened bacterial background lawn was seen in several tester strains with and without activation at the high concentration. These results were not sufficiently severe to prevent the test item being tested up to the maximum concentration of 5000 ug/plate. No precipitate of test article was seen in any plates.

No significant increase in frequency of revertant colonies was recorded for any of the strains with any concentration of the test article, with or without metabolic activation. The test article, SD10, was considered non-mutagenic under the conditions of the test.

The test article was tested in the L5178Y/TK+/- Mouse Lymphoma Assay (OECD Guideline 476 In Vitro Mammalian Cell Gene Mutation Assay) in the presence and absence of Aroclor-induced rat liver S9. Ethanol was used as the solvent. In a preliminary toxicity test, the maximum test article concentration was 2850 ug/mL (10 mM). Based on a preliminary toxicity assay, the concentrations in the initial mutagenicity assay ranged from 25 to 750 ug/mL with and without S9-mix. The exposure time was 4 hours. No precipitate was noted. Concentrations chosen for cloning were 25, 75, 150, 250 and 300 ug/mL without S9, and 25, 50, 100, 250 ug/mL with S9 activation. No cloned cultures exhibited induced mutation frequencies of 90 or higher mutants per 10E6 clonable cells. There was no concentration-dependent increase in mutant frequency.

The concentrations in the extended treatment assay ranged from 5.0 to 150 ug/mL without S9 . with a 24 hour exposure. No precipitates were noted in the treatment medium. Concentrations chosen for cloning were 5.0, 10, 15, and 25 ug/mL. No cloned cultures exhibited induced mutation frequencies of 90 or higher mutants per 10E6 clonable cells. There was no concentration-dependent increase in mutant frequency.

The trifluorothymidine-resistant colonies for the positive and solvent control cultures from both assays were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS and DMBA positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods to detect small colony mutants) and large colonies.

Under the conditions of the study, the test article was considered to be negative in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay.

The test substance was tested in a chromosome aberration assay using Chinese hamster ovary (CHO) cells both in the presence and absence of an Arocolor-induced rat liver S9 metabolic activation system to evaluate clastogenic potential of the substance (OECD guideline No. 473). 

Ethanol was used as the solvent. Based on cytotoxicity in a preliminary test, doses chosen for the chromosome aberration assay ranged from 21.9 to 2850 ug/mL for the non-activated 4-hour exposure group, from 6.5 to 1000 ug/mL for the S9-activated 4-hour exposure group, and from 6.5 to 500 ug/mL for the non-activated 20-hour continuous exposure group.

           In the initial chromosome aberration assay, the cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. No precipitates of test article were seen. Selection of doses for microscopic analysis was based on toxicity (the lowest dose with at least 50% reduction in cell growth and two lower doses) in the non-activated 4 and 20 hour exposure groups. In the S9-activated 4-hour exposure group, due to inconsistent cytotoxicity, the chromosome aberration assay was repeated.

The percentage of cells with structural or numerical aberrations in the non-activated 4- and 20-hour exposure groups was not significantly increased relative to solvent control at any dose level (p>0.05, Fisher’s Exact test).

In the repeat assay, dose levels tested ranged from 175 to 2850 ug/mL. Cells were treated for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. No precipitates were seen. Selection of doses for microscopic analysis was based on toxicity (the lowest dose with at least 50% reduction in cell growth and two lower doses). The percentage of cells with structural or numerical aberrations in the S9-activated 4 hour exposure group was not significantly increased relative to solvent control at any dose level. (P > 0.05, Fisher’s Exact test).

Based on findings in this study, the test article was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells in borth the non-activated and the S-9 activated test systems.

 

The test article was evaluated for genotoxicity potential (clastogenicity/aneugenicity) as measured by its ability to increase the incidence of micronucleated polychromatic erythrocytes (MPCEs) in bone marrow of male and female Hsd:SD rats. Methods complied with OECD Guideline 474 (Genetic Toxicology: Mammalian Erythrocyte Micronucleus Test). Test article was administered orally by single oral dose gavage of a fomulation in arachis (peanut) oil, at a dose volume of 10 mL/kg. Vehicle control was the arachis oil, and positive control substance was cyclophosphamide monohydrate (50 mg/kg).

 The dose selction was based on a preliminary study. The definitive study consisted of 7 groups with 5 rats per sex in each. Five of the groups were dosed with control (vehicle or positive) substances or with the test article at 100, 200, or 400 mg/kg, and were euthanized at 24 hours post dose. Rats in the two remaining groups were dosed with either vehicle or the high dose test article formulation and euthanized at 48 hours. An additional 5 rats/sex were dosed at the high dose to be used as replacement animals if needed, but mortality did not occur, and bone marrow was not collected from that group. At the scheduled time point, femoral bone marrow was collected, and marrow smears (slides) were prepared and stained with a nucleic acid specific stain (acridine orange stain). Bone marrow cells (polychromatic erythrocytes (2000 PCEs/animal)] were examined for presence of micronuclei (micronucleated PCEs, MPCEs). Statistical analysis of the data was performed using the Kastenbaum-Bowman Tables (binomial distribution, p </= 0.05).

           No mortality was noted in the definitive study. Piloerection was noted in the 400 mg/kg group animals, and lethargy was noted in the 400 mg/kg group sacrificed at 48 hours. Reductions of up to 15% in the PCEs/ECs ratio of polychromatic erythrocytes to total erythrocytes relative to the vehicle control groups was observed in some of the test groups without a dose response, suggesting that the substance reached the bone marrow, but did not markedly inhibit erythropoiesis. No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in test substance groups relative to the respective vehicle control groups was observed in male or female rats at 24 or 48 hours post dose administration. (p > 0.05, binomial distribution, Kastenbaum-Bowman Tables). The positive control substance (cyclophosphamide monohydrate) induced a statistically significant increase in the incidence of micronucleated PCEs in male and female rats (p < 0.05, binomial distribution, Kastenbaum-Bowman Tables). The number of MPCEs in the vehicle control groups did not exceed historical control vehicle ranges. All criteria were met for a valid test.

           Under the conditions of the study, a single oral dose of test article at doses up to and including a dose of 400 mg/kg (MTD) did not induce a significant increase in micronucleated polychromatic erythrocytes in bone marrow of male and female Hsd:SD rats. Therefore, the test article was concluded to be negative in the rat micronucleus assay.


Short description of key information:
The substance was not genotoxic and cause neither gene mutations nor chromosomal aberrations in three in vitro guideline studies for genotoxicity. Furthermore a the substance did not induce micronuclei in an in vivo rat micronucleus test according to recent guidelines and GLP and thus showed no clastogenic or aneugenic potential in this test system.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Due to the negative results in three valid in vitro genotoxicity assays and one in vivo rat micronucleus assay, the substance is not classified for mutagenicity.